Abstract
Pseudomonas aeruginosa, a versatile and antibiotic-resistant gram-negative pathogen, poses a critical threat to both immunocompromised and immunocompetent populations, underscoring the urgent need for new therapeutic targets. This study applies an extensive subtractive proteomics approach to identify viable drug targets within the core proteome of P. aeruginosa PAO1, analyzing a total of 5563 proteins. Through a rigorous, multi-stage process, we excluded human homologs, identified essential proteins, mapped functional pathways, determined subcellular localization, and assessed virulence and resistance factors. This comprehensive analysis led to the identification of three novel, druggable targets integral to P. aeruginosa's pathogenicity and multidrug resistance: preprotein translocase subunit SecD, chemotaxis-specific methyl esterase, and imidazole glycerol phosphate synthase subunit HisF2. Following this, inverse virtual screening of 464,867 compounds from the VITAS-M library, performed using Schrödinger's Glide module, initially pinpointed 15 potent hits with favorable binding affinities and pharmacokinetic profiles as confirmed by QikProp analysis. Subsequent molecular dynamics, MMPBSA and DFT calculations refined these to three promising candidates: STK417467 for imidazole glycerol phosphate synthase subunit HisF2, STL321396 for chemotaxis-specific methylesterase, and STL243336 for preprotein translocase subunit SecD. These compounds show strong potential as inhibitors and could be developed further as therapeutic agents against multidrug-resistant P. aeruginosa infections. This study provides a robust computational framework for the discovery of drug targets and candidate inhibitors, marking a significant step toward effective treatments for resistant Pseudomonas infections.
Keywords: Computational biology, Pseudomonas aeruginosa, Subtractive proteomics, Antimicrobial resistance, Drug discovery, Molecular docking, Molecular dynamics, Therapeutic inhibitors
1. Introduction
Infectious diseases continue to pose a serious and urgent threat to the health of populations across the globe [1]. The World Health Organization (WHO) designates Pseudomonas aeruginosa as a “high-priority pathogen,” emphasizing the urgent need for innovative therapeutic approaches [2]. This bacterium, known for causing significant morbidity and mortality, is notoriously difficult to treat due to its intrinsic and acquired resistance mechanisms, limited treatment options, and high transmissibility. Its increasing prevalence, coupled with rising multidrug resistance, underscores its public health importance [[3], [4], [5]]. P. aeruginosa is an opportunistic pathogen frequently associated with cystic fibrosis (CF) and ventilator-associated pneumonia. Among healthcare-associated infections (HAIs), it accounts for 7.1 %–7.3 % of cases, including nosocomial pneumonia and structural lung diseases like CF [6,7]. Over the past decade, its prevalence has surged, contributing significantly to intensive care unit (ICU)-acquired infections [8,9]. Notably, P. aeruginosa causes 16.2 % of infections and 23 % of ICU-acquired illnesses, with respiratory failure being the most common site of infection. This pathogen is also responsible for a considerable proportion of catheter-associated urinary tract infections (CAUTIs) in ICU patients [10]. Moreover, in pediatric burn patients, P. aeruginosa infections account for 86 % of burn ICU fatalities [11]. Bloodstream infections (BSIs) caused by P. aeruginosa are particularly concerning, with fatality rates ranging from 43.2 % to 58.8 % [12]. The pathogenicity and resilience of P. aeruginosa stem from its multifaceted resistance mechanisms. Innate resistance includes low outer membrane permeability and efflux pumps that expel antibiotics, while acquired resistance arises from genetic mutations and horizontal gene transfer [13]. Additionally, P. aeruginosa forms biofilms on medical devices and host tissues, creating a barrier against antibiotics. These biofilms, regulated by quorum sensing systems, enhance resistance and virulence, making treatment more challenging. Enzyme promiscuity further aids adaptation under antibiotic pressure, contributing to its survival in diverse environments [14,15]. The increasing prevalence of multidrug-resistant (MDR) P. aeruginosa is alarming [16]. Despite considerable advancements in antimicrobial therapies, including FDA-approved drugs such as ceftazidime-avibactam and ceftolozane-tazobactam, along with promising agents like cefiderocol and imipenem-cilastatin/relebactam, the relentless rise of resistance remains a pressing concern [17,18]. Compounding this issue is the decline in corporate investment in antibiotic research and the scarcity of suitable molecular targets, which have considerably hindered the development of effective treatments [19]. These challenges emphasize the critical need for innovative approaches that capitalize on recent technological advancements and integrate diverse data sources. Given the myriad strategies bacteria employ to evade antibiotics, identifying novel drug targets is essential for designing next-generation antimicrobial agents and addressing the escalating threat of antimicrobial resistance (AMR). The availability of complete genome sequences for humans and various pathogenic organisms has greatly accelerated the identification of viable therapeutic targets, opening new avenues to address AMR [20]. Recently, omics-based approaches have emerged as powerful tools for gaining comprehensive insights into pathogen biology, facilitating the discovery of novel therapeutic targets. Numerous studies have demonstrated the potential of omics data, including genomics, proteomics, and metabolomics, in advancing target identification and contributing significantly to the development of effective antimicrobial strategies [21,22]. Among these, subtractive proteomics has emerged as a robust approach for identifying novel drug targets in pathogens, particularly for targeting species-specific entities while minimizing cross-reactions [23,24]. In recent years, this method has been effectively applied to uncover potential drug targets in various pathogenic bacteria, offering a focused approach to target essential proteins crucial for bacterial survival and virulence [[25], [26], [27]]. The insights gained from subtractive proteomics can be further validated through a combination of computational, laboratory-based, and animal model experiments, providing comprehensive data on the organism's essential biological functions [28]. This technique has proven particularly advantageous in identifying druggable targets that could pave the way for the development of more effective anti-infective compounds. Traditional drug discovery methods, while effective, are often labor-intensive, time-consuming, and costly. To address these limitations, in-silico approaches have emerged as a viable alternative, enabling faster, more efficient, and cost-effective discoveries. These computational techniques provide dynamic solutions to drug development, significantly streamlining the process by predicting potential drug targets, assessing their druggability, and exploring existing compounds that may be repurposed for therapeutic use [29]. In this context, several recent studies have focused on utilizing advanced in-silico methods for drug target identification and drug repurposing against multidrug-resistant P. aeruginosa strains. A recent study integrated subtractive proteomics with ensemble docking, predicting high-affinity drug candidates for the resistance-nodulation-division (RND) superfamily proteins in P. aeruginosa. This approach highlighted several FDA-approved drugs with the potential to be repurposed for combating the resistance mechanisms in the pathogen [30]. Another study combined subtractive genomics with protein-protein interaction (PPI) network analysis to identify core essential proteins critical for the virulence and survival of P. aeruginosa. This network-based analysis helped prioritize potential drug targets based on their centrality in the bacterial survival processes [24]. A third study bridged computational predictions with experimental validation, uncovering promising antimicrobial agents, including natural product derivatives, which showed potent activity against multidrug-resistant strains of P. aeruginosa. Finally, an in-silico subtractive genomics approach was applied to a range of human bacterial pathogens, identifying conserved, essential, and druggable targets [31]. These studies collectively demonstrated the potential of subtractive proteomics and in-silico methods in advancing drug target identification. While numerous recent studies have employed subtractive proteomics and in-silico methods for drug target identification in P. aeruginosa, significant gaps remain in fully exploring the pathogen's core proteome and identifying druggable targets critical to its virulence, resistance, and multidrug-resistant mechanisms. Many studies tend to focus on specific protein subsets or fail to extensively validate the identified targets using advanced computational techniques such as molecular dynamics simulations and inverse virtual screening. Moreover, while efflux pumps and membrane-bound proteins are often prioritized as potential drug targets, other key components involved in P. aeruginosa's pathogenicity have not been explored as thoroughly. In addition, comprehensive in-silico pipelines that integrate multiple stages of drug discovery—from target identification to compound screening and optimization—are still not widely applied, particularly in addressing multidrug-resistant strains of P. aeruginosa.
Our study addresses these gaps by conducting a thorough and multi-stage subtractive proteomics analysis of P. aeruginosa PAO1's core proteome. Through a comprehensive approach, we aim to uncover innovative, underexplored druggable proteins essential for the pathogen's survival and multidrug resistance. This research distinguishes itself by integrating advanced in-silico methods with a robust validation pipeline, encompassing molecular dynamics simulations, MMPBSA, and DFT calculations. By combining these techniques, our study provides a more holistic approach to target identification and compound screening. This comprehensive methodology offers a more complete framework for discovering new drug targets and developing potential therapeutic agents. Our study fills a critical gap by addressing multidrug resistance in P. aeruginosa, offering a more extensive and rigorous pathway for the discovery of effective treatments.
2. Methodology
With a genome size ranging from 5.5 to 7 Mbp, P. aeruginosa possesses one of the largest genomes among prokaryotes. This genome encodes a substantial number of proteins involved in transport, virulence, and regulatory functions. This section outlines an all-encompassing computational strategy to pinpoint potential therapeutic targets in the P. aeruginosa PAO1 strain. We focus specifically on non-homologous, essential proteins crucial for the survival of P. aeruginosa, with particular emphasis on those absent from the human proteome. These proteins present promising opportunities for developing novel antibacterial agents, especially in light of the increasing threat of AMR. The methodology combines sequence-based analyses, druggability evaluations, protein structure predictions, and molecular dynamics simulations to prioritize drug targets distinct from human proteins.
2.1. Protein sequence retrieval and dataset preparation
2.10.1. Retrieval of Protein sequences
The complete proteome of P. aeruginosa PAO1 (ATCC 15692/DSM 22644) was obtained from the UniProt database version 2024 (https://www.uniprot.org/) [32]. Only proteins with a minimum length of 50 amino acids were selected, ensuring the inclusion of full-length functional proteins. Sequences containing non-amino acid residues, such as those with frameshift mutations or ambiguous characters, were excluded from further analysis. This selection ensured the integrity and biological relevance of the data used in subsequent steps. The selected protein sequences were then formatted into FASTA files for downstream analysis. These files contained the unique identifiers for each protein, which were cross-referenced with UniProt's gene annotation system for further analysis [33].
2.1.2. Essential gene data collection
Essential genes were identified using the Database of Essential Genes (DEG-15) (http://www.essentialgene.org/), which catalogs genes that are indispensable for the survival of organisms [34]. The essential genes of P. aeruginosa were extracted from the DEG-15 database, ensuring that only those genes critical for bacterial survival were considered. This database was specifically chosen because it provides reliable, experimentally validated data on essential genes in various species, including P. aeruginosa [35].
2.2. Sequence analysis and comparative search
2.2.1. Identification of non-homologous proteins using Basic Local Alignment Search Tool (BLAST)
To identify non-homologous proteins in P. aeruginosa that could serve as therapeutic targets, protein sequences were compared against the human proteome. Basic Local Alignment Search Tool for proteins (BLASTp) was used for the sequence similarity search. The BLASTp program was run on the NCBI BLAST server (https://blast.ncbi.nlm.nih.gov/) using the default parameters with a maximum e-value of 0.005 and a minimum bit score of 100. These parameters ensured that only significant hits were retained, filtering out proteins that might exhibit strong homology with human proteins [36]. The results from the BLASTp search were manually inspected to ensure that no P. aeruginosa proteins with significant homology to human proteins were included in the list of potential drug targets. Proteins that showed no significant similarity to human sequences were considered non-homologous and selected for further analysis.
2.2.2. Database comparison
To ensure the uniqueness of the identified non-homologous proteins, a comparative sequence analysis was conducted using the P. aeruginosa proteome and the DEG-15 database, which includes essential genes from over 66 bacterial species. Sequence alignments were performed using the Blocks Substitution Matrix (BLOSUM62) to align protein sequences and identify potential homologous proteins in other bacterial species. The cut-off values used were an e-value of 10⁻⁵ and a bit score greater than 100. These parameters were selected to focus on highly conserved essential proteins that could serve as universal targets across different bacterial species [37].
2.3. Metabolic pathway analysis and subcellular localization
2.3.1. Metabolic pathway mapping
The non-homologous essential proteins identified from P. aeruginosa were mapped to known metabolic pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database version 5.0 (https://www.genome.jp/kegg/). KEGG provides comprehensive information about molecular networks, metabolic pathways, and functional modules, making it a critical tool for understanding the metabolic role of the identified proteins [38,39].
Each protein was mapped to its corresponding metabolic pathway, and manual curation was performed to confirm the presence of essential metabolic processes unique to P. aeruginosa. Pathways not present in humans were prioritized as potential targets for drug development, as targeting these could reduce the risk of off-target effects on human cells.
2.3.2. Subcellular localization prediction
To determine the cellular compartment where each essential protein functions, subcellular localization was predicted using PSORTb version 3.0.3 (https://www.psort.org/psortb/), an online tool designed specifically for bacterial protein localization prediction [40]. PSORTb categorizes proteins into one of five compartments: cytoplasm, periplasm, outer membrane, inner membrane, and extracellular space [41]. The predicted localization provided valuable insights into the potential druggable sites within P. aeruginosa that could be targeted without affecting human cellular functions.
2.4. Druggability assessment
2.4.1. Drug target identification and prioritization
The druggability of the identified non-homologous proteins was evaluated by performing a search against the DrugBank database version 5.1.12 (https://go.drugbank.com/). DrugBank is an integrated resource containing data on approved drugs, experimental drugs, and their interactions with biological targets. Proteins that did not match any known drug targets were considered novel targets for drug development.
Additionally, druggability was assessed by considering the biological function of each protein, its involvement in essential cellular processes, and its accessibility to small molecules. Proteins involved in unique and critical pathways were prioritized for drug development [42].
2.5. Virulence factor and antibiotic resistance analysis
2.5.1. Virulence factor identification
The identification of virulence factors in P. aeruginosa was performed using the Virulence Factor Database (VFDB) (http://www.mgc.ac.cn/VFs/). VFDB is a specialized database containing information about the virulence factors of bacteria [43]. Protein sequences were cross-referenced with the VFDB to identify P. aeruginosa virulence factors. Proteins that were found to be involved in virulence were prioritized for further analysis.
2.5.2. Antibiotic resistance gene analysis
The Comprehensive Antibiotic Resistance Database (CARD) version 3.2.4 (https://card.mcmaster.ca/) was employed to identify antibiotic resistance genes in P. aeruginosa [44]. Resistance-associated proteins were further examined to assess their potential interactions with human pathways. Proteins that exhibited no interaction with human pathways were deemed promising candidates for therapeutic development [45].
2.6. Broad-spectrum target identification
A comprehensive broad-spectrum target identification analysis was conducted using BLASTp to assess the homology of P. aeruginosa PAO1-derived proteins across various pathogens. The VFDB – 2022 was used to compile a list of Pseudomonas species, including multiple strains of P. aeruginosa. To identify potential broad-spectrum drug targets, proteins exhibiting significant homology across these Pseudomonas isolates and other pathogens were carefully evaluated. Specifically, 17 medically relevant Pseudomonas strains sourced from the VFDB were examined to determine if drug targets from P. aeruginosa PAO1 were conserved in other virulent Pseudomonas strains. For the broad-spectrum analysis, BLASTp was employed with stringent parameters: e-value = 0.0001, bit score >100, and sequence identity >25 %. This homology search was extended to include 240 disease-causing bacterial species, as compiled from the scientific literature. The analysis revealed several potential broad-spectrum targets, with significant homology observed among closely related pathogens. These findings provide insight into possible targets that could be therapeutically relevant across a range of pathogenic bacteria [46,47].
2.7. Protein physicochemical property analysis
The selection of the most desirable therapeutic targets extends beyond the criteria of lacking homology to human proteins and playing a vital role in the survival of the pathogen. Several additional physicochemical characteristics play a crucial role in determining the potential of a target as a drug or vaccine candidate. These characteristics include low molecular weight, the Grand Average of Hydropathicity (GRAVY), isoelectric point (pI), and aliphatic index, all of which contribute to the druggability and immunogenicity of a protein. To evaluate these parameters, we utilized the ExPASy ProtParam tool (https://web.expasy.org/protparam/) [48], which comprehensively analyzes protein properties. Furthermore, to assess the structural feasibility of the selected proteins as therapeutic targets, we investigated whether they possess resolved three-dimensional structures. This was achieved by querying the Protein Data Bank (PDB) (https://www.rcsb.org/) [49] and ModBase [50] (https://modbase.compbio.ucsf.edu/modbase-cgi/index.cgi) databases provides access to experimentally determined and computationally modeled protein structures, respectively. This structural information is critical for understanding the potential interactions of these proteins with therapeutic agents.
2.8. Functional annotation and evolutionary analysis
2.8.1. Domain analysis
Domain characterization of the protein sequences was performed using the NCBI Conserved Domain Search Service (CD Search) version 3.21 (https://www.ncbi.nlm.nih.gov/Structure/cdd/) [51] and InterProScan 101.0 (https://www.ebi.ac.uk/interpro/) [52].The CD Search tool was employed to identify conserved domains within the protein sequences. To perform this, Reverse Position-Specific BLAST (RPS-BLAST) [53] was utilized, which compares the query sequence to position-specific score matrices derived from conserved domain alignments within the Conserved Domain Database (CDD). This allowed us to determine the presence of conserved domains and further understand their functional relevance. Additionally, we used the Pfam 37.0 database (http://pfam.xfam.org/) [54] and the SCOP-Superfamily database (https://supfam.org/) [55] to infer the evolutionary connections among the proteins. Pfam is a comprehensive resource that incorporates sequence alignments and annotations derived from hidden Markov models (HMMs), allowing for the categorization of proteins into families based on shared domains. The SCOP-Superfamily database helped classify the proteins based on structural and evolutionary relationships. Protein sequence motifs were analyzed using the MOTIF server (https://www.genome.jp/tools/motif/), which identifies recurring sequence patterns that may be important for the function or structure of the proteins.
2.8.2. Phylogenetic analysis
Phylogenetic analysis was performed to investigate the evolutionary relationships among the selected proteins further. The six protein sequences retrieved from the UniProt database were aligned using the MUSCLE program within MEGA v. 11 software [56]. The resulting alignment was subjected to clustering using the Neighbor-Joining method, generating a MEGA output file. Statistical analysis was conducted using the Maximum Likelihood approach with 1000 bootstrap replicates, employing the Jones-Taylor-Thornton (JTT) model to construct a Newick tree. The generated phylogenetic tree was analyzed and visualized using the Interactive Tree of Life (iTOL) v5 [57].
2.9. Structural prediction and validation
2.9.1. Secondary structure prediction
Structural and functional predictions are crucial in identifying novel pharmacological targets for therapeutic intervention. Two computational techniques were applied to anticipate the structural folding details of the selected proteins: PSI-BLAST-based secondary structure prediction using PSIPRED 4.0 (https://bio.tools/psipred) [58] and the Self Optimized Prediction Method with Alignment (SOPMA) (https://npsaprabi.ibcp.fr/npsa/npsa_sopma.html) [59]. PSIPRED 4.0 is a well-established tool for anticipating the secondary structure of proteins through sequence homology. At the same time, SOPMA provides an alternative method for predicting secondary structures using a self-optimized approach with sequence alignment. These methods were applied to gain insights into the structural characteristics of the proteins and aid in the discovery of possible targets for therapeutic intervention.
2.9.2. Tertiary structure prediction and druggability assessment
The PDB was initially explored to acquire three-dimensional structural data for the identified six protein targets. The PDB contains experimentally resolved 3D protein structures, offering high-quality models for structural analysis [60]. For protein targets without experimentally determined structures available in the PDB, predicted 3D structure models were retrieved using the AlphaFold Protein Structure Database v3.0 by DeepMind (https://alphafold.ebi.ac.uk) [61]. The robustness and quality of the AlphaFold models were evaluated using Ramachandran plot analysis. The Ramachandran plot assesses the phi (φ) and psi (ψ) dihedral angles of amino acid residues in a protein, categorizing them into favored, allowed, and disallowed regions. A reliable protein structure typically has over 90 % of its residues in the favored region, which ensures structural validity and alignment with experimental data. To further assess the therapeutic potential of the shortlisted proteins, the SiteMap tool within the Schrödinger Suite 2023 was employed [62]. This tool evaluated the druggability of the proteins by analyzing possible binding sites and identifying features conducive to small-molecule binding. The combined application of experimental structure databases and advanced predictive tools facilitated the selected targets' comprehensive structural and functional characterization, advancing their evaluation for potential therapeutic applications.
2.10. Virtual screening and molecular docking
Identifying inhibitors for the selected protein targets was undertaken to facilitate the discovery of novel therapeutics against multidrug-resistant P. aeruginosa. This was achieved through receptor-based virtual screening. A comprehensive collection of over 1,430,000 high-throughput screening (HTS) compounds suitable for pharmaceutical and agrochemical research was sourced from the VITAS-M laboratory. A targeted subset of approximately 464,867 molecules relevant to the study's objectives was curated to streamline the analysis. The ligand library was acquired in Structured Data File (SDF) format and subsequently imported into Schrödinger's workspace for preparation. Ligand preparation involved optimizing the geometric features and generating ionization states of the compounds to achieve the physiological pH of 7.0 ± 2.0. This was accomplished using the LigPrep module within the Schrödinger Suite 2023 [63]. Molecular structures were optimized during preparation to ensure high-quality input for subsequent docking studies. For receptor preparation, the receptor grid generation panel in Schrödinger was used to define the ligand-binding sites. Based on established coordinates, binding site grids were created around the active regions of the three target proteins. The van der Waals radii of receptor atoms were scaled with a partial charge cut-off of 0.25 Å and a default scaling factor of 1.0 Å [64]. This ensured a precise definition of the receptor's interaction potential with ligands. The prepared receptor grids and ligand library were then subjected to cross-docking analysis using Schrödinger's XGlide module. The receptor grids for the three target proteins were defined within the receptor section, while the prepared ligand dataset was specified in the ligand section. Cross-docking was performed in extra precision (XP) mode using the optimized OPLS4 force field to calculate the binding affinities of the ligand-receptor interactions [65]. The docking process identified potential inhibitors by evaluating the binding affinities of ligand-protein complexes. Detailed interaction profiles were generated for the best-docked complexes, providing insights into their binding modes. These profiles facilitated the identification of high-affinity ligands and poorly bound complexes, which may serve as starting points for further investigation.
2.11. Druggability analysis of hit compounds
The druggability of hit compounds identified through molecular docking was further evaluated using the QikProp module integrated into Maestro 13.1 [66]. This computational tool predicts an array of physicochemical and pharmacokinetic parameters, enabling a comprehensive assessment of the drug-likeness of the identified leads. By providing insights into key attributes critical for drug development, this analysis supports the identification of compounds with favorable profiles for further investigation. The evaluation encompassed several physicochemical parameters, including molecular weight (mol_MW), the number of hydrogen bond acceptors (accptHB), hydrogen bond donors (donorHB), and the predicted percentage of human oral absorption. These parameters are essential for determining the potential oral bioavailability of the compounds. Additionally, pharmacokinetic properties were assessed to predict the leads' behavior in a biological system. This included predicted aqueous solubility (QPlogS), which evaluates the solubility of compounds in water, and the predicted octanol/water partition coefficient (QPlogPo/w), which estimates lipophilicity—a critical determinant for membrane permeability and drug distribution. The predicted apparent Caco-2 cell permeability (QPPCaco) provided insights into intestinal absorption, an important aspect of oral drug delivery. Furthermore, the estimated IC50 values for the inhibition of human ether-à-go-go-related gene (HERG) K+ channels (QPlogHERG) were calculated to assess potential cardiotoxicity risks associated with the compounds. By integrating these evaluations, the QikProp analysis provided a robust framework for understanding the physicochemical and pharmacokinetic profiles of the hit compounds, guiding the prioritization of promising candidates for subsequent validation and optimization in the drug discovery pipeline.
2.12. Molecular dynamics simulation
To investigate the protein's real-time dynamics and conformational stability upon ligand binding, 100-ns (ns) molecular dynamics (MD) simulations were conducted on the docked complexes using the Desmond module integrated within the Schrödinger Suite. This computational approach facilitated a detailed assessment of molecular interactions and structural fluctuations over time, providing insights into the stability and dynamics of the protein-ligand complexes. The initial configuration for MD simulations was prepared using the System Builder Panel, where each protein-ligand complex was positioned within a 10 Å orthorhombic simulation box to ensure sufficient space for solvent molecules and other system components. The system was solvated using the Single-Point-Charge (SPC) explicit solvent model to represent water molecules accurately. To maintain an isosmotic environment and ensure charge neutrality, sodium (Na⁺) and chloride (Cl⁻) counter-ions were introduced at a physiological concentration of 0.15 M, mimicking biological conditions. Energy minimization and equilibration were performed using Desmond's default protocol under the isothermal-isobaric (NPT) ensemble, with the temperature set at 300 K using the Nose-Hoover thermostat and pressure controlled at 1 atm using the Martyna-Tobias-Klein barostat. The MD simulations were conducted for a duration of 100 ns, and the resulting trajectory data were analyzed using the Simulation Interaction Diagram (SID) module. Several key structural and dynamic stability metrics, including Root Mean Square Deviation (RMSD), Protein Root Mean Square Fluctuation (P-RMSF), Ligand Root Mean Square Fluctuation (L-RMSF), Radius of Gyration (Rg), Molecular Surface Area (MolSA), Solvent Accessible Surface Area (SASA), and Polar Surface Area (PSA), were assessed to evaluate the system's stability and conformational changes. The RMSD provided valuable insights into the overall stability of the complex and its conformational deviations over time. Meanwhile, P-RMSF revealed the flexibility of individual amino acid residues, pinpointing regions of high mobility or structural rigidity. The L-RMSF analysis examined the stability and binding dynamics of the ligand within the active site, quantifying its positional fluctuations to infer binding affinity and the stability of ligand-receptor interactions. The Rg reflects the 'extendedness' of the ligand, corresponding to its principal moment of inertia during binding. MolSA represents the van der Waals surface area of the molecule. SASA indicates the surface area accessible to water molecules, while PSA refers explicitly to the solvent-accessible surface area contributed by oxygen and nitrogen atoms, highlighting polar interactions. These analyses collectively provided a comprehensive understanding of the protein-ligand interactions, conformational dynamics, and stability of the complexes, contributing to identifying potential therapeutic targets [67,68].
2.13. Molecular Mechanics-Poisson Boltzmann surface area (MMPBSA) analysis
Binding free energy (ΔG) calculations for the final frames of stable protein-ligand complexes obtained from the MD simulations were performed using the Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) methodology implemented in the farPPI (Fast Amber Rescoring for Protein-Protein Interaction Inhibitors) web server (http://cadd.zju.edu.cn/farppi/) [69]. The MM-PBSA method integrates molecular mechanics predictions with implicit solvent models to provide a reliable assessment of interaction energies in docked configurations [70]. In this study, the General Amber Force Field 2 (GAFF2) was employed for the ligands, while the ff14SB force field was applied to the proteins, ensuring accurate energy computations. The PB3 method within the farPPI framework was employed for MM-PBSA analysis, as it offers enhanced accuracy compared to alternative computational approaches [71]. By leveraging this methodology, the binding free energy of protein-ligand complexes was estimated, providing an essential factor for assessing the effectiveness of small molecule inhibitors. These calculations were instrumental in prioritizing the most potential candidates for future experimental verification, thereby advancing the development of therapeutics targeting multidrug-resistant P. aeruginosa.
2.14. Density Functional Theory calculations
To evaluate the reactivity and stability of the selected candidate compounds, Density Functional Theory (DFT) studies were conducted. DFT is a widely adopted computational approach for investigating the properties of compounds with inhibitory characteristics. The hybrid DFT method, utilizing Becke's three-parameter exchange potential in combination with the 6–31 G∗∗ functional and Lee-Yang-Parr correlation theory (B3LYP), was applied in Jaguar to optimize generated molecules geometrically [72]. The electronic properties of these molecules were analyzed in electron volts, and the global reactivity, along with the frontier molecular orbitals, was determined (eV) [73]. DFT calculations were further employed to assess the quantum mechanical properties of the ligands using the Jaguar module within the Schrödinger Interface [74]. This methodology enabled a thorough DFT analysis, providing insights into ligand binding characteristics and enhancing the overall understanding of their reactivity and stability.
3. Results
A computational hierarchical method was employed to pinpoint potential therapeutic targets for Pseudomonas species (Fig. 1). This process utilizes functionally essential proteins from the pathogen's genome as input to identify several viable therapeutic targets for P. aeruginosa.
Fig. 1.
Experimental Workflow for therapeutic target identification in multi-drug resistant P. aeruginosa.
3.1. Protein sequence retrieval and dataset preparation
3.1.1. Protein data retrieval and sequence analysis
The complete proteome of P. aeruginosa PAO1 strain (ATCC 15692/DSM 22644) was retrieved from the UniProt database. The initial dataset contained 5563 protein sequences (Supplementary Data). Proteins with non-amino acid residues, ambiguous characters, or lengths below 50 amino acids were excluded to maintain data integrity and biological relevance. This filtering process resulted in a refined dataset of 5549 proteins formatted into FASTA files for downstream analysis. Each protein was annotated using UniProt identifiers for sequence comparisons and functional analyses.
3.2. Sequence analysis and comparative search
3.2.1. Comparative analysis and identification of non-homologous proteins using BLAST
To identify proteins with therapeutic potential, a non-homology analysis was conducted to exclude proteins similar to those in the human proteome. The BLASTp tool was used with parameters set to an e-value threshold of 0.005 and a bit score of at least 100. Among the 5549 proteins analyzed, 394 showed significant homology to human proteins and were excluded from further analysis. The remaining 5155 proteins were identified as non-homologous and selected for subsequent evaluations of their essentiality and druggability (Table 1).
Table 1.
Identified Non-Homologous Proteins of P. aeruginosa PAO1 using BLASTp Analysis.
| S.No | Uniprot ID | S.No | Uniprot ID | S.No | Uniprot ID | S.No | Uniprot ID | S.No | Uniprot ID |
|---|---|---|---|---|---|---|---|---|---|
| 1 | G3XCV0 | 1083 | G3XD73 | 2165 | Q9I6T4 | 3247 | Q9HXS1 | 4329 | Q9I2J1 |
| 2 | G3XCX3 | 1084 | G3XD77 | 2166 | Q9I6T9 | 3248 | Q9HXS2 | 4330 | Q9I2J3 |
| 3 | G3XCY4 | 1085 | G3XD84 | 2167 | Q9I6U4 | 3249 | Q9HXS3 | 4331 | Q9I2J5 |
| 4 | G3XCY6 | 1086 | G3XD87 | 2168 | Q9I6V4 | 3250 | Q9HXS5 | 4332 | Q9I2J6 |
| 5 | G3XD01 | 1087 | G3XD90 | 2169 | Q9I6V8 | 3251 | Q9HXS6 | 4333 | Q9I2J7 |
| 6 | G3XD23 | 1088 | G3XDA5 | 2170 | Q9I6W0 | 3252 | Q9HXS7 | 4334 | Q9I2J8 |
| 7 | G3XD24 | 1089 | G3XDA7 | 2171 | Q9I6Y1 | 3253 | Q9HXS8 | 4335 | Q9I2K0 |
| 8 | G3XD94 | 1090 | G3XDB2 | 2172 | Q9I6Y3 | 3254 | Q9HXS9 | 4336 | Q9I2K1 |
| 9 | G3XD97 | 1091 | O31038 | 2173 | Q9I6Y9 | 3255 | Q9HXT0 | 4337 | Q9I2K2 |
| 10 | G3XDA8 | 1092 | O52658 | 2174 | Q9I6Z8 | 3256 | Q9HXT1 | 4338 | Q9I2K3 |
| 11 | O05927 | 1093 | O52759 | 2175 | Q9I701 | 3257 | Q9HXT2 | 4339 | Q9I2K5 |
| 12 | O30508 | 1094 | O52761 | 2176 | Q9I713 | 3258 | Q9HXT3 | 4340 | Q9I2K6 |
| 13 | O33407 | 1095 | O68281 | 2177 | Q9I714 | 3259 | Q9HXT4 | 4341 | Q9I2K7 |
| 14 | O50274 | 1096 | O68283 | 2178 | Q9I724 | 3260 | Q9HXT6 | 4342 | Q9I2K8 |
| 15 | O69078 | 1097 | O68560 | 2179 | Q9I725 | 3261 | Q9HXU1 | 4343 | Q9I2K9 |
| 16 | P00282 | 1098 | O68823 | 2180 | Q9I729 | 3262 | Q9HXU2 | 4344 | Q9I2L0 |
| 17 | P04739 | 1099 | O68826 | 2181 | Q9I730 | 3263 | Q9HXU3 | 4345 | Q9I2L2 |
| 18 | P07874 | 1100 | O68877 | 2182 | Q9I732 | 3264 | Q9HXU4 | 4346 | Q9I2L3 |
| 19 | P08308 | 1101 | O69753 | 2183 | Q9I738 | 3265 | Q9HXU5 | 4347 | Q9I2L6 |
| 20 | P09785 | 1102 | O82850 | 2184 | Q9I740 | 3266 | Q9HXU6 | 4348 | Q9I2L7 |
| 21 | P0DPC1 | 1103 | O82851 | 2185 | Q9I744 | 3267 | Q9HXU8 | 4349 | Q9I2L8 |
| 22 | P11439 | 1104 | O82853 | 2186 | Q9I746 | 3268 | Q9HXU9 | 4350 | Q9I2L9 |
| 23 | P11720 | 1105 | O85732 | 2187 | Q9I753 | 3269 | Q9HXV1 | 4351 | Q9I2M0 |
| 24 | P11759 | 1106 | O87005 | 2188 | Q9I755 | 3270 | Q9HXV2 | 4352 | Q9I2M2 |
| 25 | P14532 | 1107 | O87014 | 2189 | Q9I757 | 3271 | Q9HXV5 | 4353 | Q9I2M3 |
| 26 | P14756 | 1108 | O87131 | 2190 | Q9I765 | 3272 | Q9HXV6 | 4354 | Q9I2M6 |
| 27 | P14789 | 1109 | P05384 | 2191 | Q9I778 | 3273 | Q9HXV7 | 4355 | Q9I2M8 |
| 28 | P20582 | 1110 | P07345 | 2192 | Q9I781 | 3274 | Q9HXV8 | 4356 | Q9I2N1 |
| 29 | P20586 | 1111 | P09852 | 2193 | Q9I787 | 3275 | Q9HXV9 | 4357 | Q9I2N5 |
| 30 | P22608 | 1112 | P0C2B2 | 2194 | Q9I792 | 3276 | Q9HXW1 | 4358 | Q9I2N6 |
| 31 | P22610 | 1113 | P0DTB6 | 2195 | Q9I7A0 | 3277 | Q9HXW4 | 4359 | Q9I2N7 |
| 32 | P23747 | 1114 | P11221 | 2196 | Q9I7A4 | 3278 | Q9HXW5 | 4360 | Q9I2N8 |
| 33 | P24474 | 1115 | P17323 | 2197 | Q9I7B3 | 3279 | Q9HXW6 | 4361 | Q9I2P0 |
| 34 | P24559 | 1116 | P20577 | 2198 | Q9I7B5 | 3280 | Q9HXW8 | 4362 | Q9I2P1 |
| 35 | P25084 | 1117 | P21628 | 2199 | Q9I7B7 | 3281 | Q9HXW9 | 4363 | Q9I2P2 |
| 36 | P26275 | 1118 | P24562 | 2200 | Q9I7B8 | 3282 | Q9HXX0 | 4364 | Q9I2P3 |
| 37 | P26276 | 1119 | P24908 | 2201 | Q9I7C3 | 3283 | Q9HXX1 | 4365 | Q9I2P5 |
| 38 | P26876 | 1120 | P28809 | 2202 | Q9I7C5 | 3284 | Q9HXX2 | 4366 | Q9I2P6 |
| 39 | P26993 | 1121 | P29363 | 2203 | Q9RMT1 | 3285 | Q9HXX4 | 4367 | Q9I2P7 |
| 41 | P32722 | 1122 | P29436 | 2204 | Q9RPF2 | 3286 | Q9HXX5 | 4368 | Q9I2P9 |
| 1123 | P33641 | 2205 | Q9RPF3 | 3287 | Q9HXX6 | 4369 | Q9I2Q3 | ||
| 42 | P33639 | 1124 | P34750 | 2206 | Q9RPT0 | 3288 | Q9HXX7 | 4370 | Q9I2Q4 |
| 43 | P35482 | 1125 | P37799 | 2207 | Q9S508 | 3289 | Q9HXX8 | 4371 | Q9I2Q5 |
| 44 | P35483 | 1126 | P37860 | 2208 | Q9X2T1 | 3290 | Q9HXY0 | 4372 | Q9I2Q8 |
| 45 | P35818 | 1127 | P38102 | 2209 | Q9X4P2 | 3291 | Q9HXZ0 | 4373 | Q9I2Q9 |
| 46 | P38100 | 1128 | P40695 | 2210 | Q9X6W6 | 3292 | Q9HXZ8 | 4374 | Q9I2R0 |
| 47 | P45684 | 1129 | P42378 | 2211 | Q9XCX7 | 3293 | Q9HXZ9 | 4375 | Q9I2R1 |
| 48 | P48636 | 1130 | P42513 | 2212 | Q9ZAA0 | 3294 | Q9HY00 | 4376 | Q9I2R2 |
| 49 | P51691 | 1131 | P43335 | 2213 | Q9ZIJ1 | 3295 | Q9HY09 | 4377 | Q9I2R3 |
| 50 | P52002 | 1132 | P43501 | 2214 | B5WWL1 | 3296 | Q9HY10 | 4378 | Q9I2R4 |
| 51 | P52003 | 1133 | P43502 | 2215 | E1JGJ6 | 3297 | Q9HY11 | 4379 | Q9I2R5 |
| 52 | P52477 | 1134 | P43903 | 2216 | E1JGJ7 | 3298 | Q9HY12 | 4380 | Q9I2R6 |
| 53 | P54292 | 1135 | P45682 | 2217 | E1JGJ9 | 3299 | Q9HY13 | 4381 | Q9I2R7 |
| 54 | P55222 | 1136 | P47203 | 2218 | E1JGK0 | 3300 | Q9HY14 | 4382 | Q9I2R8 |
| 55 | P72131 | 1137 | P50598 | 2219 | G3XCT4 | 3301 | Q9HY15 | 4383 | Q9I2R9 |
| 56 | P77915 | 1138 | P50599 | 2220 | G3XCT5 | 3302 | Q9HY17 | 4384 | Q9I2S0 |
| 57 | P95434 | 1139 | P50601 | 2221 | G3XCT8 | 3303 | Q9HY18 | 4385 | Q9I2S1 |
| 58 | Q00512 | 1140 | P50987 | 2222 | G3XCT9 | 3304 | Q9HY20 | 4386 | Q9I2S4 |
| 59 | Q00514 | 1141 | P52024 | 2223 | G3XCU0 | 3305 | Q9HY21 | 4387 | Q9I2S6 |
| 60 | Q00515 | 1142 | P52111 | 2224 | G3XCU1 | 3306 | Q9HY27 | 4388 | Q9I2S8 |
| 61 | Q00516 | 1143 | P52112 | 2225 | G3XCU3 | 3307 | Q9HY28 | 4389 | Q9I2T0 |
| 62 | Q00517 | 1144 | P57109 | 2226 | G3XCU4 | 3308 | Q9HY29 | 4390 | Q9I2T2 |
| 63 | Q03023 | 1145 | P57683 | 2227 | G3XCU5 | 3309 | Q9HY31 | 4391 | Q9I2T3 |
| 64 | Q03456 | 1146 | P57686 | 2228 | G3XCU7 | 3310 | Q9HY32 | 4392 | Q9I2T4 |
| 65 | Q05098 | 1147 | P57701 | 2229 | G3XCU8 | 3311 | Q9HY33 | 4393 | Q9I2T5 |
| 66 | Q06198 | 1148 | P57707 | 2230 | G3XCV3 | 3312 | Q9HY34 | 4394 | Q9I2T6 |
| 67 | Q06749 | 1149 | P65116 | 2231 | G3XCV5 | 3313 | Q9HY35 | 4395 | Q9I2U5 |
| 68 | Q51366 | 1150 | P72157 | 2232 | G3XCV8 | 3314 | Q9HY36 | 4396 | Q9I2V1 |
| 69 | Q51371 | 1151 | P72171 | 2233 | G3XCV9 | 3315 | Q9HY37 | 4397 | Q9I2V2 |
| 70 | Q51372 | 1152 | Q01602 | 2234 | G3XCW0 | 3316 | Q9HY38 | 4398 | Q9I2V3 |
| 71 | Q51424 | 1153 | Q01610 | 2235 | G3XCW1 | 3317 | Q9HY40 | 4399 | Q9I2V4 |
| 72 | Q51487 | 1154 | Q03026 | 2236 | G3XCW4 | 3318 | Q9HY43 | 4400 | Q9I2V6 |
| 73 | Q51507 | 1155 | Q04633 | 2237 | G3XCW5 | 3319 | Q9HY44 | 4401 | Q9I2V7 |
| 74 | Q51548 | 1156 | Q06552 | 2238 | G3XCW7 | 3320 | Q9HY48 | 4402 | Q9I2V8 |
| 75 | Q59636 | 1157 | Q06553 | 2239 | G3XCX0 | 3321 | Q9HY50 | 4403 | Q9I2W2 |
| 76 | Q59638 | 1158 | Q06579 | 2240 | G3XCX2 | 3322 | Q9HY51 | 4404 | Q9I2W6 |
| 77 | Q59643 | 1159 | Q14T74 | 2241 | G3XCX4 | 3323 | Q9HY52 | 4405 | Q9I2W8 |
| 78 | Q9HTC0 | 1160 | Q51382 | 2242 | G3XCX5 | 3324 | Q9HY53 | 4406 | Q9I2X1 |
| 79 | Q9HTH8 | 1161 | Q51389 | 2243 | G3XCX8 | 3325 | Q9HY54 | 4407 | Q9I2X2 |
| 80 | Q9HTI8 | 1162 | Q51391 | 2244 | G3XCX9 | 3326 | Q9HY72 | 4408 | Q9I2X3 |
| 82 | Q9HTN2 | 1163 | Q51416 | 2245 | G3XCY0 | 3327 | Q9HY73 | 4409 | Q9I2X4 |
| 1164 | Q51417 | 2246 | G3XCY1 | 3328 | Q9HY74 | 4410 | Q9I2X5 | ||
| 83 | Q9HTQ0 | 1165 | Q51423 | 2247 | G3XCY3 | 3329 | Q9HY76 | 4411 | Q9I2X6 |
| 84 | Q9HTR2 | 1166 | Q51462 | 2248 | G3XCY9 | 3330 | Q9HY78 | 4412 | Q9I2X7 |
| 85 | Q9HU05 | 1167 | Q51463 | 2249 | G3XCZ1 | 3331 | Q9HY83 | 4413 | Q9I2X8 |
| 86 | Q9HU15 | 1168 | Q51464 | 2250 | G3XCZ3 | 3332 | Q9HY86 | 4414 | Q9I2X9 |
| 87 | Q9HU21 | 1169 | Q51465 | 2251 | G3XCZ4 | 3333 | Q9HY89 | 4415 | Q9I2Y0 |
| 88 | Q9HU77 | 1170 | Q51466 | 2252 | G3XCZ7 | 3334 | Q9HY90 | 4416 | Q9I2Y1 |
| 89 | Q9HUF7 | 1171 | Q51468 | 2253 | G3XCZ9 | 3335 | Q9HY91 | 4417 | Q9I2Y3 |
| 90 | Q9HUI9 | 1172 | Q51480 | 2254 | G3XD02 | 3336 | Q9HY93 | 4418 | Q9I2Y4 |
| 91 | Q9HUK9 | 1173 | Q51484 | 2255 | G3XD03 | 3337 | Q9HY94 | 4419 | Q9I2Y6 |
| 92 | Q9HUU1 | 1174 | Q51551 | 2256 | G3XD06 | 3338 | Q9HY95 | 4420 | Q9I2Y8 |
| 93 | Q9HV27 | 1175 | Q51553 | 2257 | G3XD07 | 3339 | Q9HY96 | 4421 | Q9I2Y9 |
| 94 | Q9HVD1 | 1176 | Q51576 | 2258 | G3XD08 | 3340 | Q9HY97 | 4422 | Q9I2Z0 |
| 95 | Q9HVM5 | 1177 | Q59637 | 2259 | G3XD09 | 3341 | Q9HY98 | 4423 | Q9I2Z1 |
| 96 | Q9HVW0 | 1178 | Q59649 | 2260 | G3XD10 | 3342 | Q9HY99 | 4424 | Q9I2Z2 |
| 97 | Q9HW91 | 1179 | Q7DC81 | 2261 | G3XD22 | 3343 | Q9HYA0 | 4425 | Q9I2Z3 |
| 98 | Q9HWC1 | 1180 | Q7DC82 | 2262 | G3XD25 | 3344 | Q9HYA1 | 4426 | Q9I2Z4 |
| 99 | Q9HWH2 | 1181 | Q820A5 | 2263 | G3XD26 | 3345 | Q9HYA2 | 4427 | Q9I2Z5 |
| 100 | Q9HWK6 | 1182 | Q9HI36 | 2264 | G3XD27 | 3346 | Q9HYA3 | 4428 | Q9I2Z6 |
| 101 | Q9HWS0 | 1183 | Q9HI37 | 2265 | G3XD32 | 3347 | Q9HYA5 | 4429 | Q9I2Z7 |
| 102 | Q9HWT6 | 1184 | Q9HT05 | 2266 | G3XD33 | 3348 | Q9HYA6 | 4430 | Q9I2Z8 |
| 103 | Q9HX69 | 1185 | Q9HT10 | 2267 | G3XD37 | 3349 | Q9HYA7 | 4431 | Q9I2Z9 |
| 104 | Q9HXE3 | 1186 | Q9HT12 | 2268 | G3XD38 | 3350 | Q9HYA8 | 4432 | Q9I300 |
| 105 | Q9HXI4 | 1187 | Q9HT13 | 2269 | G3XD39 | 3351 | Q9HYB0 | 4433 | Q9I301 |
| 106 | Q9HXJ2 | 1188 | Q9HT14 | 2270 | G3XD41 | 3352 | Q9HYB1 | 4434 | Q9I302 |
| 107 | Q9HXM1 | 1189 | Q9HT16 | 2271 | G3XD42 | 3353 | Q9HYB2 | 4435 | Q9I303 |
| 108 | Q9HYC5 | 1190 | Q9HT17 | 2272 | G3XD44 | 3354 | Q9HYB3 | 4436 | Q9I304 |
| 109 | Q9HYF1 | 1191 | Q9HT19 | 2273 | G3XD45 | 3355 | Q9HYC1 | 4437 | Q9I305 |
| 110 | Q9HZ76 | 1192 | Q9HT24 | 2274 | G3XD48 | 3356 | Q9HYC6 | 4438 | Q9I306 |
| 111 | Q9HZE0 | 1193 | Q9HT32 | 2275 | G3XD52 | 3357 | Q9HYD1 | 4439 | Q9I307 |
| 112 | Q9HZJ2 | 1194 | Q9HT33 | 2276 | G3XD55 | 3358 | Q9HYD4 | 4440 | Q9I308 |
| 113 | Q9HZK0 | 1195 | Q9HT35 | 2277 | G3XD56 | 3359 | Q9HYD6 | 4441 | Q9I309 |
| 114 | Q9HZP8 | 1196 | Q9HT36 | 2278 | G3XD57 | 3360 | Q9HYD7 | 4442 | Q9I313 |
| 115 | Q9HZQ8 | 1197 | Q9HT38 | 2279 | G3XD59 | 3361 | Q9HYD8 | 4443 | Q9I316 |
| 116 | Q9I060 | 1198 | Q9HT45 | 2280 | G3XD60 | 3362 | Q9HYD9 | 4444 | Q9I318 |
| 117 | Q9I0F2 | 1199 | Q9HT50 | 2281 | G3XD62 | 3363 | Q9HYE0 | 4445 | Q9I320 |
| 119 | Q9I191 | 1200 | Q9HT62 | 2282 | G3XD65 | 3364 | Q9HYE1 | 4446 | Q9I323 |
| 1201 | Q9HT72 | 2283 | G3XD68 | 3365 | Q9HYE2 | 4447 | Q9I325 | ||
| 120 | Q9I194 | 1202 | Q9HT75 | 2284 | G3XD69 | 3366 | Q9HYE3 | 4448 | Q9I326 |
| 121 | Q9I1H3 | 1203 | Q9HT76 | 2285 | G3XD70 | 3367 | Q9HYE5 | 4449 | Q9I328 |
| 122 | Q9I1K1 | 1204 | Q9HT81 | 2286 | G3XD71 | 3368 | Q9HYE6 | 4450 | Q9I329 |
| 123 | Q9I1X7 | 1205 | Q9HT86 | 2287 | G3XD72 | 3369 | Q9HYE8 | 4451 | Q9I331 |
| 124 | Q9I234 | 1206 | Q9HT87 | 2288 | G3XD75 | 3370 | Q9HYE9 | 4452 | Q9I332 |
| 126 | Q9I2C5 | 1207 | Q9HT89 | 2289 | G3XD79 | 3371 | Q9HYF3 | 4453 | Q9I333 |
| 1208 | Q9HT91 | 2290 | G3XD81 | 3372 | Q9HYF5 | 4454 | Q9I334 | ||
| 127 | Q9I2N0 | 1209 | Q9HT95 | 2291 | G3XD82 | 3373 | Q9HYF8 | 4455 | Q9I335 |
| 128 | Q9I2T9 | 1210 | Q9HT97 | 2292 | G3XD83 | 3374 | Q9HYG0 | 4456 | Q9I336 |
| 129 | Q9I2Y2 | 1211 | Q9HT99 | 2293 | G3XD85 | 3375 | Q9HYG3 | 4457 | Q9I337 |
| 130 | Q9I3T5 | 1212 | Q9HTB1 | 2294 | G3XD86 | 3376 | Q9HYG5 | 4458 | Q9I343 |
| 131 | Q9I433 | 1213 | Q9HTB2 | 2295 | G3XD89 | 3377 | Q9HYG6 | 4459 | Q9I345 |
| 132 | Q9I485 | 1214 | Q9HTB4 | 2296 | G3XD91 | 3378 | Q9HYH0 | 4460 | Q9I346 |
| 133 | Q9I492 | 1215 | Q9HTB5 | 2297 | G3XD92 | 3379 | Q9HYH2 | 4461 | Q9I348 |
| 134 | Q9I4E3 | 1216 | Q9HTB8 | 2298 | G3XD93 | 3380 | Q9HYH3 | 4462 | Q9I349 |
| 135 | Q9I4G8 | 1217 | Q9HTC2 | 2299 | G3XD95 | 3381 | Q9HYH4 | 4463 | Q9I350 |
| 136 | Q9I4K0 | 1218 | Q9HTD0 | 2300 | G3XD96 | 3382 | Q9HYH6 | 4464 | Q9I353 |
| 137 | Q9I4L4 | 1219 | Q9HTD2 | 2301 | G3XD99 | 3383 | Q9HYH7 | 4465 | Q9I355 |
| 138 | Q9I4L5 | 1220 | Q9HTD7 | 2302 | G3XDA0 | 3384 | Q9HYH9 | 4466 | Q9I356 |
| 139 | Q9I4L6 | 1221 | Q9HTD9 | 2303 | G3XDA4 | 3385 | Q9HYI0 | 4467 | Q9I358 |
| 141 | Q9I509 | 1222 | Q9HTE2 | 2304 | G3XDA6 | 3386 | Q9HYI1 | 4468 | Q9I359 |
| 1223 | Q9HTE4 | 2305 | G3XDA9 | 3387 | Q9HYI2 | 4469 | Q9I360 | ||
| 142 | Q9I527 | 1224 | Q9HTE6 | 2306 | G3XDB1 | 3388 | Q9HYI3 | 4470 | Q9I361 |
| 143 | Q9I596 | 1225 | Q9HTG5 | 2307 | O30372 | 3389 | Q9HYI4 | 4471 | Q9I362 |
| 144 | Q9I5F3 | 1226 | Q9HTG6 | 2308 | O68561 | 3390 | Q9HYI5 | 4472 | Q9I364 |
| 145 | Q9I5I6 | 1227 | Q9HTG8 | 2309 | O68827 | 3391 | Q9HYI6 | 4473 | Q9I365 |
| 146 | Q9I5I9 | 1228 | Q9HTH4 | 2310 | P23205 | 3392 | Q9HYI9 | 4474 | Q9I366 |
| 147 | Q9I5W4 | 1229 | Q9HTI5 | 2311 | P25254 | 3393 | Q9HYJ1 | 4475 | Q9I367 |
| 148 | Q9I609 | 1230 | Q9HTI9 | 2312 | P29370 | 3394 | Q9HYJ2 | 4476 | Q9I368 |
| 149 | Q9I6H0 | 1231 | Q9HTJ0 | 2313 | P39879 | 3395 | Q9HYJ3 | 4477 | Q9I369 |
| 150 | Q9I6M7 | 1232 | Q9HTJ3 | 2314 | P40882 | 3396 | Q9HYJ4 | 4478 | Q9I370 |
| 151 | Q9I6N5 | 1233 | Q9HTJ5 | 2315 | P42514 | 3397 | Q9HYJ6 | 4479 | Q9I371 |
| 152 | Q9I6V6 | 1234 | Q9HTK1 | 2316 | P42810 | 3398 | Q9HYK0 | 4480 | Q9I372 |
| 153 | Q9I6V9 | 1235 | Q9HTK5 | 2317 | P58040 | 3399 | Q9HYK1 | 4481 | Q9I373 |
| 154 | Q9I700 | 1236 | Q9HTL0 | 2318 | P95453 | 3400 | Q9HYK2 | 4482 | Q9I374 |
| 155 | Q9Z4J7 | 1237 | Q9HTM0 | 2319 | Q01609 | 3401 | Q9HYK3 | 4483 | Q9I375 |
| 156 | Q9ZN70 | 1238 | Q9HTM1 | 2320 | Q03268 | 3402 | Q9HYK4 | 4484 | Q9I376 |
| 157 | G3XCV2 | 1239 | Q9HTN0 | 2321 | Q03381 | 3403 | Q9HYK5 | 4485 | Q9I377 |
| 158 | G3XCX7 | 1240 | Q9HTN1 | 2322 | Q04628 | 3404 | Q9HYK6 | 4486 | Q9I378 |
| 159 | G3XCY8 | 1241 | Q9HTN5 | 2323 | Q51384 | 3405 | Q9HYK8 | 4487 | Q9I379 |
| 160 | G3XCZ8 | 1242 | Q9HTN8 | 2324 | Q51483 | 3406 | Q9HYL0 | 4488 | Q9I380 |
| 161 | G3XD12 | 1243 | Q9HTN9 | 2325 | Q9HT08 | 3407 | Q9HYL4 | 4489 | Q9I381 |
| 162 | G3XD28 | 1244 | Q9HTP6 | 2326 | Q9HT11 | 3408 | Q9HYL5 | 4490 | Q9I384 |
| 163 | G3XD30 | 1245 | Q9HTR0 | 2327 | Q9HT23 | 3409 | Q9HYL6 | 4491 | Q9I385 |
| 164 | G3XD43 | 1246 | Q9HTR3 | 2328 | Q9HT26 | 3410 | Q9HYL8 | 4492 | Q9I386 |
| 165 | G3XD46 | 1247 | Q9HTR4 | 2329 | Q9HT27 | 3411 | Q9HYL9 | 4493 | Q9I390 |
| 166 | G3XD51 | 1248 | Q9HTR5 | 2330 | Q9HT28 | 3412 | Q9HYM0 | 4494 | Q9I391 |
| 167 | G3XD61 | 1249 | Q9HTR6 | 2331 | Q9HT29 | 3413 | Q9HYM1 | 4495 | Q9I392 |
| 168 | G3XD64 | 1250 | Q9HTR7 | 2332 | Q9HT30 | 3414 | Q9HYM2 | 4496 | Q9I393 |
| 169 | G3XD67 | 1251 | Q9HTS5 | 2333 | Q9HT31 | 3415 | Q9HYM3 | 4497 | Q9I394 |
| 170 | G3XD76 | 1252 | Q9HTT2 | 2334 | Q9HT34 | 3416 | Q9HYM5 | 4498 | Q9I395 |
| 171 | G3XD78 | 1253 | Q9HTT8 | 2335 | Q9HT37 | 3417 | Q9HYM6 | 4499 | Q9I396 |
| 172 | G3XDA1 | 1254 | Q9HTU3 | 2336 | Q9HT39 | 3418 | Q9HYM7 | 4500 | Q9I397 |
| 173 | O33877 | 1255 | Q9HTU5 | 2337 | Q9HT40 | 3419 | Q9HYM9 | 4501 | Q9I399 |
| 175 | O68282 | 1256 | Q9HTU6 | 2338 | Q9HT41 | 3420 | Q9HYN0 | 4502 | Q9I3A0 |
| 1257 | Q9HTU8 | 2339 | Q9HT42 | 3421 | Q9HYN1 | 4503 | Q9I3A1 | ||
| 176 | O86062 | 1258 | Q9HTV7 | 2340 | Q9HT43 | 3422 | Q9HYN2 | 4504 | Q9I3A2 |
| 177 | O86428 | 1259 | Q9HTW2 | 2341 | Q9HT44 | 3423 | Q9HYN3 | 4505 | Q9I3A3 |
| 178 | O87125 | 1260 | Q9HTW3 | 2342 | Q9HT46 | 3424 | Q9HYN4 | 4506 | Q9I3A4 |
| 179 | P00099 | 1261 | Q9HTX5 | 2343 | Q9HT47 | 3425 | Q9HYN5 | 4507 | Q9I3A5 |
| 180 | P06200 | 1262 | Q9HTX6 | 2344 | Q9HT48 | 3426 | Q9HYN6 | 4508 | Q9I3A6 |
| 181 | P09786 | 1263 | Q9HTY2 | 2345 | Q9HT51 | 3427 | Q9HYN7 | 4509 | Q9I3A7 |
| 182 | P0DPB9 | 1264 | Q9HTY3 | 2346 | Q9HT52 | 3428 | Q9HYN8 | 4510 | Q9I3B0 |
| 183 | P11436 | 1265 | Q9HTY5 | 2347 | Q9HT53 | 3429 | Q9HYP0 | 4511 | Q9I3B3 |
| 184 | P11724 | 1266 | Q9HTY6 | 2348 | Q9HT54 | 3430 | Q9HYP1 | 4512 | Q9I3B4 |
| 185 | P13794 | 1267 | Q9HTY7 | 2349 | Q9HT55 | 3431 | Q9HYP2 | 4513 | Q9I3B5 |
| 186 | P13981 | 1268 | Q9HTY8 | 2350 | Q9HT56 | 3432 | Q9HYP3 | 4514 | Q9I3B6 |
| 187 | P13982 | 1269 | Q9HTY9 | 2351 | Q9HT58 | 3433 | Q9HYP6 | 4515 | Q9I3B7 |
| 188 | P14165 | 1270 | Q9HTZ0 | 2352 | Q9HT60 | 3434 | Q9HYP7 | 4516 | Q9I3B8 |
| 189 | P18275 | 1271 | Q9HTZ2 | 2353 | Q9HT61 | 3435 | Q9HYP8 | 4517 | Q9I3B9 |
| 190 | P20576 | 1272 | Q9HTZ4 | 2354 | Q9HT63 | 3436 | Q9HYP9 | 4518 | Q9I3C0 |
| 191 | P20580 | 1273 | Q9HTZ6 | 2355 | Q9HT64 | 3437 | Q9HYQ3 | 4519 | Q9I3C2 |
| 192 | P20581 | 1274 | Q9HU09 | 2356 | Q9HT65 | 3438 | Q9HYQ4 | 4520 | Q9I3C4 |
| 193 | P21175 | 1275 | Q9HU23 | 2357 | Q9HT66 | 3439 | Q9HYQ7 | 4521 | Q9I3C6 |
| 194 | P21629 | 1276 | Q9HU24 | 2358 | Q9HT67 | 3440 | Q9HYQ9 | 4522 | Q9I3C7 |
| 195 | P22609 | 1277 | Q9HU26 | 2359 | Q9HT68 | 3441 | Q9HYR0 | 4523 | Q9I3C8 |
| 196 | P23189 | 1278 | Q9HU29 | 2360 | Q9HT69 | 3442 | Q9HYR1 | 4524 | Q9I3C9 |
| 197 | P23620 | 1279 | Q9HU36 | 2361 | Q9HT71 | 3443 | Q9HYR3 | 4525 | Q9I3D0 |
| 198 | P23926 | 1280 | Q9HU41 | 2362 | Q9HT77 | 3444 | Q9HYR4 | 4526 | Q9I3D9 |
| 199 | P24734 | 1281 | Q9HU43 | 2363 | Q9HT79 | 3445 | Q9HYR7 | 4527 | Q9I3E0 |
| 200 | P25060 | 1282 | Q9HU51 | 2364 | Q9HT82 | 3446 | Q9HYR8 | 4528 | Q9I3E1 |
| 201 | P25061 | 1283 | Q9HU55 | 2365 | Q9HT83 | 3447 | Q9HYS0 | 4529 | Q9I3E2 |
| 202 | P26995 | 1284 | Q9HU56 | 2366 | Q9HT85 | 3448 | Q9HYS1 | 4530 | Q9I3E4 |
| 203 | P29365 | 1285 | Q9HU59 | 2367 | Q9HT88 | 3449 | Q9HYS2 | 4531 | Q9I3E5 |
| 204 | P38098 | 1286 | Q9HU60 | 2368 | Q9HT90 | 3450 | Q9HYS3 | 4532 | Q9I3E6 |
| 205 | P47205 | 1287 | Q9HU63 | 2369 | Q9HT92 | 3451 | Q9HYS4 | 4533 | Q9I3E7 |
| 206 | P49988 | 1288 | Q9HU76 | 2370 | Q9HT93 | 3452 | Q9HYS5 | 4534 | Q9I3E8 |
| 207 | P50597 | 1289 | Q9HU78 | 2371 | Q9HT94 | 3453 | Q9HYS6 | 4535 | Q9I3E9 |
| 208 | P53593 | 1290 | Q9HU86 | 2372 | Q9HT96 | 3454 | Q9HYS7 | 4536 | Q9I3F0 |
| 209 | P53652 | 1291 | Q9HU88 | 2373 | Q9HT98 | 3455 | Q9HYS8 | 4537 | Q9I3F2 |
| 210 | P57112 | 1292 | Q9HU99 | 2374 | Q9HTA1 | 3456 | Q9HYS9 | 4538 | Q9I3F7 |
| 211 | P57714 | 1293 | Q9HUA3 | 2375 | Q9HTA2 | 3457 | Q9HYT1 | 4539 | Q9I3F8 |
| 212 | P72138 | 1294 | Q9HUA5 | 2376 | Q9HTA3 | 3458 | Q9HYT2 | 4540 | Q9I3F9 |
| 213 | P95435 | 1295 | Q9HUA6 | 2377 | Q9HTA4 | 3459 | Q9HYT4 | 4541 | Q9I3G1 |
| 214 | P96956 | 1296 | Q9HUB2 | 2378 | Q9HTA5 | 3460 | Q9HYT5 | 4542 | Q9I3G4 |
| 215 | Q00518 | 1297 | Q9HUB3 | 2379 | Q9HTA6 | 3461 | Q9HYT7 | 4543 | Q9I3G6 |
| 216 | Q00934 | 1298 | Q9HUB5 | 2380 | Q9HTA7 | 3462 | Q9HYT8 | 4544 | Q9I3G7 |
| 217 | Q01269 | 1299 | Q9HUB6 | 2381 | Q9HTA8 | 3463 | Q9HYT9 | 4545 | Q9I3G9 |
| 218 | Q06584 | 1300 | Q9HUB9 | 2382 | Q9HTA9 | 3464 | Q9HYU1 | 4546 | Q9I3H0 |
| 219 | Q07806 | 1301 | Q9HUC5 | 2383 | Q9HTB0 | 3465 | Q9HYU2 | 4547 | Q9I3H1 |
| 220 | Q51344 | 1302 | Q9HUC8 | 2384 | Q9HTB3 | 3466 | Q9HYU5 | 4548 | Q9I3H4 |
| 221 | Q51368 | 1303 | Q9HUD0 | 2385 | Q9HTB9 | 3467 | Q9HYU8 | 4549 | Q9I3H6 |
| 222 | Q51385 | 1304 | Q9HUD2 | 2386 | Q9HTC1 | 3468 | Q9HYU9 | 4550 | Q9I3H7 |
| 223 | Q51393 | 1305 | Q9HUD3 | 2387 | Q9HTC3 | 3469 | Q9HYV0 | 4551 | Q9I3I2 |
| 224 | Q51404 | 1306 | Q9HUD5 | 2388 | Q9HTC4 | 3470 | Q9HYV1 | 4552 | Q9I3I3 |
| 225 | Q51422 | 1307 | Q9HUE7 | 2389 | Q9HTC5 | 3471 | Q9HYV2 | 4553 | Q9I3I7 |
| 226 | Q51426 | 1308 | Q9HUG5 | 2390 | Q9HTC6 | 3472 | Q9HYV3 | 4554 | Q9I3I9 |
| 227 | Q51472 | 1309 | Q9HUK5 | 2391 | Q9HTC8 | 3473 | Q9HYV4 | 4555 | Q9I3J0 |
| 228 | Q51559 | 1310 | Q9HUK6 | 2392 | Q9HTC9 | 3474 | Q9HYV5 | 4556 | Q9I3J1 |
| 229 | Q51566 | 1311 | Q9HUL1 | 2393 | Q9HTD1 | 3475 | Q9HYV8 | 4557 | Q9I3J3 |
| 230 | Q59635 | 1312 | Q9HUL2 | 2394 | Q9HTD3 | 3476 | Q9HYV9 | 4558 | Q9I3J4 |
| 231 | Q59647 | 1313 | Q9HUL6 | 2395 | Q9HTD4 | 3477 | Q9HYW0 | 4559 | Q9I3J6 |
| 232 | Q59650 | 1314 | Q9HUL7 | 2396 | Q9HTD5 | 3478 | Q9HYW1 | 4560 | Q9I3K0 |
| 233 | Q9HT22 | 1315 | Q9HUL8 | 2397 | Q9HTD6 | 3479 | Q9HYW2 | 4561 | Q9I3K3 |
| 234 | Q9HT84 | 1316 | Q9HUM1 | 2398 | Q9HTD8 | 3480 | Q9HYW3 | 4562 | Q9I3K4 |
| 235 | Q9HTB6 | 1317 | Q9HUM2 | 2399 | Q9HTE0 | 3481 | Q9HYW4 | 4563 | Q9I3K5 |
| 236 | Q9HTE9 | 1318 | Q9HUM3 | 2400 | Q9HTE1 | 3482 | Q9HYW5 | 4564 | Q9I3K6 |
| 237 | Q9HTF3 | 1319 | Q9HUM5 | 2401 | Q9HTE5 | 3483 | Q9HYW6 | 4565 | Q9I3K8 |
| 238 | Q9HTF4 | 1320 | Q9HUM7 | 2402 | Q9HTE7 | 3484 | Q9HYW7 | 4566 | Q9I3K9 |
| 239 | Q9HTI4 | 1321 | Q9HUM8 | 2403 | Q9HTF0 | 3485 | Q9HYW8 | 4567 | Q9I3L1 |
| 240 | Q9HTI6 | 1322 | Q9HUM9 | 2404 | Q9HTF2 | 3486 | Q9HYW9 | 4568 | Q9I3L2 |
| 241 | Q9HTI7 | 1323 | Q9HUN0 | 2405 | Q9HTF5 | 3487 | Q9HYX1 | 4569 | Q9I3L3 |
| 242 | Q9HTK8 | 1324 | Q9HUN2 | 2406 | Q9HTF6 | 3488 | Q9HYX2 | 4570 | Q9I3L5 |
| 243 | Q9HTK9 | 1325 | Q9HUN3 | 2407 | Q9HTF7 | 3489 | Q9HYX3 | 4571 | Q9I3L6 |
| 244 | Q9HTL3 | 1326 | Q9HUN6 | 2408 | Q9HTF8 | 3490 | Q9HYX4 | 4572 | Q9I3L7 |
| 245 | Q9HTN4 | 1327 | Q9HUN9 | 2409 | Q9HTF9 | 3491 | Q9HYX6 | 4573 | Q9I3L8 |
| 246 | Q9HTQ2 | 1328 | Q9HUP4 | 2410 | Q9HTG0 | 3492 | Q9HYX9 | 4574 | Q9I3M0 |
| 247 | Q9HTZ1 | 1329 | Q9HUP5 | 2411 | Q9HTG1 | 3493 | Q9HYY0 | 4575 | Q9I3M1 |
| 248 | Q9HU66 | 1330 | Q9HUP6 | 2412 | Q9HTG2 | 3494 | Q9HYY1 | 4576 | Q9I3M2 |
| 249 | Q9HU67 | 1331 | Q9HUQ0 | 2413 | Q9HTG3 | 3495 | Q9HYY2 | 4577 | Q9I3M3 |
| 250 | Q9HUB1 | 1332 | Q9HUQ1 | 2414 | Q9HTG4 | 3496 | Q9HYY4 | 4578 | Q9I3M4 |
| 251 | Q9HUC0 | 1333 | Q9HUQ3 | 2415 | Q9HTG7 | 3497 | Q9HYY5 | 4579 | Q9I3M5 |
| 252 | Q9HUG6 | 1334 | Q9HUR6 | 2416 | Q9HTG9 | 3498 | Q9HYY6 | 4580 | Q9I3M6 |
| 253 | Q9HUG9 | 1335 | Q9HUR7 | 2417 | Q9HTH1 | 3499 | Q9HYY7 | 4581 | Q9I3M8 |
| 254 | Q9HUH4 | 1336 | Q9HUS0 | 2418 | Q9HTH2 | 3500 | Q9HYY8 | 4582 | Q9I3N8 |
| 255 | Q9HUI3 | 1337 | Q9HUS1 | 2419 | Q9HTH3 | 3501 | Q9HYY9 | 4583 | Q9I3N9 |
| 256 | Q9HUJ8 | 1338 | Q9HUS2 | 2420 | Q9HTH7 | 3502 | Q9HYZ0 | 4584 | Q9I3P0 |
| 257 | Q9HUK1 | 1339 | Q9HUS3 | 2421 | Q9HTH9 | 3503 | Q9HYZ1 | 4585 | Q9I3P1 |
| 258 | Q9HUK7 | 1340 | Q9HUS7 | 2422 | Q9HTI0 | 3504 | Q9HYZ2 | 4586 | Q9I3P2 |
| 259 | Q9HUL5 | 1341 | Q9HUS8 | 2423 | Q9HTI1 | 3505 | Q9HYZ9 | 4587 | Q9I3P4 |
| 260 | Q9HUN4 | 1342 | Q9HUT0 | 2424 | Q9HTI2 | 3506 | Q9HZ01 | 4588 | Q9I3P5 |
| 261 | Q9HUX5 | 1343 | Q9HUT3 | 2425 | Q9HTI3 | 3507 | Q9HZ02 | 4589 | Q9I3P6 |
| 262 | Q9HV48 | 1344 | Q9HUT5 | 2426 | Q9HTJ4 | 3508 | Q9HZ03 | 4590 | Q9I3P7 |
| 263 | Q9HV50 | 1345 | Q9HUU6 | 2427 | Q9HTJ6 | 3509 | Q9HZ05 | 4591 | Q9I3Q1 |
| 264 | Q9HVA2 | 1346 | Q9HUU8 | 2428 | Q9HTJ7 | 3510 | Q9HZ07 | 4592 | Q9I3Q6 |
| 265 | Q9HVC5 | 1347 | Q9HUU9 | 2429 | Q9HTJ8 | 3511 | Q9HZ08 | 4593 | Q9I3Q7 |
| 266 | Q9HVI1 | 1348 | Q9HUV7 | 2430 | Q9HTJ9 | 3512 | Q9HZ09 | 4594 | Q9I3Q8 |
| 267 | Q9HVI7 | 1349 | Q9HUW0 | 2431 | Q9HTK2 | 3513 | Q9HZ10 | 4595 | Q9I3R0 |
| 268 | Q9HVM8 | 1350 | Q9HUW3 | 2432 | Q9HTK3 | 3514 | Q9HZ11 | 4596 | Q9I3R1 |
| 269 | Q9HVV7 | 1351 | Q9HUW9 | 2433 | Q9HTK4 | 3515 | Q9HZ12 | 4597 | Q9I3R2 |
| 270 | Q9HVZ0 | 1352 | Q9HUX7 | 2434 | Q9HTK6 | 3516 | Q9HZ14 | 4598 | Q9I3R3 |
| 271 | Q9HW04 | 1353 | Q9HUY8 | 2435 | Q9HTL1 | 3517 | Q9HZ15 | 4599 | Q9I3R4 |
| 272 | Q9HW93 | 1354 | Q9HUZ2 | 2436 | Q9HTL2 | 3518 | Q9HZ16 | 4600 | Q9I3R5 |
| 273 | Q9HWA4 | 1355 | Q9HUZ7 | 2437 | Q9HTL5 | 3519 | Q9HZ18 | 4601 | Q9I3R6 |
| 274 | Q9HWB6 | 1356 | Q9HUZ8 | 2438 | Q9HTL6 | 3520 | Q9HZ19 | 4602 | Q9I3R7 |
| 275 | Q9HWG4 | 1357 | Q9HUZ9 | 2439 | Q9HTL7 | 3521 | Q9HZ20 | 4603 | Q9I3R8 |
| 276 | Q9HWG9 | 1358 | Q9HV00 | 2440 | Q9HTL8 | 3522 | Q9HZ21 | 4604 | Q9I3R9 |
| 277 | Q9HWH9 | 1359 | Q9HV02 | 2441 | Q9HTL9 | 3523 | Q9HZ22 | 4605 | Q9I3S0 |
| 278 | Q9HWR3 | 1360 | Q9HV18 | 2442 | Q9HTM3 | 3524 | Q9HZ24 | 4606 | Q9I3S2 |
| 279 | Q9HWT7 | 1361 | Q9HV28 | 2443 | Q9HTM4 | 3525 | Q9HZ25 | 4607 | Q9I3S4 |
| 280 | Q9HXC7 | 1362 | Q9HV30 | 2444 | Q9HTM5 | 3526 | Q9HZ26 | 4608 | Q9I3S5 |
| 281 | Q9HXM5 | 1363 | Q9HV36 | 2445 | Q9HTM6 | 3527 | Q9HZ27 | 4609 | Q9I3S6 |
| 282 | Q9HXZ4 | 1364 | Q9HV38 | 2446 | Q9HTM7 | 3528 | Q9HZ31 | 4610 | Q9I3S7 |
| 283 | Q9HY63 | 1365 | Q9HV39 | 2447 | Q9HTM8 | 3529 | Q9HZ32 | 4611 | Q9I3S8 |
| 284 | Q9HY69 | 1366 | Q9HV40 | 2448 | Q9HTM9 | 3530 | Q9HZ33 | 4612 | Q9I3S9 |
| 285 | Q9HY82 | 1367 | Q9HV41 | 2449 | Q9HTN6 | 3531 | Q9HZ36 | 4613 | Q9I3T1 |
| 286 | Q9HYG7 | 1368 | Q9HV42 | 2450 | Q9HTN7 | 3532 | Q9HZ37 | 4614 | Q9I3T2 |
| 287 | Q9HYL2 | 1369 | Q9HV46 | 2451 | Q9HTP0 | 3533 | Q9HZ38 | 4615 | Q9I3T3 |
| 288 | Q9HZ17 | 1370 | Q9HV52 | 2452 | Q9HTP1 | 3534 | Q9HZ40 | 4616 | Q9I3T4 |
| 289 | Q9HZ47 | 1371 | Q9HV53 | 2453 | Q9HTP2 | 3535 | Q9HZ41 | 4617 | Q9I3T7 |
| 290 | Q9HZ59 | 1372 | Q9HV54 | 2454 | Q9HTP3 | 3536 | Q9HZ42 | 4618 | Q9I3T8 |
| 291 | Q9HZ62 | 1373 | Q9HV56 | 2455 | Q9HTP4 | 3537 | Q9HZ43 | 4619 | Q9I3U0 |
| 292 | Q9HZA6 | 1374 | Q9HV58 | 2456 | Q9HTP5 | 3538 | Q9HZ44 | 4620 | Q9I3U1 |
| 293 | Q9HZA7 | 1375 | Q9HV75 | 2457 | Q9HTP7 | 3539 | Q9HZ48 | 4621 | Q9I3U2 |
| 294 | Q9HZJ5 | 1376 | Q9HV76 | 2458 | Q9HTP8 | 3540 | Q9HZ49 | 4622 | Q9I3U3 |
| 295 | Q9HZK1 | 1377 | Q9HV82 | 2459 | Q9HTP9 | 3541 | Q9HZ50 | 4623 | Q9I3U4 |
| 296 | Q9HZM8 | 1378 | Q9HV88 | 2460 | Q9HTQ1 | 3542 | Q9HZ51 | 4624 | Q9I3U5 |
| 297 | Q9HZX3 | 1379 | Q9HVA8 | 2461 | Q9HTQ3 | 3543 | Q9HZ54 | 4625 | Q9I3U6 |
| 298 | Q9HZZ1 | 1380 | Q9HVB7 | 2462 | Q9HTQ4 | 3544 | Q9HZ56 | 4626 | Q9I3U7 |
| 299 | Q9I055 | 1381 | Q9HVB8 | 2463 | Q9HTQ5 | 3545 | Q9HZ74 | 4627 | Q9I3U9 |
| 300 | Q9I0F3 | 1382 | Q9HVB9 | 2464 | Q9HTQ7 | 3546 | Q9HZ78 | 4628 | Q9I3V0 |
| 301 | Q9I0H4 | 1383 | Q9HVC0 | 2465 | Q9HTQ9 | 3547 | Q9HZ80 | 4629 | Q9I3V2 |
| 302 | Q9I0I4 | 1384 | Q9HVC2 | 2466 | Q9HTR1 | 3548 | Q9HZ81 | 4630 | Q9I3V3 |
| 303 | Q9I0I6 | 1385 | Q9HVC3 | 2467 | Q9HTR8 | 3549 | Q9HZ83 | 4631 | Q9I3V4 |
| 304 | Q9I0M3 | 1386 | Q9HVC4 | 2468 | Q9HTR9 | 3550 | Q9HZ84 | 4632 | Q9I3V5 |
| 305 | Q9I0M7 | 1387 | Q9HVC9 | 2469 | Q9HTS0 | 3551 | Q9HZ85 | 4633 | Q9I3V6 |
| 306 | Q9I0N5 | 1388 | Q9HVD0 | 2470 | Q9HTS1 | 3552 | Q9HZ87 | 4634 | Q9I3V7 |
| 307 | Q9I138 | 1389 | Q9HVD2 | 2471 | Q9HTS2 | 3553 | Q9HZ88 | 4635 | Q9I3V9 |
| 308 | Q9I189 | 1390 | Q9HVE0 | 2472 | Q9HTS3 | 3554 | Q9HZ89 | 4636 | Q9I3W1 |
| 309 | Q9I1H0 | 1391 | Q9HVE7 | 2473 | Q9HTS6 | 3555 | Q9HZ90 | 4637 | Q9I3W2 |
| 310 | Q9I1L4 | 1392 | Q9HVF6 | 2474 | Q9HTS7 | 3556 | Q9HZ91 | 4638 | Q9I3W3 |
| 311 | Q9I1M0 | 1393 | Q9HVF7 | 2475 | Q9HTS8 | 3557 | Q9HZ92 | 4639 | Q9I3W4 |
| 312 | Q9I235 | 1394 | Q9HVF9 | 2476 | Q9HTS9 | 3558 | Q9HZ93 | 4640 | Q9I3W5 |
| 313 | Q9I2E2 | 1395 | Q9HVG2 | 2477 | Q9HTT0 | 3559 | Q9HZ94 | 4641 | Q9I3W6 |
| 314 | Q9I2Q1 | 1396 | Q9HVG4 | 2478 | Q9HTT1 | 3560 | Q9HZ96 | 4642 | Q9I3W7 |
| 315 | Q9I2V0 | 1397 | Q9HVG7 | 2479 | Q9HTT3 | 3561 | Q9HZ97 | 4643 | Q9I3X0 |
| 316 | Q9I2V5 | 1398 | Q9HVH2 | 2480 | Q9HTT4 | 3562 | Q9HZ98 | 4644 | Q9I3X2 |
| 317 | Q9I317 | 1399 | Q9HVH4 | 2481 | Q9HTT5 | 3563 | Q9HZ99 | 4645 | Q9I3X3 |
| 318 | Q9I322 | 1400 | Q9HVH7 | 2482 | Q9HTT7 | 3564 | Q9HZA0 | 4646 | Q9I3X4 |
| 319 | Q9I341 | 1401 | Q9HVI8 | 2483 | Q9HTU2 | 3565 | Q9HZA1 | 4647 | Q9I3X5 |
| 320 | Q9I3E3 | 1402 | Q9HVI9 | 2484 | Q9HTU4 | 3566 | Q9HZA2 | 4648 | Q9I3X6 |
| 321 | Q9I3F6 | 1403 | Q9HVJ4 | 2485 | Q9HTU7 | 3567 | Q9HZB0 | 4649 | Q9I3X7 |
| 322 | Q9I3N5 | 1404 | Q9HVJ7 | 2486 | Q9HTU9 | 3568 | Q9HZB4 | 4650 | Q9I3X9 |
| 323 | Q9I3W9 | 1405 | Q9HVJ9 | 2487 | Q9HTV0 | 3569 | Q9HZB5 | 4651 | Q9I3Y0 |
| 324 | Q9I406 | 1406 | Q9HVK8 | 2488 | Q9HTV2 | 3570 | Q9HZB6 | 4652 | Q9I3Y1 |
| 325 | Q9I426 | 1407 | Q9HVL2 | 2489 | Q9HTV4 | 3571 | Q9HZB7 | 4653 | Q9I3Y2 |
| 326 | Q9I434 | 1408 | Q9HVL3 | 2490 | Q9HTV5 | 3572 | Q9HZB8 | 4654 | Q9I3Y4 |
| 327 | Q9I466 | 1409 | Q9HVL6 | 2491 | Q9HTV6 | 3573 | Q9HZB9 | 4655 | Q9I3Y5 |
| 328 | Q9I476 | 1410 | Q9HVL7 | 2492 | Q9HTV8 | 3574 | Q9HZC1 | 4656 | Q9I3Y6 |
| 329 | Q9I489 | 1411 | Q9HVM2 | 2493 | Q9HTV9 | 3575 | Q9HZC2 | 4657 | Q9I3Y7 |
| 330 | Q9I4D4 | 1412 | Q9HVM6 | 2494 | Q9HTW0 | 3576 | Q9HZC3 | 4658 | Q9I3Y8 |
| 331 | Q9I4F5 | 1413 | Q9HVM9 | 2495 | Q9HTW1 | 3577 | Q9HZC4 | 4659 | Q9I3Y9 |
| 332 | Q9I4G3 | 1414 | Q9HVN0 | 2496 | Q9HTW4 | 3578 | Q9HZC6 | 4660 | Q9I3Z0 |
| 333 | Q9I4K6 | 1415 | Q9HVP7 | 2497 | Q9HTW5 | 3579 | Q9HZC7 | 4661 | Q9I3Z1 |
| 334 | Q9I4N3 | 1416 | Q9HVQ5 | 2498 | Q9HTW6 | 3580 | Q9HZC8 | 4662 | Q9I3Z2 |
| 335 | Q9I4U2 | 1417 | Q9HVQ7 | 2499 | Q9HTW7 | 3581 | Q9HZC9 | 4663 | Q9I3Z3 |
| 336 | Q9I4W3 | 1418 | Q9HVQ8 | 2500 | Q9HTW8 | 3582 | Q9HZD2 | 4664 | Q9I3Z4 |
| 337 | Q9I4X0 | 1419 | Q9HVR0 | 2501 | Q9HTW9 | 3583 | Q9HZD3 | 4665 | Q9I3Z5 |
| 338 | Q9I4X1 | 1420 | Q9HVS1 | 2502 | Q9HTX0 | 3584 | Q9HZD5 | 4666 | Q9I3Z6 |
| 339 | Q9I4X3 | 1421 | Q9HVS4 | 2503 | Q9HTX1 | 3585 | Q9HZD6 | 4667 | Q9I3Z7 |
| 340 | Q9I589 | 1422 | Q9HVS7 | 2504 | Q9HTX2 | 3586 | Q9HZD7 | 4668 | Q9I3Z8 |
| 341 | Q9I5A5 | 1423 | Q9HVT6 | 2505 | Q9HTX3 | 3587 | Q9HZD8 | 4669 | Q9I3Z9 |
| 342 | Q9I5F6 | 1424 | Q9HVT7 | 2506 | Q9HTX4 | 3588 | Q9HZD9 | 4670 | Q9I566 |
| 343 | Q9I5F9 | 1425 | Q9HVT8 | 2507 | Q9HTX8 | 3589 | Q9HZE1 | 4671 | Q9I567 |
| 344 | Q9I5N9 | 1426 | Q9HVU0 | 2508 | Q9HTX9 | 3590 | Q9HZE2 | 4672 | Q9I568 |
| 345 | Q9I5Q5 | 1427 | Q9HVU1 | 2509 | Q9HTY0 | 3591 | Q9HZE3 | 4673 | Q9I569 |
| 346 | Q9I5U0 | 1428 | Q9HVU2 | 2510 | Q9HTY1 | 3592 | Q9HZE8 | 4674 | Q9I570 |
| 347 | Q9I5U1 | 1429 | Q9HVU4 | 2511 | Q9HTY4 | 3593 | Q9HZE9 | 4675 | Q9I571 |
| 348 | Q9I5U4 | 1430 | Q9HVV2 | 2512 | Q9HTZ3 | 3594 | Q9HZF1 | 4676 | Q9I577 |
| 349 | Q9I5V3 | 1431 | Q9HVV3 | 2513 | Q9HTZ5 | 3595 | Q9HZF2 | 4677 | Q9I578 |
| 350 | Q9I6A8 | 1432 | Q9HVV4 | 2514 | Q9HTZ8 | 3596 | Q9HZF3 | 4678 | Q9I579 |
| 351 | Q9I6G4 | 1433 | Q9HVV5 | 2515 | Q9HTZ9 | 3597 | Q9HZF5 | 4679 | Q9I580 |
| 352 | Q9I6H1 | 1434 | Q9HVV6 | 2516 | Q9HU00 | 3598 | Q9HZF6 | 4680 | Q9I581 |
| 353 | Q9I6J0 | 1435 | Q9HVV8 | 2517 | Q9HU01 | 3599 | Q9HZF7 | 4681 | Q9I582 |
| 354 | Q9I6J1 | 1436 | Q9HVV9 | 2518 | Q9HU02 | 3600 | Q9HZG2 | 4682 | Q9I584 |
| 355 | Q9I6J2 | 1437 | Q9HVW2 | 2519 | Q9HU03 | 3601 | Q9HZG3 | 4683 | Q9I585 |
| 356 | Q9I6J9 | 1438 | Q9HVW8 | 2520 | Q9HU04 | 3602 | Q9HZG6 | 4684 | Q9I586 |
| 357 | Q9I6K2 | 1439 | Q9HVX0 | 2521 | Q9HU06 | 3603 | Q9HZG7 | 4685 | Q9I588 |
| 358 | Q9I6P6 | 1440 | Q9HVX1 | 2522 | Q9HU07 | 3604 | Q9HZG8 | 4686 | Q9I591 |
| 359 | Q9I6Y4 | 1441 | Q9HVX2 | 2523 | Q9HU08 | 3605 | Q9HZG9 | 4687 | Q9I598 |
| 360 | Q9I6Z1 | 1442 | Q9HVX4 | 2524 | Q9HU10 | 3606 | Q9HZH1 | 4688 | Q9I599 |
| 361 | Q9I702 | 1443 | Q9HVX7 | 2525 | Q9HU11 | 3607 | Q9HZH2 | 4689 | Q9I5A0 |
| 362 | Q9I739 | 1444 | Q9HVY2 | 2526 | Q9HU12 | 3608 | Q9HZH3 | 4690 | Q9I5A1 |
| 363 | Q9I788 | 1445 | Q9HVY3 | 2527 | Q9HU13 | 3609 | Q9HZH4 | 4691 | Q9I5A2 |
| 364 | Q9I7C1 | 1446 | Q9HVY4 | 2528 | Q9HU25 | 3610 | Q9HZH5 | 4692 | Q9I5A6 |
| 365 | Q9L7T2 | 1447 | Q9HVY5 | 2529 | Q9HU27 | 3611 | Q9HZH9 | 4693 | Q9I5A7 |
| 366 | G3XCU2 | 1448 | Q9HVY6 | 2530 | Q9HU28 | 3612 | Q9HZI1 | 4694 | Q9I5A8 |
| 367 | G3XCV1 | 1449 | Q9HVZ3 | 2531 | Q9HU30 | 3613 | Q9HZI2 | 4695 | Q9I5A9 |
| 368 | G3XCV7 | 1450 | Q9HVZ4 | 2532 | Q9HU31 | 3614 | Q9HZI4 | 4696 | Q9I5B0 |
| 369 | G3XCW3 | 1451 | Q9HVZ5 | 2533 | Q9HU33 | 3615 | Q9HZI5 | 4697 | Q9I5B1 |
| 370 | G3XCW9 | 1452 | Q9HVZ6 | 2534 | Q9HU34 | 3616 | Q9HZI6 | 4698 | Q9I5B2 |
| 371 | G3XCX1 | 1453 | Q9HW07 | 2535 | Q9HU35 | 3617 | Q9HZI8 | 4699 | Q9I5B3 |
| 372 | G3XCZ0 | 1454 | Q9HW08 | 2536 | Q9HU38 | 3618 | Q9HZI9 | 4700 | Q9I5B4 |
| 373 | G3XD04 | 1455 | Q9HW12 | 2537 | Q9HU39 | 3619 | Q9HZJ0 | 4701 | Q9I5B5 |
| 374 | G3XD14 | 1456 | Q9HW22 | 2538 | Q9HU40 | 3620 | Q9HZJ1 | 4702 | Q9I5B6 |
| 375 | G3XD19 | 1457 | Q9HW26 | 2539 | Q9HU45 | 3621 | Q9HZJ4 | 4703 | Q9I5B7 |
| 376 | G3XD20 | 1458 | Q9HW34 | 2540 | Q9HU46 | 3622 | Q9HZJ6 | 4704 | Q9I5B8 |
| 377 | G3XD29 | 1459 | Q9HW35 | 2541 | Q9HU47 | 3623 | Q9HZJ7 | 4705 | Q9I5B9 |
| 378 | G3XD31 | 1460 | Q9HW38 | 2542 | Q9HU48 | 3624 | Q9HZK2 | 4706 | Q9I5C0 |
| 379 | G3XD47 | 1461 | Q9HW43 | 2543 | Q9HU49 | 3625 | Q9HZL3 | 4707 | Q9I5C1 |
| 380 | G3XD74 | 1462 | Q9HW51 | 2544 | Q9HU52 | 3626 | Q9HZL4 | 4708 | Q9I5C2 |
| 381 | G3XD80 | 1463 | Q9HW68 | 2545 | Q9HU54 | 3627 | Q9HZL5 | 4709 | Q9I5C3 |
| 382 | G3XD88 | 1464 | Q9HW85 | 2546 | Q9HU58 | 3628 | Q9HZL9 | 4710 | Q9I5C4 |
| 383 | G3XD98 | 1465 | Q9HW86 | 2547 | Q9HU61 | 3629 | Q9HZM0 | 4711 | Q9I5C5 |
| 384 | G3XDA3 | 1466 | Q9HW87 | 2548 | Q9HU62 | 3630 | Q9HZM1 | 4712 | Q9I5C6 |
| 385 | G3XDB0 | 1467 | Q9HWA1 | 2549 | Q9HU64 | 3631 | Q9HZN0 | 4713 | Q9I5C7 |
| 386 | O30557 | 1468 | Q9HWA8 | 2550 | Q9HU68 | 3632 | Q9HZN1 | 4714 | Q9I5C8 |
| 387 | O50174 | 1469 | Q9HWB9 | 2551 | Q9HU69 | 3633 | Q9HZN3 | 4715 | Q9I5D0 |
| 388 | O50175 | 1470 | Q9HWC3 | 2552 | Q9HU70 | 3634 | Q9HZN9 | 4716 | Q9I5D2 |
| 389 | O50177 | 1471 | Q9HWC4 | 2553 | Q9HU71 | 3635 | Q9HZP0 | 4717 | Q9I5D3 |
| 390 | O50273 | 1472 | Q9HWC5 | 2554 | Q9HU74 | 3636 | Q9HZP1 | 4718 | Q9I5D4 |
| 391 | O52760 | 1473 | Q9HWC6 | 2555 | Q9HU75 | 3637 | Q9HZP2 | 4719 | Q9I5D5 |
| 392 | O54438 | 1474 | Q9HWC7 | 2556 | Q9HU79 | 3638 | Q9HZP3 | 4720 | Q9I5D6 |
| 393 | O54439 | 1475 | Q9HWC8 | 2557 | Q9HU80 | 3639 | Q9HZP4 | 4721 | Q9I5D7 |
| 394 | O68562 | 1476 | Q9HWD1 | 2558 | Q9HU81 | 3640 | Q9HZQ0 | 4722 | Q9I5D8 |
| 395 | O68799 | 1477 | Q9HWD2 | 2559 | Q9HU82 | 3641 | Q9HZQ1 | 4723 | Q9I5E0 |
| 396 | O68822 | 1478 | Q9HWD4 | 2560 | Q9HU84 | 3642 | Q9HZQ5 | 4724 | Q9I5E1 |
| 397 | O69077 | 1479 | Q9HWD5 | 2561 | Q9HU87 | 3643 | Q9HZQ6 | 4725 | Q9I5E5 |
| 398 | O69754 | 1480 | Q9HWD7 | 2562 | Q9HU89 | 3644 | Q9HZQ7 | 4726 | Q9I5E7 |
| 399 | O82852 | 1481 | Q9HWD8 | 2563 | Q9HU90 | 3645 | Q9HZQ9 | 4727 | Q9I5E8 |
| 400 | O86422 | 1482 | Q9HWD9 | 2564 | Q9HU95 | 3646 | Q9HZR0 | 4728 | Q9I5E9 |
| 401 | O87016 | 1483 | Q9HWE0 | 2565 | Q9HU96 | 3647 | Q9HZR2 | 4729 | Q9I5F1 |
| 402 | P00106 | 1484 | Q9HWE1 | 2566 | Q9HU97 | 3648 | Q9HZR4 | 4730 | Q9I5F4 |
| 403 | P05695 | 1485 | Q9HWE2 | 2567 | Q9HU98 | 3649 | Q9HZR5 | 4731 | Q9I5F8 |
| 404 | P07344 | 1486 | Q9HWE3 | 2568 | Q9HUA0 | 3650 | Q9HZR7 | 4732 | Q9I5G0 |
| 405 | P08280 | 1487 | Q9HWE4 | 2569 | Q9HUA1 | 3651 | Q9HZR8 | 4733 | Q9I5G1 |
| 406 | P0DP44 | 1488 | Q9HWE5 | 2570 | Q9HUA2 | 3652 | Q9HZR9 | 4734 | Q9I5G2 |
| 407 | P10932 | 1489 | Q9HWE6 | 2571 | Q9HUA7 | 3653 | Q9HZS0 | 4735 | Q9I5G6 |
| 408 | P15275 | 1490 | Q9HWE7 | 2572 | Q9HUA8 | 3654 | Q9HZS4 | 4736 | Q9I5H0 |
| 409 | P15276 | 1491 | Q9HWE8 | 2573 | Q9HUA9 | 3655 | Q9HZS5 | 4737 | Q9I5H1 |
| 410 | P15713 | 1492 | Q9HWE9 | 2574 | Q9HUB0 | 3656 | Q9HZS6 | 4738 | Q9I5H3 |
| 411 | P18895 | 1493 | Q9HWF0 | 2575 | Q9HUC1 | 3657 | Q9HZS7 | 4739 | Q9I5H4 |
| 412 | P19072 | 1494 | Q9HWF1 | 2576 | Q9HUC2 | 3658 | Q9HZS8 | 4740 | Q9I5H5 |
| 413 | P19572 | 1495 | Q9HWF3 | 2577 | Q9HUC3 | 3659 | Q9HZS9 | 4741 | Q9I5H6 |
| 414 | P20574 | 1496 | Q9HWF4 | 2578 | Q9HUC4 | 3660 | Q9HZT0 | 4742 | Q9I5H7 |
| 415 | P21482 | 1497 | Q9HWF5 | 2579 | Q9HUC7 | 3661 | Q9HZT2 | 4743 | Q9I5H8 |
| 416 | P21627 | 1498 | Q9HWF6 | 2580 | Q9HUD1 | 3662 | Q9HZT3 | 4744 | Q9I5I0 |
| 417 | P21630 | 1499 | Q9HWF7 | 2581 | Q9HUD4 | 3663 | Q9HZT4 | 4745 | Q9I5I4 |
| 418 | P22008 | 1500 | Q9HWF8 | 2582 | Q9HUD6 | 3664 | Q9HZT6 | 4746 | Q9I5I5 |
| 419 | P22567 | 1501 | Q9HWG8 | 2583 | Q9HUD7 | 3665 | Q9HZT8 | 4747 | Q9I5I7 |
| 420 | P23621 | 1502 | Q9HWH1 | 2584 | Q9HUD8 | 3666 | Q9HZU1 | 4748 | Q9I5I8 |
| 421 | P24735 | 1503 | Q9HWH3 | 2585 | Q9HUD9 | 3667 | Q9HZU4 | 4749 | Q9I5J0 |
| 422 | P25185 | 1504 | Q9HWI4 | 2586 | Q9HUE0 | 3668 | Q9HZU5 | 4750 | Q9I5J1 |
| 423 | P26480 | 1505 | Q9HWJ3 | 2587 | Q9HUE1 | 3669 | Q9HZU6 | 4751 | Q9I5J2 |
| 424 | P26841 | 1506 | Q9HWK9 | 2588 | Q9HUE2 | 3670 | Q9HZU7 | 4752 | Q9I5J3 |
| 425 | P26994 | 1507 | Q9HWM5 | 2589 | Q9HUE3 | 3671 | Q9HZU8 | 4753 | Q9I5J4 |
| 426 | P27017 | 1508 | Q9HWM7 | 2590 | Q9HUE4 | 3672 | Q9HZU9 | 4754 | Q9I5J5 |
| 427 | P27726 | 1509 | Q9HWN7 | 2591 | Q9HUE5 | 3673 | Q9HZV0 | 4755 | Q9I5J8 |
| 428 | P28811 | 1510 | Q9HWN8 | 2592 | Q9HUE9 | 3674 | Q9HZV1 | 4756 | Q9I5J9 |
| 429 | P29248 | 1511 | Q9HWP5 | 2593 | Q9HUF2 | 3675 | Q9HZV2 | 4757 | Q9I5K0 |
| 430 | P29364 | 1512 | Q9HWP9 | 2594 | Q9HUF6 | 3676 | Q9HZV3 | 4758 | Q9I5K1 |
| 431 | P29369 | 1513 | Q9HWQ1 | 2595 | Q9HUF8 | 3677 | Q9HZV6 | 4759 | Q9I5K2 |
| 432 | P30417 | 1514 | Q9HWR2 | 2596 | Q9HUF9 | 3678 | Q9HZV7 | 4760 | Q9I5K3 |
| 433 | P30720 | 1515 | Q9HWR7 | 2597 | Q9HUG0 | 3679 | Q9HZV9 | 4761 | Q9I5K4 |
| 434 | P30819 | 1516 | Q9HWR8 | 2598 | Q9HUG1 | 3680 | Q9HZW1 | 4762 | Q9I5K5 |
| 435 | P31961 | 1517 | Q9HWS1 | 2599 | Q9HUG2 | 3681 | Q9HZW3 | 4763 | Q9I5K6 |
| 436 | P32265 | 1518 | Q9HWS6 | 2600 | Q9HUG3 | 3682 | Q9HZW4 | 4764 | Q9I5K7 |
| 437 | P32977 | 1519 | Q9HWS7 | 2601 | Q9HUG4 | 3683 | Q9HZW5 | 4765 | Q9I5K8 |
| 438 | P33640 | 1520 | Q9HWU0 | 2602 | Q9HUG7 | 3684 | Q9HZW6 | 4766 | Q9I5K9 |
| 439 | P33642 | 1521 | Q9HWU4 | 2603 | Q9HUH0 | 3685 | Q9HZW7 | 4767 | Q9I5L0 |
| 440 | P33663 | 1522 | Q9HWV9 | 2604 | Q9HUH1 | 3686 | Q9HZW8 | 4768 | Q9I5L1 |
| 441 | P33883 | 1523 | Q9HWX1 | 2605 | Q9HUH2 | 3687 | Q9HZW9 | 4769 | Q9I5L2 |
| 442 | P34002 | 1524 | Q9HWX3 | 2606 | Q9HUH3 | 3688 | Q9HZX0 | 4770 | Q9I5L4 |
| 443 | P34003 | 1525 | Q9HWX6 | 2607 | Q9HUH6 | 3689 | Q9HZX1 | 4771 | Q9I5L5 |
| 444 | P37452 | 1526 | Q9HWX8 | 2608 | Q9HUH8 | 3690 | Q9HZX2 | 4772 | Q9I5L6 |
| 445 | P37798 | 1527 | Q9HWY5 | 2609 | Q9HUH9 | 3691 | Q9HZX4 | 4773 | Q9I5L7 |
| 446 | P38103 | 1528 | Q9HWZ1 | 2610 | Q9HUI0 | 3692 | Q9HZX5 | 4774 | Q9I5L9 |
| 447 | P38107 | 1529 | Q9HWZ3 | 2611 | Q9HUI1 | 3693 | Q9HZX7 | 4775 | Q9I5M0 |
| 448 | P38108 | 1530 | Q9HX04 | 2612 | Q9HUI4 | 3694 | Q9HZX8 | 4776 | Q9I5M2 |
| 449 | P40883 | 1531 | Q9HX11 | 2613 | Q9HUI5 | 3695 | Q9HZX9 | 4777 | Q9I5M3 |
| 450 | P40947 | 1532 | Q9HX17 | 2614 | Q9HUI6 | 3696 | Q9HZY0 | 4778 | Q9I5M5 |
| 451 | P42257 | 1533 | Q9HX20 | 2615 | Q9HUI7 | 3697 | Q9HZY1 | 4779 | Q9I5M6 |
| 452 | P42512 | 1534 | Q9HX22 | 2616 | Q9HUJ0 | 3698 | Q9HZY2 | 4780 | Q9I5M7 |
| 453 | P42805 | 1535 | Q9HX23 | 2617 | Q9HUJ1 | 3699 | Q9HZY4 | 4781 | Q9I5M8 |
| 454 | P42807 | 1536 | Q9HX24 | 2618 | Q9HUJ3 | 3700 | Q9HZY6 | 4782 | Q9I5M9 |
| 455 | P42812 | 1537 | Q9HX28 | 2619 | Q9HUJ5 | 3701 | Q9HZY9 | 4783 | Q9I5N0 |
| 456 | P43334 | 1538 | Q9HX31 | 2620 | Q9HUJ6 | 3702 | Q9HZZ3 | 4784 | Q9I5N1 |
| 457 | P43336 | 1539 | Q9HX32 | 2621 | Q9HUJ7 | 3703 | Q9HZZ5 | 4785 | Q9I5N2 |
| 458 | P43898 | 1540 | Q9HX33 | 2622 | Q9HUJ9 | 3704 | Q9HZZ6 | 4786 | Q9I5N3 |
| 459 | P43904 | 1541 | Q9HX37 | 2623 | Q9HUK0 | 3705 | Q9HZZ7 | 4787 | Q9I5N5 |
| 460 | P45683 | 1542 | Q9HX41 | 2624 | Q9HUK2 | 3706 | Q9HZZ8 | 4788 | Q9I5N8 |
| 461 | P46384 | 1543 | Q9HX42 | 2625 | Q9HUK3 | 3707 | Q9HZZ9 | 4789 | Q9I5P1 |
| 462 | P47204 | 1544 | Q9HX45 | 2626 | Q9HUK4 | 3708 | Q9I001 | 4790 | Q9I5P2 |
| 463 | P48246 | 1545 | Q9HX46 | 2627 | Q9HUL0 | 3709 | Q9I002 | 4791 | Q9I5P3 |
| 464 | P48247 | 1546 | Q9HX48 | 2628 | Q9HUM4 | 3710 | Q9I005 | 4792 | Q9I5P6 |
| 465 | P48372 | 1547 | Q9HX70 | 2629 | Q9HUN1 | 3711 | Q9I006 | 4793 | Q9I5P8 |
| 466 | P48632 | 1548 | Q9HX72 | 2630 | Q9HUN5 | 3712 | Q9I007 | 4794 | Q9I5Q0 |
| 467 | P50587 | 1549 | Q9HX83 | 2631 | Q9HUN7 | 3713 | Q9I008 | 4795 | Q9I5Q1 |
| 468 | P50600 | 1550 | Q9HX93 | 2632 | Q9HUN8 | 3714 | Q9I009 | 4796 | Q9I5Q7 |
| 469 | P54291 | 1551 | Q9HX98 | 2633 | Q9HUP0 | 3715 | Q9I010 | 4797 | Q9I5Q8 |
| 470 | P55218 | 1552 | Q9HX99 | 2634 | Q9HUP2 | 3716 | Q9I012 | 4798 | Q9I5R1 |
| 471 | P57668 | 1553 | Q9HXA0 | 2635 | Q9HUP7 | 3717 | Q9I014 | 4799 | Q9I5R2 |
| 472 | P57698 | 1554 | Q9HXA1 | 2636 | Q9HUP8 | 3718 | Q9I018 | 4800 | Q9I5R3 |
| 473 | P57703 | 1555 | Q9HXA2 | 2637 | Q9HUP9 | 3719 | Q9I020 | 4801 | Q9I5R4 |
| 474 | P57708 | 1556 | Q9HXB0 | 2638 | Q9HUQ2 | 3720 | Q9I021 | 4802 | Q9I5R5 |
| 475 | P72139 | 1557 | Q9HXB2 | 2639 | Q9HUQ5 | 3721 | Q9I022 | 4803 | Q9I5R8 |
| 476 | P72151 | 1558 | Q9HXB9 | 2640 | Q9HUQ6 | 3722 | Q9I023 | 4804 | Q9I5R9 |
| 477 | P72154 | 1559 | Q9HXC3 | 2641 | Q9HUQ7 | 3723 | Q9I024 | 4805 | Q9I5S0 |
| 478 | P72158 | 1560 | Q9HXC5 | 2642 | Q9HUQ8 | 3724 | Q9I026 | 4806 | Q9I5S1 |
| 479 | P72161 | 1561 | Q9HXD2 | 2643 | Q9HUQ9 | 3725 | Q9I027 | 4807 | Q9I5S2 |
| 480 | P72170 | 1562 | Q9HXD3 | 2644 | Q9HUR0 | 3726 | Q9I029 | 4808 | Q9I5S3 |
| 481 | P72173 | 1563 | Q9HXD4 | 2645 | Q9HUR1 | 3727 | Q9I030 | 4809 | Q9I5S4 |
| 482 | P72174 | 1564 | Q9HXD9 | 2646 | Q9HUR3 | 3728 | Q9I031 | 4810 | Q9I5S5 |
| 483 | P80357 | 1565 | Q9HXE0 | 2647 | Q9HUR4 | 3729 | Q9I038 | 4811 | Q9I5S6 |
| 484 | P80358 | 1566 | Q9HXE2 | 2648 | Q9HUR5 | 3730 | Q9I039 | 4812 | Q9I5S7 |
| 485 | P95412 | 1567 | Q9HXF3 | 2649 | Q9HUR8 | 3731 | Q9I040 | 4813 | Q9I5S8 |
| 486 | P95413 | 1568 | Q9HXF5 | 2650 | Q9HUR9 | 3732 | Q9I041 | 4814 | Q9I5S9 |
| 487 | P95414 | 1569 | Q9HXF7 | 2651 | Q9HUS4 | 3733 | Q9I042 | 4815 | Q9I5T0 |
| 488 | P95415 | 1570 | Q9HXG4 | 2652 | Q9HUS5 | 3734 | Q9I043 | 4816 | Q9I5T2 |
| 489 | P95454 | 1571 | Q9HXH4 | 2653 | Q9HUS6 | 3735 | Q9I044 | 4817 | Q9I5T3 |
| 490 | P95458 | 1572 | Q9HXH5 | 2654 | Q9HUS9 | 3736 | Q9I045 | 4818 | Q9I5T4 |
| 491 | P96963 | 1573 | Q9HXH7 | 2655 | Q9HUT1 | 3737 | Q9I046 | 4819 | Q9I5T6 |
| 492 | Q00513 | 1574 | Q9HXH8 | 2656 | Q9HUT2 | 3738 | Q9I050 | 4820 | Q9I5T7 |
| 493 | Q01725 | 1575 | Q9HXI1 | 2657 | Q9HUT4 | 3739 | Q9I051 | 4821 | Q9I5T9 |
| 494 | Q03024 | 1576 | Q9HXI2 | 2658 | Q9HUT6 | 3740 | Q9I052 | 4822 | Q9I5U6 |
| 495 | Q03025 | 1577 | Q9HXI6 | 2659 | Q9HUT7 | 3741 | Q9I053 | 4823 | Q9I5U9 |
| 496 | Q03027 | 1578 | Q9HXJ0 | 2660 | Q9HUT8 | 3742 | Q9I054 | 4824 | Q9I5V0 |
| 497 | Q04803 | 1579 | Q9HXJ1 | 2661 | Q9HUT9 | 3743 | Q9I056 | 4825 | Q9I5V1 |
| 498 | Q04804 | 1580 | Q9HXJ3 | 2662 | Q9HUU0 | 3744 | Q9I057 | 4826 | Q9I5V2 |
| 499 | Q05097 | 1581 | Q9HXJ5 | 2663 | Q9HUU2 | 3745 | Q9I058 | 4827 | Q9I5V9 |
| 500 | Q06062 | 1582 | Q9HXJ7 | 2664 | Q9HUU3 | 3746 | Q9I059 | 4828 | Q9I5W2 |
| 501 | Q51342 | 1583 | Q9HXL4 | 2665 | Q9HUU4 | 3747 | Q9I062 | 4829 | Q9I5W3 |
| 502 | Q51363 | 1584 | Q9HXL5 | 2666 | Q9HUV0 | 3748 | Q9I064 | 4830 | Q9I5W5 |
| 503 | Q51373 | 1585 | Q9HXL6 | 2667 | Q9HUV1 | 3749 | Q9I065 | 4831 | Q9I5W6 |
| 504 | Q51375 | 1586 | Q9HXL8 | 2668 | Q9HUV2 | 3750 | Q9I067 | 4832 | Q9I5W7 |
| 505 | Q51383 | 1587 | Q9HXM8 | 2669 | Q9HUV3 | 3751 | Q9I070 | 4833 | Q9I5W8 |
| 506 | Q51390 | 1588 | Q9HXN4 | 2670 | Q9HUV4 | 3752 | Q9I071 | 4834 | Q9I5W9 |
| 507 | Q51392 | 1589 | Q9HXN5 | 2671 | Q9HUV5 | 3753 | Q9I072 | 4835 | Q9I5X0 |
| 508 | Q51397 | 1590 | Q9HXN7 | 2672 | Q9HUW2 | 3754 | Q9I074 | 4836 | Q9I5X1 |
| 509 | Q51425 | 1591 | Q9HXN9 | 2673 | Q9HUW4 | 3755 | Q9I075 | 4837 | Q9I5X2 |
| 510 | Q51434 | 1592 | Q9HXP9 | 2674 | Q9HUW8 | 3756 | Q9I076 | 4838 | Q9I5X3 |
| 511 | Q51455 | 1593 | Q9HXQ0 | 2675 | Q9HUX0 | 3757 | Q9I077 | 4839 | Q9I5X4 |
| 512 | Q51467 | 1594 | Q9HXQ2 | 2676 | Q9HUX2 | 3758 | Q9I078 | 4840 | Q9I5X5 |
| 513 | Q51470 | 1595 | Q9HXQ3 | 2677 | Q9HUX8 | 3759 | Q9I079 | 4841 | Q9I5X6 |
| 514 | Q51473 | 1596 | Q9HXQ6 | 2678 | Q9HUX9 | 3760 | Q9I080 | 4842 | Q9I5X7 |
| 515 | Q51479 | 1597 | Q9HXR7 | 2679 | Q9HUY0 | 3761 | Q9I081 | 4843 | Q9I5X8 |
| 516 | Q51481 | 1598 | Q9HXS0 | 2680 | Q9HUY2 | 3762 | Q9I082 | 4844 | Q9I5X9 |
| 517 | Q51485 | 1599 | Q9HXT5 | 2681 | Q9HUY3 | 3763 | Q9I083 | 4845 | Q9I5Y0 |
| 518 | Q51506 | 1600 | Q9HXT7 | 2682 | Q9HUY4 | 3764 | Q9I084 | 4846 | Q9I5Y2 |
| 519 | Q51508 | 1601 | Q9HXT9 | 2683 | Q9HUY6 | 3765 | Q9I085 | 4847 | Q9I5Y3 |
| 520 | Q51546 | 1602 | Q9HXV0 | 2684 | Q9HUY7 | 3766 | Q9I086 | 4848 | Q9I5Y6 |
| 521 | Q51547 | 1603 | Q9HXV3 | 2685 | Q9HUY9 | 3767 | Q9I087 | 4849 | Q9I5Y7 |
| 522 | Q51561 | 1604 | Q9HXW0 | 2686 | Q9HUZ0 | 3768 | Q9I089 | 4850 | Q9I5Y9 |
| 523 | Q51564 | 1605 | Q9HXW2 | 2687 | Q9HUZ1 | 3769 | Q9I090 | 4851 | Q9I5Z1 |
| 524 | Q51575 | 1606 | Q9HXW3 | 2688 | Q9HUZ3 | 3770 | Q9I091 | 4852 | Q9I5Z2 |
| 525 | Q52463 | 1607 | Q9HXW7 | 2689 | Q9HUZ4 | 3771 | Q9I092 | 4853 | Q9I5Z3 |
| 526 | Q59640 | 1608 | Q9HXX3 | 2690 | Q9HUZ5 | 3772 | Q9I093 | 4854 | Q9I5Z4 |
| 527 | Q59641 | 1609 | Q9HXX9 | 2691 | Q9HUZ6 | 3773 | Q9I094 | 4855 | Q9I5Z6 |
| 528 | Q59646 | 1610 | Q9HXY3 | 2692 | Q9HV03 | 3774 | Q9I096 | 4856 | Q9I5Z7 |
| 529 | Q59653 | 1611 | Q9HXY4 | 2693 | Q9HV04 | 3775 | Q9I097 | 4857 | Q9I5Z9 |
| 530 | Q59654 | 1612 | Q9HXY5 | 2694 | Q9HV05 | 3776 | Q9I098 | 4858 | Q9I600 |
| 531 | Q60169 | 1613 | Q9HXY6 | 2695 | Q9HV06 | 3777 | Q9I0A5 | 4859 | Q9I601 |
| 532 | Q6H941 | 1614 | Q9HXY8 | 2696 | Q9HV07 | 3778 | Q9I0A6 | 4860 | Q9I602 |
| 533 | Q9HT06 | 1615 | Q9HXZ1 | 2697 | Q9HV08 | 3779 | Q9I0A8 | 4861 | Q9I603 |
| 534 | Q9HT07 | 1616 | Q9HXZ3 | 2698 | Q9HV09 | 3780 | Q9I0A9 | 4862 | Q9I604 |
| 535 | Q9HT15 | 1617 | Q9HXZ6 | 2699 | Q9HV10 | 3781 | Q9I0B1 | 4863 | Q9I605 |
| 536 | Q9HT21 | 1618 | Q9HY07 | 2700 | Q9HV11 | 3782 | Q9I0B2 | 4864 | Q9I606 |
| 537 | Q9HT25 | 1619 | Q9HY08 | 2701 | Q9HV12 | 3783 | Q9I0B3 | 4865 | Q9I607 |
| 538 | Q9HT57 | 1620 | Q9HY16 | 2702 | Q9HV13 | 3784 | Q9I0B4 | 4866 | Q9I608 |
| 539 | Q9HT70 | 1621 | Q9HY22 | 2703 | Q9HV14 | 3785 | Q9I0B6 | 4867 | Q9I610 |
| 540 | Q9HT73 | 1622 | Q9HY24 | 2704 | Q9HV15 | 3786 | Q9I0B7 | 4868 | Q9I611 |
| 541 | Q9HT74 | 1623 | Q9HY25 | 2705 | Q9HV16 | 3787 | Q9I0B8 | 4869 | Q9I612 |
| 542 | Q9HT80 | 1624 | Q9HY30 | 2706 | Q9HV17 | 3788 | Q9I0B9 | 4870 | Q9I613 |
| 543 | Q9HTB7 | 1625 | Q9HY39 | 2707 | Q9HV19 | 3789 | Q9I0C0 | 4871 | Q9I616 |
| 544 | Q9HTC7 | 1626 | Q9HY42 | 2708 | Q9HV20 | 3790 | Q9I0C1 | 4872 | Q9I619 |
| 545 | Q9HTE3 | 1627 | Q9HY45 | 2709 | Q9HV21 | 3791 | Q9I0C2 | 4873 | Q9I620 |
| 546 | Q9HTE8 | 1628 | Q9HY46 | 2710 | Q9HV22 | 3792 | Q9I0C3 | 4874 | Q9I621 |
| 547 | Q9HTF1 | 1629 | Q9HY47 | 2711 | Q9HV23 | 3793 | Q9I0C5 | 4875 | Q9I622 |
| 548 | Q9HTH0 | 1630 | Q9HY55 | 2712 | Q9HV24 | 3794 | Q9I0C6 | 4876 | Q9I623 |
| 549 | Q9HTH5 | 1631 | Q9HY56 | 2713 | Q9HV25 | 3795 | Q9I0C8 | 4877 | Q9I624 |
| 550 | Q9HTH6 | 1632 | Q9HY57 | 2714 | Q9HV26 | 3796 | Q9I0C9 | 4878 | Q9I625 |
| 551 | Q9HTJ2 | 1633 | Q9HY58 | 2715 | Q9HV29 | 3797 | Q9I0D0 | 4879 | Q9I627 |
| 552 | Q9HTK0 | 1634 | Q9HY75 | 2716 | Q9HV33 | 3798 | Q9I0D1 | 4880 | Q9I628 |
| 553 | Q9HTK7 | 1635 | Q9HY77 | 2717 | Q9HV47 | 3799 | Q9I0D2 | 4881 | Q9I629 |
| 554 | Q9HTL4 | 1636 | Q9HY80 | 2718 | Q9HV60 | 3800 | Q9I0D4 | 4882 | Q9I630 |
| 555 | Q9HTM2 | 1637 | Q9HY87 | 2719 | Q9HV61 | 3801 | Q9I0D5 | 4883 | Q9I631 |
| 556 | Q9HTN3 | 1638 | Q9HY88 | 2720 | Q9HV62 | 3802 | Q9I0D6 | 4884 | Q9I634 |
| 557 | Q9HTQ6 | 1639 | Q9HYA9 | 2721 | Q9HV63 | 3803 | Q9I0D7 | 4885 | Q9I635 |
| 558 | Q9HTQ8 | 1640 | Q9HYB5 | 2722 | Q9HV64 | 3804 | Q9I0D8 | 4886 | Q9I637 |
| 559 | Q9HTV1 | 1641 | Q9HYB6 | 2723 | Q9HV65 | 3805 | Q9I0E1 | 4887 | Q9I638 |
| 560 | Q9HTV3 | 1642 | Q9HYB7 | 2724 | Q9HV77 | 3806 | Q9I0E2 | 4888 | Q9I639 |
| 561 | Q9HTZ7 | 1643 | Q9HYC0 | 2725 | Q9HV79 | 3807 | Q9I0E3 | 4889 | Q9I640 |
| 562 | Q9HU14 | 1644 | Q9HYE4 | 2726 | Q9HV80 | 3808 | Q9I0E4 | 4890 | Q9I641 |
| 563 | Q9HU16 | 1645 | Q9HYE7 | 2727 | Q9HV81 | 3809 | Q9I0E5 | 4891 | Q9I642 |
| 564 | Q9HU17 | 1646 | Q9HYF4 | 2728 | Q9HV83 | 3810 | Q9I0E6 | 4892 | Q9I643 |
| 565 | Q9HU18 | 1647 | Q9HYF7 | 2729 | Q9HV84 | 3811 | Q9I0E7 | 4893 | Q9I644 |
| 566 | Q9HU19 | 1648 | Q9HYG1 | 2730 | Q9HV85 | 3812 | Q9I0E8 | 4894 | Q9I645 |
| 567 | Q9HU20 | 1649 | Q9HYG4 | 2731 | Q9HV86 | 3813 | Q9I0E9 | 4895 | Q9I647 |
| 568 | Q9HU22 | 1650 | Q9HYG9 | 2732 | Q9HV87 | 3814 | Q9I0F0 | 4896 | Q9I649 |
| 569 | Q9HU37 | 1651 | Q9HYH1 | 2733 | Q9HV89 | 3815 | Q9I0F1 | 4897 | Q9I651 |
| 570 | Q9HU42 | 1652 | Q9HYH5 | 2734 | Q9HV90 | 3816 | Q9I0F5 | 4898 | Q9I652 |
| 571 | Q9HU44 | 1653 | Q9HYI7 | 2735 | Q9HV91 | 3817 | Q9I0F6 | 4899 | Q9I653 |
| 572 | Q9HU50 | 1654 | Q9HYI8 | 2736 | Q9HV92 | 3818 | Q9I0F7 | 4900 | Q9I656 |
| 573 | Q9HU53 | 1655 | Q9HYJ0 | 2737 | Q9HV93 | 3819 | Q9I0F8 | 4901 | Q9I657 |
| 574 | Q9HU57 | 1656 | Q9HYJ5 | 2738 | Q9HV94 | 3820 | Q9I0F9 | 4902 | Q9I658 |
| 575 | Q9HU65 | 1657 | Q9HYJ7 | 2739 | Q9HV95 | 3821 | Q9I0G1 | 4903 | Q9I660 |
| 576 | Q9HU73 | 1658 | Q9HYJ8 | 2740 | Q9HV96 | 3822 | Q9I0G2 | 4904 | Q9I661 |
| 577 | Q9HU83 | 1659 | Q9HYJ9 | 2741 | Q9HV97 | 3823 | Q9I0G4 | 4905 | Q9I662 |
| 578 | Q9HU85 | 1660 | Q9HYK7 | 2742 | Q9HV98 | 3824 | Q9I0G5 | 4906 | Q9I663 |
| 579 | Q9HU91 | 1661 | Q9HYK9 | 2743 | Q9HV99 | 3825 | Q9I0G6 | 4907 | Q9I664 |
| 580 | Q9HU92 | 1662 | Q9HYL1 | 2744 | Q9HVA6 | 3826 | Q9I0G7 | 4908 | Q9I665 |
| 581 | Q9HU93 | 1663 | Q9HYL3 | 2745 | Q9HVA7 | 3827 | Q9I0G8 | 4909 | Q9I667 |
| 582 | Q9HUA4 | 1664 | Q9HYM4 | 2746 | Q9HVA9 | 3828 | Q9I0G9 | 4910 | Q9I670 |
| 583 | Q9HUB4 | 1665 | Q9HYM8 | 2747 | Q9HVB0 | 3829 | Q9I0H0 | 4911 | Q9I672 |
| 584 | Q9HUB7 | 1666 | Q9HYN9 | 2748 | Q9HVB1 | 3830 | Q9I0H1 | 4912 | Q9I673 |
| 585 | Q9HUB8 | 1667 | Q9HYP4 | 2749 | Q9HVB2 | 3831 | Q9I0H3 | 4913 | Q9I674 |
| 586 | Q9HUC6 | 1668 | Q9HYP5 | 2750 | Q9HVB3 | 3832 | Q9I0H5 | 4914 | Q9I675 |
| 587 | Q9HUC9 | 1669 | Q9HYQ0 | 2751 | Q9HVB4 | 3833 | Q9I0H6 | 4915 | Q9I677 |
| 588 | Q9HUE6 | 1670 | Q9HYQ2 | 2752 | Q9HVB5 | 3834 | Q9I0H7 | 4916 | Q9I679 |
| 589 | Q9HUE8 | 1671 | Q9HYQ5 | 2753 | Q9HVB6 | 3835 | Q9I0H8 | 4917 | Q9I680 |
| 590 | Q9HUF0 | 1672 | Q9HYQ6 | 2754 | Q9HVC1 | 3836 | Q9I0H9 | 4918 | Q9I681 |
| 591 | Q9HUF4 | 1673 | Q9HYR6 | 2755 | Q9HVD3 | 3837 | Q9I0I0 | 4919 | Q9I682 |
| 592 | Q9HUF5 | 1674 | Q9HYT3 | 2756 | Q9HVD4 | 3838 | Q9I0I3 | 4920 | Q9I684 |
| 593 | Q9HUH5 | 1675 | Q9HYU0 | 2757 | Q9HVD6 | 3839 | Q9I0I5 | 4921 | Q9I686 |
| 594 | Q9HUH7 | 1676 | Q9HYU3 | 2758 | Q9HVD8 | 3840 | Q9I0I7 | 4922 | Q9I688 |
| 595 | Q9HUI2 | 1677 | Q9HYU4 | 2759 | Q9HVD9 | 3841 | Q9I0I8 | 4923 | Q9I695 |
| 596 | Q9HUI8 | 1678 | Q9HYU6 | 2760 | Q9HVE1 | 3842 | Q9I0K2 | 4924 | Q9I6A2 |
| 597 | Q9HUJ2 | 1679 | Q9HYX5 | 2761 | Q9HVE2 | 3843 | Q9I0K3 | 4925 | Q9I6A3 |
| 598 | Q9HUJ4 | 1680 | Q9HYX7 | 2762 | Q9HVE3 | 3844 | Q9I0K5 | 4926 | Q9I6A4 |
| 599 | Q9HUK8 | 1681 | Q9HYX8 | 2763 | Q9HVE4 | 3845 | Q9I0K6 | 4927 | Q9I6A6 |
| 600 | Q9HUL3 | 1682 | Q9HYY3 | 2764 | Q9HVE5 | 3846 | Q9I0K7 | 4928 | Q9I6A7 |
| 601 | Q9HUL4 | 1683 | Q9HYZ3 | 2765 | Q9HVE8 | 3847 | Q9I0K8 | 4929 | Q9I6B0 |
| 602 | Q9HUL9 | 1684 | Q9HYZ5 | 2766 | Q9HVE9 | 3848 | Q9I0L0 | 4930 | Q9I6B1 |
| 603 | Q9HUM0 | 1685 | Q9HYZ6 | 2767 | Q9HVF0 | 3849 | Q9I0M8 | 4931 | Q9I6B2 |
| 604 | Q9HUM6 | 1686 | Q9HYZ7 | 2768 | Q9HVF2 | 3850 | Q9I0M9 | 4932 | Q9I6B5 |
| 605 | Q9HUP3 | 1687 | Q9HZ00 | 2769 | Q9HVF3 | 3851 | Q9I0N1 | 4933 | Q9I6B6 |
| 606 | Q9HUR2 | 1688 | Q9HZ04 | 2770 | Q9HVF4 | 3852 | Q9I0N2 | 4934 | Q9I6B8 |
| 607 | Q9HUU5 | 1689 | Q9HZ06 | 2771 | Q9HVF5 | 3853 | Q9I0N4 | 4935 | Q9I6C3 |
| 608 | Q9HUU7 | 1690 | Q9HZ23 | 2772 | Q9HVF8 | 3854 | Q9I0N6 | 4936 | Q9I6C5 |
| 609 | Q9HUV6 | 1691 | Q9HZ29 | 2773 | Q9HVG0 | 3855 | Q9I0N7 | 4937 | Q9I6C6 |
| 610 | Q9HUV9 | 1692 | Q9HZ30 | 2774 | Q9HVG1 | 3856 | Q9I0N9 | 4938 | Q9I6C7 |
| 611 | Q9HUW1 | 1693 | Q9HZ34 | 2775 | Q9HVG3 | 3857 | Q9I0P0 | 4939 | Q9I6C9 |
| 612 | Q9HUW5 | 1694 | Q9HZ35 | 2776 | Q9HVG5 | 3858 | Q9I0P2 | 4940 | Q9I6D0 |
| 613 | Q9HUW6 | 1695 | Q9HZ39 | 2777 | Q9HVG6 | 3859 | Q9I0P3 | 4941 | Q9I6D2 |
| 614 | Q9HUW7 | 1696 | Q9HZ46 | 2778 | Q9HVG8 | 3860 | Q9I0P4 | 4942 | Q9I6D4 |
| 615 | Q9HUX1 | 1697 | Q9HZ52 | 2779 | Q9HVG9 | 3861 | Q9I0P8 | 4943 | Q9I6D5 |
| 616 | Q9HUX3 | 1698 | Q9HZ57 | 2780 | Q9HVH0 | 3862 | Q9I0P9 | 4944 | Q9I6D6 |
| 617 | Q9HUX4 | 1699 | Q9HZ58 | 2781 | Q9HVH1 | 3863 | Q9I0Q5 | 4945 | Q9I6D7 |
| 618 | Q9HUX6 | 1700 | Q9HZ60 | 2782 | Q9HVH3 | 3864 | Q9I0Q9 | 4946 | Q9I6D9 |
| 619 | Q9HUY5 | 1701 | Q9HZ64 | 2783 | Q9HVH5 | 3865 | Q9I0R0 | 4947 | Q9I6E1 |
| 620 | Q9HV01 | 1702 | Q9HZ68 | 2784 | Q9HVH9 | 3866 | Q9I0R1 | 4948 | Q9I6E2 |
| 621 | Q9HV31 | 1703 | Q9HZ71 | 2785 | Q9HVI0 | 3867 | Q9I0R3 | 4949 | Q9I6E4 |
| 622 | Q9HV32 | 1704 | Q9HZ86 | 2786 | Q9HVI2 | 3868 | Q9I0R4 | 4950 | Q9I6E5 |
| 623 | Q9HV34 | 1705 | Q9HZA4 | 2787 | Q9HVI3 | 3869 | Q9I0R5 | 4951 | Q9I6E7 |
| 624 | Q9HV35 | 1706 | Q9HZA8 | 2788 | Q9HVI4 | 3870 | Q9I0R6 | 4952 | Q9I6E8 |
| 625 | Q9HV37 | 1707 | Q9HZA9 | 2789 | Q9HVI5 | 3871 | Q9I0R7 | 4953 | Q9I6E9 |
| 626 | Q9HV49 | 1708 | Q9HZC5 | 2790 | Q9HVI6 | 3872 | Q9I0R9 | 4954 | Q9I6F0 |
| 627 | Q9HV51 | 1709 | Q9HZD0 | 2791 | Q9HVJ0 | 3873 | Q9I0S0 | 4955 | Q9I6F3 |
| 628 | Q9HV55 | 1710 | Q9HZD1 | 2792 | Q9HVJ3 | 3874 | Q9I0S2 | 4956 | Q9I6F4 |
| 629 | Q9HV59 | 1711 | Q9HZD4 | 2793 | Q9HVJ5 | 3875 | Q9I0S3 | 4957 | Q9I6F5 |
| 630 | Q9HV68 | 1712 | Q9HZE4 | 2794 | Q9HVJ6 | 3876 | Q9I0S4 | 4958 | Q9I6F7 |
| 631 | Q9HV69 | 1713 | Q9HZE5 | 2795 | Q9HVJ8 | 3877 | Q9I0S5 | 4959 | Q9I6F8 |
| 632 | Q9HV70 | 1714 | Q9HZE6 | 2796 | Q9HVK0 | 3878 | Q9I0S7 | 4960 | Q9I6F9 |
| 633 | Q9HV71 | 1715 | Q9HZE7 | 2797 | Q9HVK2 | 3879 | Q9I0S8 | 4961 | Q9I6G2 |
| 634 | Q9HV72 | 1716 | Q9HZF0 | 2798 | Q9HVK3 | 3880 | Q9I0S9 | 4962 | Q9I6G6 |
| 635 | Q9HV73 | 1717 | Q9HZF4 | 2799 | Q9HVK4 | 3881 | Q9I0T2 | 4963 | Q9I6G8 |
| 636 | Q9HV74 | 1718 | Q9HZF9 | 2800 | Q9HVK5 | 3882 | Q9I0T3 | 4964 | Q9I6G9 |
| 637 | Q9HV78 | 1719 | Q9HZG1 | 2801 | Q9HVK6 | 3883 | Q9I0T5 | 4965 | Q9I6H2 |
| 638 | Q9HVA0 | 1720 | Q9HZG4 | 2802 | Q9HVK7 | 3884 | Q9I0T6 | 4966 | Q9I6H3 |
| 639 | Q9HVA1 | 1721 | Q9HZG5 | 2803 | Q9HVK9 | 3885 | Q9I0T7 | 4967 | Q9I6H6 |
| 640 | Q9HVA3 | 1722 | Q9HZH0 | 2804 | Q9HVL0 | 3886 | Q9I0T8 | 4968 | Q9I6H7 |
| 641 | Q9HVA4 | 1723 | Q9HZH6 | 2805 | Q9HVL1 | 3887 | Q9I0U0 | 4969 | Q9I6H8 |
| 642 | Q9HVA5 | 1724 | Q9HZH7 | 2806 | Q9HVL4 | 3888 | Q9I0U2 | 4970 | Q9I6H9 |
| 643 | Q9HVC8 | 1725 | Q9HZH8 | 2807 | Q9HVL5 | 3889 | Q9I0U3 | 4971 | Q9I6I0 |
| 644 | Q9HVD5 | 1726 | Q9HZI0 | 2808 | Q9HVM0 | 3890 | Q9I0U4 | 4972 | Q9I6I1 |
| 645 | Q9HVD7 | 1727 | Q9HZI7 | 2809 | Q9HVN6 | 3891 | Q9I0U6 | 4973 | Q9I6I2 |
| 646 | Q9HVE6 | 1728 | Q9HZJ9 | 2810 | Q9HVN7 | 3892 | Q9I0U7 | 4974 | Q9I6I3 |
| 647 | Q9HVF1 | 1729 | Q9HZK4 | 2811 | Q9HVN8 | 3893 | Q9I0U8 | 4975 | Q9I6I4 |
| 648 | Q9HVH6 | 1730 | Q9HZK5 | 2812 | Q9HVN9 | 3894 | Q9I0U9 | 4976 | Q9I6I5 |
| 649 | Q9HVJ1 | 1731 | Q9HZK6 | 2813 | Q9HVP0 | 3895 | Q9I0V0 | 4977 | Q9I6J3 |
| 650 | Q9HVK1 | 1732 | Q9HZK9 | 2814 | Q9HVP1 | 3896 | Q9I0V1 | 4978 | Q9I6J6 |
| 651 | Q9HVL8 | 1733 | Q9HZL2 | 2815 | Q9HVP2 | 3897 | Q9I0V3 | 4979 | Q9I6J7 |
| 652 | Q9HVL9 | 1734 | Q9HZL6 | 2816 | Q9HVP3 | 3898 | Q9I0V4 | 4980 | Q9I6J8 |
| 653 | Q9HVM1 | 1735 | Q9HZL8 | 2817 | Q9HVP4 | 3899 | Q9I0V6 | 4981 | Q9I6K0 |
| 654 | Q9HVM3 | 1736 | Q9HZM2 | 2818 | Q9HVP5 | 3900 | Q9I0V7 | 4982 | Q9I6K1 |
| 655 | Q9HVM4 | 1737 | Q9HZM6 | 2819 | Q9HVP6 | 3901 | Q9I0V8 | 4983 | Q9I6K4 |
| 656 | Q9HVM7 | 1738 | Q9HZN2 | 2820 | Q9HVQ1 | 3902 | Q9I0W0 | 4984 | Q9I6K5 |
| 657 | Q9HVN5 | 1739 | Q9HZN4 | 2821 | Q9HVQ2 | 3903 | Q9I0W1 | 4985 | Q9I6K6 |
| 658 | Q9HVP8 | 1740 | Q9HZN6 | 2822 | Q9HVQ4 | 3904 | Q9I0W2 | 4986 | Q9I6K7 |
| 659 | Q9HVQ3 | 1741 | Q9HZN7 | 2823 | Q9HVQ6 | 3905 | Q9I0W3 | 4987 | Q9I6K9 |
| 660 | Q9HVT9 | 1742 | Q9HZP9 | 2824 | Q9HVQ9 | 3906 | Q9I0W4 | 4988 | Q9I6L1 |
| 661 | Q9HVU3 | 1743 | Q9HZQ4 | 2825 | Q9HVR1 | 3907 | Q9I0W5 | 4989 | Q9I6L2 |
| 662 | Q9HVW7 | 1744 | Q9HZR1 | 2826 | Q9HVR2 | 3908 | Q9I0W6 | 4990 | Q9I6L4 |
| 663 | Q9HVW9 | 1745 | Q9HZR3 | 2827 | Q9HVR3 | 3909 | Q9I0W7 | 4991 | Q9I6L5 |
| 664 | Q9HVX6 | 1746 | Q9HZR6 | 2828 | Q9HVR4 | 3910 | Q9I0W8 | 4992 | Q9I6L6 |
| 665 | Q9HVZ7 | 1747 | Q9HZS2 | 2829 | Q9HVR5 | 3911 | Q9I0W9 | 4993 | Q9I6L7 |
| 666 | Q9HVZ8 | 1748 | Q9HZS3 | 2830 | Q9HVR7 | 3912 | Q9I0X0 | 4994 | Q9I6L8 |
| 667 | Q9HVZ9 | 1749 | Q9HZT1 | 2831 | Q9HVR8 | 3913 | Q9I0X1 | 4995 | Q9I6L9 |
| 668 | Q9HW00 | 1750 | Q9HZT7 | 2832 | Q9HVR9 | 3914 | Q9I0X2 | 4996 | Q9I6M0 |
| 669 | Q9HW01 | 1751 | Q9HZT9 | 2833 | Q9HVS0 | 3915 | Q9I0X6 | 4997 | Q9I6M1 |
| 670 | Q9HW02 | 1752 | Q9HZU0 | 2834 | Q9HVS2 | 3916 | Q9I0X7 | 4998 | Q9I6M2 |
| 671 | Q9HW06 | 1753 | Q9HZU3 | 2835 | Q9HVS3 | 3917 | Q9I0X8 | 4999 | Q9I6M6 |
| 672 | Q9HW09 | 1754 | Q9HZV8 | 2836 | Q9HVS5 | 3918 | Q9I0X9 | 5000 | Q9I6M8 |
| 673 | Q9HW19 | 1755 | Q9HZW2 | 2837 | Q9HVS6 | 3919 | Q9I0Y0 | 5001 | Q9I6M9 |
| 674 | Q9HW50 | 1756 | Q9HZX6 | 2838 | Q9HVS8 | 3920 | Q9I0Y1 | 5002 | Q9I6N1 |
| 675 | Q9HW69 | 1757 | Q9HZY3 | 2839 | Q9HVS9 | 3921 | Q9I0Y2 | 5003 | Q9I6N2 |
| 676 | Q9HW72 | 1758 | Q9HZY5 | 2840 | Q9HVT0 | 3922 | Q9I0Y5 | 5004 | Q9I6N3 |
| 677 | Q9HWA7 | 1759 | Q9HZZ0 | 2841 | Q9HVT1 | 3923 | Q9I0Y6 | 5005 | Q9I6N4 |
| 678 | Q9HWB5 | 1760 | Q9HZZ4 | 2842 | Q9HVT2 | 3924 | Q9I0Y9 | 5006 | Q9I6N6 |
| 679 | Q9HWB7 | 1761 | Q9I000 | 2843 | Q9HVT3 | 3925 | Q9I0Z1 | 5007 | Q9I6N7 |
| 680 | Q9HWB8 | 1762 | Q9I004 | 2844 | Q9HVT4 | 3926 | Q9I0Z2 | 5008 | Q9I6N8 |
| 681 | Q9HWC0 | 1763 | Q9I011 | 2845 | Q9HVT5 | 3927 | Q9I0Z3 | 5009 | Q9I6N9 |
| 682 | Q9HWD0 | 1764 | Q9I013 | 2846 | Q9HVU5 | 3928 | Q9I0Z4 | 5010 | Q9I6P0 |
| 683 | Q9HWD6 | 1765 | Q9I015 | 2847 | Q9HVU6 | 3929 | Q9I0Z5 | 5011 | Q9I6P1 |
| 684 | Q9HWF2 | 1766 | Q9I019 | 2848 | Q9HVU7 | 3930 | Q9I0Z6 | 5012 | Q9I6P2 |
| 685 | Q9HWF9 | 1767 | Q9I025 | 2849 | Q9HVU8 | 3931 | Q9I0Z7 | 5013 | Q9I6P5 |
| 686 | Q9HWG0 | 1768 | Q9I032 | 2850 | Q9HVU9 | 3932 | Q9I0Z8 | 5014 | Q9I6P7 |
| 687 | Q9HWG3 | 1769 | Q9I033 | 2851 | Q9HVV0 | 3933 | Q9I0Z9 | 5015 | Q9I6P8 |
| 688 | Q9HWH8 | 1770 | Q9I034 | 2852 | Q9HVV1 | 3934 | Q9I100 | 5016 | Q9I6P9 |
| 689 | Q9HWI0 | 1771 | Q9I035 | 2853 | Q9HVW1 | 3935 | Q9I103 | 5017 | Q9I6Q0 |
| 690 | Q9HWJ0 | 1772 | Q9I061 | 2854 | Q9HVW3 | 3936 | Q9I105 | 5018 | Q9I6Q1 |
| 691 | Q9HWP3 | 1773 | Q9I066 | 2855 | Q9HVW4 | 3937 | Q9I107 | 5019 | Q9I6Q2 |
| 692 | Q9HWP8 | 1774 | Q9I068 | 2856 | Q9HVW5 | 3938 | Q9I108 | 5020 | Q9I6Q4 |
| 693 | Q9HWX2 | 1775 | Q9I073 | 2857 | Q9HVW6 | 3939 | Q9I109 | 5021 | Q9I6Q5 |
| 694 | Q9HWX4 | 1776 | Q9I088 | 2858 | Q9HVX3 | 3940 | Q9I110 | 5022 | Q9I6Q6 |
| 695 | Q9HWX5 | 1777 | Q9I0A0 | 2859 | Q9HVX5 | 3941 | Q9I111 | 5023 | Q9I6Q7 |
| 696 | Q9HWX7 | 1778 | Q9I0A1 | 2860 | Q9HVX8 | 3942 | Q9I112 | 5024 | Q9I6Q9 |
| 697 | Q9HWY1 | 1779 | Q9I0A2 | 2861 | Q9HVX9 | 3943 | Q9I113 | 5025 | Q9I6R3 |
| 698 | Q9HWZ6 | 1780 | Q9I0A7 | 2862 | Q9HVY1 | 3944 | Q9I115 | 5026 | Q9I6R4 |
| 699 | Q9HX02 | 1781 | Q9I0B0 | 2863 | Q9HVY7 | 3945 | Q9I117 | 5027 | Q9I6R5 |
| 700 | Q9HX03 | 1782 | Q9I0B5 | 2864 | Q9HVY8 | 3946 | Q9I118 | 5028 | Q9I6R7 |
| 701 | Q9HX07 | 1783 | Q9I0C4 | 2865 | Q9HVY9 | 3947 | Q9I120 | 5029 | Q9I6R8 |
| 702 | Q9HX08 | 1784 | Q9I0C7 | 2866 | Q9HVZ1 | 3948 | Q9I121 | 5030 | Q9I6R9 |
| 703 | Q9HX25 | 1785 | Q9I0D3 | 2867 | Q9HVZ2 | 3949 | Q9I122 | 5031 | Q9I6S0 |
| 704 | Q9HX40 | 1786 | Q9I0G0 | 2868 | Q9HW03 | 3950 | Q9I123 | 5032 | Q9I6S1 |
| 705 | Q9HX97 | 1787 | Q9I0G3 | 2869 | Q9HW05 | 3951 | Q9I124 | 5033 | Q9I6S2 |
| 706 | Q9HXB1 | 1788 | Q9I0H2 | 2870 | Q9HW10 | 3952 | Q9I125 | 5034 | Q9I6S3 |
| 707 | Q9HXC2 | 1789 | Q9I0J3 | 2871 | Q9HW11 | 3953 | Q9I126 | 5035 | Q9I6S6 |
| 708 | Q9HXC4 | 1790 | Q9I0L1 | 2872 | Q9HW13 | 3954 | Q9I127 | 5036 | Q9I6S7 |
| 709 | Q9HXD6 | 1791 | Q9I0L3 | 2873 | Q9HW14 | 3955 | Q9I128 | 5037 | Q9I6T0 |
| 710 | Q9HXE4 | 1792 | Q9I0L4 | 2874 | Q9HW15 | 3956 | Q9I129 | 5038 | Q9I6T1 |
| 711 | Q9HXE5 | 1793 | Q9I0L6 | 2875 | Q9HW16 | 3957 | Q9I131 | 5039 | Q9I6T3 |
| 712 | Q9HXE9 | 1794 | Q9I0L7 | 2876 | Q9HW17 | 3958 | Q9I132 | 5040 | Q9I6T5 |
| 713 | Q9HXI5 | 1795 | Q9I0L8 | 2877 | Q9HW18 | 3959 | Q9I134 | 5041 | Q9I6T6 |
| 714 | Q9HXJ4 | 1796 | Q9I0M0 | 2878 | Q9HW20 | 3960 | Q9I135 | 5042 | Q9I6T7 |
| 715 | Q9HXJ8 | 1797 | Q9I0M2 | 2879 | Q9HW21 | 3961 | Q9I141 | 5043 | Q9I6T8 |
| 716 | Q9HXK5 | 1798 | Q9I0M5 | 2880 | Q9HW23 | 3962 | Q9I142 | 5044 | Q9I6U0 |
| 717 | Q9HXM6 | 1799 | Q9I0N0 | 2881 | Q9HW24 | 3963 | Q9I145 | 5045 | Q9I6U1 |
| 718 | Q9HXN1 | 1800 | Q9I0N3 | 2882 | Q9HW25 | 3964 | Q9I146 | 5046 | Q9I6U2 |
| 719 | Q9HXN2 | 1801 | Q9I0N8 | 2883 | Q9HW27 | 3965 | Q9I148 | 5047 | Q9I6U3 |
| 720 | Q9HXP3 | 1802 | Q9I0P1 | 2884 | Q9HW28 | 3966 | Q9I149 | 5048 | Q9I6U5 |
| 721 | Q9HXP8 | 1803 | Q9I0P5 | 2885 | Q9HW29 | 3967 | Q9I150 | 5049 | Q9I6U6 |
| 722 | Q9HXQ1 | 1804 | Q9I0P6 | 2886 | Q9HW30 | 3968 | Q9I151 | 5050 | Q9I6U7 |
| 723 | Q9HXT8 | 1805 | Q9I0P7 | 2887 | Q9HW31 | 3969 | Q9I152 | 5051 | Q9I6U8 |
| 724 | Q9HXU0 | 1806 | Q9I0Q1 | 2888 | Q9HW32 | 3970 | Q9I153 | 5052 | Q9I6U9 |
| 725 | Q9HXY2 | 1807 | Q9I0Q2 | 2889 | Q9HW33 | 3971 | Q9I155 | 5053 | Q9I6V0 |
| 726 | Q9HXY7 | 1808 | Q9I0Q3 | 2890 | Q9HW36 | 3972 | Q9I156 | 5054 | Q9I6V1 |
| 727 | Q9HXY9 | 1809 | Q9I0Q4 | 2891 | Q9HW37 | 3973 | Q9I158 | 5055 | Q9I6V3 |
| 728 | Q9HXZ2 | 1810 | Q9I0Q6 | 2892 | Q9HW39 | 3974 | Q9I159 | 5056 | Q9I6V5 |
| 729 | Q9HY04 | 1811 | Q9I0S1 | 2893 | Q9HW40 | 3975 | Q9I160 | 5057 | Q9I6W1 |
| 730 | Q9HY05 | 1812 | Q9I0S6 | 2894 | Q9HW41 | 3976 | Q9I161 | 5058 | Q9I6W2 |
| 731 | Q9HY23 | 1813 | Q9I0T9 | 2895 | Q9HW42 | 3977 | Q9I162 | 5059 | Q9I6W3 |
| 732 | Q9HY41 | 1814 | Q9I0U1 | 2896 | Q9HW44 | 3978 | Q9I164 | 5060 | Q9I6W4 |
| 733 | Q9HY59 | 1815 | Q9I0V2 | 2897 | Q9HW45 | 3979 | Q9I166 | 5061 | Q9I6W5 |
| 734 | Q9HY60 | 1816 | Q9I0V5 | 2898 | Q9HW46 | 3980 | Q9I167 | 5062 | Q9I6W6 |
| 735 | Q9HY61 | 1817 | Q9I0V9 | 2899 | Q9HW47 | 3981 | Q9I169 | 5063 | Q9I6W7 |
| 736 | Q9HY62 | 1818 | Q9I0X3 | 2900 | Q9HW48 | 3982 | Q9I170 | 5064 | Q9I6W8 |
| 737 | Q9HY64 | 1819 | Q9I0X4 | 2901 | Q9HW49 | 3983 | Q9I171 | 5065 | Q9I6W9 |
| 738 | Q9HY65 | 1820 | Q9I0X5 | 2902 | Q9HW52 | 3984 | Q9I172 | 5066 | Q9I6X0 |
| 739 | Q9HY79 | 1821 | Q9I0Y3 | 2903 | Q9HW53 | 3985 | Q9I173 | 5067 | Q9I6X1 |
| 740 | Q9HY84 | 1822 | Q9I0Y7 | 2904 | Q9HW54 | 3986 | Q9I174 | 5068 | Q9I6X2 |
| 741 | Q9HY85 | 1823 | Q9I0Y8 | 2905 | Q9HW55 | 3987 | Q9I175 | 5069 | Q9I6X3 |
| 742 | Q9HY92 | 1824 | Q9I101 | 2906 | Q9HW56 | 3988 | Q9I177 | 5070 | Q9I6X4 |
| 743 | Q9HYA4 | 1825 | Q9I102 | 2907 | Q9HW57 | 3989 | Q9I178 | 5071 | Q9I6X5 |
| 744 | Q9HYB4 | 1826 | Q9I106 | 2908 | Q9HW58 | 3990 | Q9I179 | 5072 | Q9I6X6 |
| 745 | Q9HYB8 | 1827 | Q9I114 | 2909 | Q9HW59 | 3991 | Q9I185 | 5073 | Q9I6X7 |
| 746 | Q9HYB9 | 1828 | Q9I119 | 2910 | Q9HW60 | 3992 | Q9I187 | 5074 | Q9I6X8 |
| 747 | Q9HYC3 | 1829 | Q9I130 | 2911 | Q9HW61 | 3993 | Q9I192 | 5075 | Q9I6X9 |
| 748 | Q9HYC4 | 1830 | Q9I136 | 2912 | Q9HW62 | 3994 | Q9I193 | 5076 | Q9I6Y0 |
| 749 | Q9HYC7 | 1831 | Q9I139 | 2913 | Q9HW63 | 3995 | Q9I195 | 5077 | Q9I6Y2 |
| 750 | Q9HYC9 | 1832 | Q9I140 | 2914 | Q9HW64 | 3996 | Q9I196 | 5078 | Q9I6Y6 |
| 751 | Q9HYD5 | 1833 | Q9I143 | 2915 | Q9HW65 | 3997 | Q9I198 | 5079 | Q9I6Y7 |
| 752 | Q9HYF0 | 1834 | Q9I144 | 2916 | Q9HW66 | 3998 | Q9I199 | 5080 | Q9I6Y8 |
| 753 | Q9HYG2 | 1835 | Q9I147 | 2917 | Q9HW70 | 3999 | Q9I1A0 | 5081 | Q9I6Z4 |
| 754 | Q9HYL7 | 1836 | Q9I154 | 2918 | Q9HW71 | 4000 | Q9I1A1 | 5082 | Q9I6Z5 |
| 755 | Q9HYQ1 | 1837 | Q9I157 | 2919 | Q9HW73 | 4001 | Q9I1A2 | 5083 | Q9I6Z6 |
| 756 | Q9HYQ8 | 1838 | Q9I163 | 2920 | Q9HW74 | 4002 | Q9I1A3 | 5084 | Q9I6Z7 |
| 757 | Q9HYR2 | 1839 | Q9I168 | 2921 | Q9HW75 | 4003 | Q9I1A4 | 5085 | Q9I704 |
| 758 | Q9HYR9 | 1840 | Q9I181 | 2922 | Q9HW76 | 4004 | Q9I1A5 | 5086 | Q9I705 |
| 759 | Q9HYT0 | 1841 | Q9I182 | 2923 | Q9HW77 | 4005 | Q9I1A7 | 5087 | Q9I706 |
| 760 | Q9HYT6 | 1842 | Q9I183 | 2924 | Q9HW78 | 4006 | Q9I1A8 | 5088 | Q9I707 |
| 761 | Q9HYV7 | 1843 | Q9I184 | 2925 | Q9HW79 | 4007 | Q9I1A9 | 5089 | Q9I708 |
| 762 | Q9HYX0 | 1844 | Q9I188 | 2926 | Q9HW80 | 4008 | Q9I1B0 | 5090 | Q9I709 |
| 763 | Q9HYZ4 | 1845 | Q9I197 | 2927 | Q9HW81 | 4009 | Q9I1B1 | 5091 | Q9I710 |
| 764 | Q9HYZ8 | 1846 | Q9I1A6 | 2928 | Q9HW82 | 4010 | Q9I1B2 | 5092 | Q9I711 |
| 765 | Q9HZ55 | 1847 | Q9I1E0 | 2929 | Q9HW83 | 4011 | Q9I1B3 | 5093 | Q9I712 |
| 766 | Q9HZ63 | 1848 | Q9I1F4 | 2930 | Q9HW84 | 4012 | Q9I1B4 | 5094 | Q9I715 |
| 767 | Q9HZ65 | 1849 | Q9I1G9 | 2931 | Q9HW88 | 4013 | Q9I1B5 | 5095 | Q9I716 |
| 768 | Q9HZ67 | 1850 | Q9I1H5 | 2932 | Q9HW89 | 4014 | Q9I1B6 | 5096 | Q9I717 |
| 769 | Q9HZ70 | 1851 | Q9I1I6 | 2933 | Q9HW90 | 4015 | Q9I1B7 | 5097 | Q9I718 |
| 770 | Q9HZ95 | 1852 | Q9I1I9 | 2934 | Q9HW92 | 4016 | Q9I1B8 | 5098 | Q9I720 |
| 771 | Q9HZA3 | 1853 | Q9I1J2 | 2935 | Q9HW94 | 4017 | Q9I1B9 | 5099 | Q9I721 |
| 772 | Q9HZC0 | 1854 | Q9I1J6 | 2936 | Q9HW95 | 4018 | Q9I1C0 | 5100 | Q9I722 |
| 773 | Q9HZF8 | 1855 | Q9I1K2 | 2937 | Q9HW96 | 4019 | Q9I1C1 | 5101 | Q9I723 |
| 774 | Q9HZG0 | 1856 | Q9I1K5 | 2938 | Q9HW97 | 4020 | Q9I1C3 | 5102 | Q9I728 |
| 775 | Q9HZI3 | 1857 | Q9I1K7 | 2939 | Q9HW98 | 4021 | Q9I1C4 | 5103 | Q9I731 |
| 776 | Q9HZJ3 | 1858 | Q9I1M1 | 2940 | Q9HW99 | 4022 | Q9I1C5 | 5104 | Q9I734 |
| 777 | Q9HZJ8 | 1859 | Q9I1M2 | 2941 | Q9HWA0 | 4023 | Q9I1C6 | 5105 | Q9I735 |
| 778 | Q9HZK3 | 1860 | Q9I1M3 | 2942 | Q9HWA2 | 4024 | Q9I1C7 | 5106 | Q9I736 |
| 779 | Q9HZK7 | 1861 | Q9I1M5 | 2943 | Q9HWA3 | 4025 | Q9I1C9 | 5107 | Q9I743 |
| 780 | Q9HZK8 | 1862 | Q9I1M7 | 2944 | Q9HWA5 | 4026 | Q9I1D0 | 5108 | Q9I751 |
| 781 | Q9HZL0 | 1863 | Q9I1M8 | 2945 | Q9HWA6 | 4027 | Q9I1D1 | 5109 | Q9I752 |
| 782 | Q9HZL1 | 1864 | Q9I1N4 | 2946 | Q9HWA9 | 4028 | Q9I1D2 | 5110 | Q9I754 |
| 783 | Q9HZL7 | 1865 | Q9I1N5 | 2947 | Q9HWB0 | 4029 | Q9I1D3 | 5111 | Q9I756 |
| 784 | Q9HZM3 | 1866 | Q9I1N8 | 2948 | Q9HWB1 | 4030 | Q9I1D4 | 5112 | Q9I760 |
| 785 | Q9HZM5 | 1867 | Q9I1R7 | 2949 | Q9HWB2 | 4031 | Q9I1D6 | 5113 | Q9I761 |
| 786 | Q9HZM7 | 1868 | Q9I1T4 | 2950 | Q9HWB3 | 4032 | Q9I1D7 | 5114 | Q9I762 |
| 787 | Q9HZM9 | 1869 | Q9I1T8 | 2951 | Q9HWB4 | 4033 | Q9I1D8 | 5115 | Q9I763 |
| 788 | Q9HZN5 | 1870 | Q9I1U3 | 2952 | Q9HWC2 | 4034 | Q9I1D9 | 5116 | Q9I764 |
| 789 | Q9HZN8 | 1871 | Q9I1V0 | 2953 | Q9HWG1 | 4035 | Q9I1E1 | 5117 | Q9I766 |
| 790 | Q9HZQ3 | 1872 | Q9I1V1 | 2954 | Q9HWG2 | 4036 | Q9I1E2 | 5118 | Q9I768 |
| 791 | Q9HZS1 | 1873 | Q9I1V2 | 2955 | Q9HWG5 | 4037 | Q9I1E3 | 5119 | Q9I769 |
| 792 | Q9HZU2 | 1874 | Q9I1W0 | 2956 | Q9HWG6 | 4038 | Q9I1E4 | 5120 | Q9I770 |
| 793 | Q9HZW0 | 1875 | Q9I1W3 | 2957 | Q9HWG7 | 4039 | Q9I1E5 | 5121 | Q9I771 |
| 794 | Q9HZY8 | 1876 | Q9I1W9 | 2958 | Q9HWH4 | 4040 | Q9I1E6 | 5122 | Q9I772 |
| 795 | Q9HZZ2 | 1877 | Q9I1Y3 | 2959 | Q9HWH5 | 4041 | Q9I1E7 | 5123 | Q9I773 |
| 796 | Q9I003 | 1878 | Q9I1Y5 | 2960 | Q9HWH6 | 4042 | Q9I1E8 | 5124 | Q9I774 |
| 797 | Q9I028 | 1879 | Q9I1Y6 | 2961 | Q9HWH7 | 4043 | Q9I1E9 | 5125 | Q9I775 |
| 798 | Q9I036 | 1880 | Q9I1Y7 | 2962 | Q9HWI1 | 4044 | Q9I1F0 | 5126 | Q9I776 |
| 799 | Q9I037 | 1881 | Q9I1Z7 | 2963 | Q9HWI2 | 4045 | Q9I1F1 | 5127 | Q9I777 |
| 800 | Q9I048 | 1882 | Q9I203 | 2964 | Q9HWI3 | 4046 | Q9I1F2 | 5128 | Q9I779 |
| 801 | Q9I063 | 1883 | Q9I209 | 2965 | Q9HWI5 | 4047 | Q9I1F3 | 5129 | Q9I780 |
| 802 | Q9I069 | 1884 | Q9I210 | 2966 | Q9HWI6 | 4048 | Q9I1F5 | 5130 | Q9I782 |
| 803 | Q9I095 | 1885 | Q9I211 | 2967 | Q9HWI7 | 4049 | Q9I1F7 | 5131 | Q9I783 |
| 804 | Q9I099 | 1886 | Q9I222 | 2968 | Q9HWI8 | 4050 | Q9I1F8 | 5132 | Q9I784 |
| 805 | Q9I0A3 | 1887 | Q9I231 | 2969 | Q9HWI9 | 4051 | Q9I1F9 | 5133 | Q9I785 |
| 806 | Q9I0A4 | 1888 | Q9I237 | 2970 | Q9HWJ1 | 4052 | Q9I1G0 | 5134 | Q9I786 |
| 807 | Q9I0D9 | 1889 | Q9I238 | 2971 | Q9HWJ2 | 4053 | Q9I1G1 | 5135 | Q9I789 |
| 808 | Q9I0E0 | 1890 | Q9I245 | 2972 | Q9HWJ4 | 4054 | Q9I1G2 | 5136 | Q9I790 |
| 809 | Q9I0F4 | 1891 | Q9I262 | 2973 | Q9HWJ5 | 4055 | Q9I1G3 | 5137 | Q9I791 |
| 810 | Q9I0I1 | 1892 | Q9I263 | 2974 | Q9HWJ6 | 4056 | Q9I1G4 | 5138 | Q9I793 |
| 811 | Q9I0I2 | 1893 | Q9I265 | 2975 | Q9HWJ7 | 4057 | Q9I1G5 | 5139 | Q9I794 |
| 812 | Q9I0I9 | 1894 | Q9I273 | 2976 | Q9HWJ8 | 4058 | Q9I1G6 | 5140 | Q9I797 |
| 813 | Q9I0J0 | 1895 | Q9I282 | 2977 | Q9HWJ9 | 4059 | Q9I1G7 | 5141 | Q9I798 |
| 814 | Q9I0J1 | 1896 | Q9I291 | 2978 | Q9HWK0 | 4060 | Q9I1G8 | 5142 | Q9I799 |
| 815 | Q9I0J2 | 1897 | Q9I298 | 2979 | Q9HWK1 | 4061 | Q9I1H1 | 5143 | Q9I7A1 |
| 816 | Q9I0J4 | 1898 | Q9I299 | 2980 | Q9HWK2 | 4062 | Q9I1H2 | 5144 | Q9I7A2 |
| 817 | Q9I0J5 | 1899 | Q9I2B1 | 2981 | Q9HWK3 | 4063 | Q9I1H4 | 5145 | Q9I7A3 |
| 818 | Q9I0J6 | 1900 | Q9I2B3 | 2982 | Q9HWK4 | 4064 | Q9I1H6 | 5146 | Q9I7A6 |
| 819 | Q9I0J7 | 1901 | Q9I2B7 | 2983 | Q9HWK5 | 4065 | Q9I1H7 | 5147 | Q9I7A7 |
| 820 | Q9I0J8 | 1902 | Q9I2C1 | 2984 | Q9HWK7 | 4066 | Q9I1H8 | 5148 | Q9I7B0 |
| 821 | Q9I0J9 | 1903 | Q9I2C2 | 2985 | Q9HWK8 | 4067 | Q9I1H9 | 5149 | Q9I7B1 |
| 822 | Q9I0K1 | 1904 | Q9I2C3 | 2986 | Q9HWL0 | 4068 | Q9I1I0 | 5150 | Q9I7B2 |
| 823 | Q9I0K4 | 1905 | Q9I2C9 | 2987 | Q9HWL1 | 4069 | Q9I1I1 | 5151 | Q9I7B4 |
| 824 | Q9I0K9 | 1906 | Q9I2E4 | 2988 | Q9HWL2 | 4070 | Q9I1I2 | 5152 | Q9I7B6 |
| 825 | Q9I0L5 | 1907 | Q9I2E6 | 2989 | Q9HWL3 | 4071 | Q9I1I3 | 5153 | Q9I7B9 |
| 826 | Q9I0M1 | 1908 | Q9I2F1 | 2990 | Q9HWL4 | 4072 | Q9I1I4 | 5154 | Q9RQ16 |
| 827 | Q9I0M4 | 1909 | Q9I2F6 | 2991 | Q9HWL5 | 4073 | Q9I1I5 | 5155 | Q9X6V8 |
| 828 | Q9I0M6 | 1910 | Q9I2F8 | 2992 | Q9HWL6 | 4074 | Q9I1I7 | ||
| 829 | Q9I0Q0 | 1911 | Q9I2G3 | 2993 | Q9HWL7 | 4075 | Q9I1I8 | ||
| 830 | Q9I0Q7 | 1912 | Q9I2H6 | 2994 | Q9HWL8 | 4076 | Q9I1J0 | ||
| 831 | Q9I0Q8 | 1913 | Q9I2I2 | 2995 | Q9HWL9 | 4077 | Q9I1J1 | ||
| 832 | Q9I0R2 | 1914 | Q9I2J2 | 2996 | Q9HWM0 | 4078 | Q9I1J3 | ||
| 833 | Q9I0R8 | 1915 | Q9I2J4 | 2997 | Q9HWM1 | 4079 | Q9I1J4 | ||
| 834 | Q9I0Z0 | 1916 | Q9I2J9 | 2998 | Q9HWM2 | 4080 | Q9I1J5 | ||
| 835 | Q9I104 | 1917 | Q9I2K4 | 2999 | Q9HWM3 | 4081 | Q9I1J7 | ||
| 836 | Q9I116 | 1918 | Q9I2L1 | 3000 | Q9HWM4 | 4082 | Q9I1J8 | ||
| 837 | Q9I133 | 1919 | Q9I2L5 | 3001 | Q9HWM6 | 4083 | Q9I1J9 | ||
| 838 | Q9I165 | 1920 | Q9I2M1 | 3002 | Q9HWM8 | 4084 | Q9I1K0 | ||
| 839 | Q9I1C2 | 1921 | Q9I2M4 | 3003 | Q9HWM9 | 4085 | Q9I1K3 | ||
| 840 | Q9I1D5 | 1922 | Q9I2M7 | 3004 | Q9HWN0 | 4086 | Q9I1K4 | ||
| 841 | Q9I1F6 | 1923 | Q9I2M9 | 3005 | Q9HWN1 | 4087 | Q9I1K6 | ||
| 842 | Q9I1L5 | 1924 | Q9I2N3 | 3006 | Q9HWN2 | 4088 | Q9I1K8 | ||
| 843 | Q9I1L9 | 1925 | Q9I2P4 | 3007 | Q9HWN3 | 4089 | Q9I1K9 | ||
| 844 | Q9I1N7 | 1926 | Q9I2P8 | 3008 | Q9HWN4 | 4090 | Q9I1L0 | ||
| 845 | Q9I1P2 | 1927 | Q9I2Q0 | 3009 | Q9HWN5 | 4091 | Q9I1L1 | ||
| 846 | Q9I1Q5 | 1928 | Q9I2Q7 | 3010 | Q9HWN6 | 4092 | Q9I1L2 | ||
| 847 | Q9I1S2 | 1929 | Q9I2U3 | 3011 | Q9HWN9 | 4093 | Q9I1L3 | ||
| 848 | Q9I1W2 | 1930 | Q9I2U4 | 3012 | Q9HWP0 | 4094 | Q9I1L6 | ||
| 849 | Q9I1W4 | 1931 | Q9I2U9 | 3013 | Q9HWP1 | 4095 | Q9I1L7 | ||
| 850 | Q9I1W5 | 1932 | Q9I2V9 | 3014 | Q9HWP2 | 4096 | Q9I1L8 | ||
| 851 | Q9I1W8 | 1933 | Q9I2W1 | 3015 | Q9HWP4 | 4097 | Q9I1M4 | ||
| 852 | Q9I1X1 | 1934 | Q9I2W3 | 3016 | Q9HWP6 | 4098 | Q9I1M6 | ||
| 853 | Q9I244 | 1935 | Q9I2W5 | 3017 | Q9HWP7 | 4099 | Q9I1M9 | ||
| 854 | Q9I2C0 | 1936 | Q9I2W9 | 3018 | Q9HWQ0 | 4100 | Q9I1N0 | ||
| 855 | Q9I2D2 | 1937 | Q9I2Y5 | 3019 | Q9HWQ2 | 4101 | Q9I1N1 | ||
| 856 | Q9I2E5 | 1938 | Q9I2Y7 | 3020 | Q9HWQ3 | 4102 | Q9I1N2 | ||
| 857 | Q9I2F4 | 1939 | Q9I310 | 3021 | Q9HWQ4 | 4103 | Q9I1N3 | ||
| 858 | Q9I2L4 | 1940 | Q9I311 | 3022 | Q9HWQ5 | 4104 | Q9I1N6 | ||
| 859 | Q9I2N2 | 1941 | Q9I312 | 3023 | Q9HWQ6 | 4105 | Q9I1N9 | ||
| 860 | Q9I2N4 | 1942 | Q9I314 | 3024 | Q9HWQ7 | 4106 | Q9I1P0 | ||
| 861 | Q9I2N9 | 1943 | Q9I319 | 3025 | Q9HWQ8 | 4107 | Q9I1P1 | ||
| 862 | Q9I2Q6 | 1944 | Q9I324 | 3026 | Q9HWQ9 | 4108 | Q9I1P3 | ||
| 863 | Q9I2S3 | 1945 | Q9I327 | 3027 | Q9HWR0 | 4109 | Q9I1P4 | ||
| 864 | Q9I2S5 | 1946 | Q9I330 | 3028 | Q9HWR1 | 4110 | Q9I1P5 | ||
| 865 | Q9I2S7 | 1947 | Q9I338 | 3029 | Q9HWR4 | 4111 | Q9I1P6 | ||
| 866 | Q9I2S9 | 1948 | Q9I339 | 3030 | Q9HWR5 | 4112 | Q9I1P7 | ||
| 867 | Q9I2T1 | 1949 | Q9I347 | 3031 | Q9HWR6 | 4113 | Q9I1P8 | ||
| 868 | Q9I2U2 | 1950 | Q9I352 | 3032 | Q9HWR9 | 4114 | Q9I1Q0 | ||
| 869 | Q9I2U8 | 1951 | Q9I357 | 3033 | Q9HWS2 | 4115 | Q9I1Q1 | ||
| 870 | Q9I2W4 | 1952 | Q9I363 | 3034 | Q9HWS3 | 4116 | Q9I1Q2 | ||
| 871 | Q9I2W7 | 1953 | Q9I387 | 3035 | Q9HWS4 | 4117 | Q9I1Q3 | ||
| 872 | Q9I2X0 | 1954 | Q9I388 | 3036 | Q9HWS5 | 4118 | Q9I1Q4 | ||
| 873 | Q9I315 | 1955 | Q9I389 | 3037 | Q9HWS8 | 4119 | Q9I1Q6 | ||
| 874 | Q9I321 | 1956 | Q9I3A9 | 3038 | Q9HWS9 | 4120 | Q9I1Q7 | ||
| 875 | Q9I340 | 1957 | Q9I3B1 | 3039 | Q9HWT0 | 4121 | Q9I1Q8 | ||
| 876 | Q9I342 | 1958 | Q9I3C1 | 3040 | Q9HWT1 | 4122 | Q9I1Q9 | ||
| 877 | Q9I344 | 1959 | Q9I3D3 | 3041 | Q9HWT2 | 4123 | Q9I1R0 | ||
| 878 | Q9I382 | 1960 | Q9I3D7 | 3042 | Q9HWT3 | 4124 | Q9I1R1 | ||
| 879 | Q9I383 | 1961 | Q9I3D8 | 3043 | Q9HWT4 | 4125 | Q9I1R2 | ||
| 880 | Q9I398 | 1962 | Q9I3F1 | 3044 | Q9HWT5 | 4126 | Q9I1R3 | ||
| 881 | Q9I3A8 | 1963 | Q9I3F3 | 3045 | Q9HWT8 | 4127 | Q9I1R4 | ||
| 882 | Q9I3B2 | 1964 | Q9I3G8 | 3046 | Q9HWU1 | 4128 | Q9I1R5 | ||
| 883 | Q9I3C3 | 1965 | Q9I3H2 | 3047 | Q9HWU2 | 4129 | Q9I1R6 | ||
| 884 | Q9I3D6 | 1966 | Q9I3H3 | 3048 | Q9HWU3 | 4130 | Q9I1R8 | ||
| 885 | Q9I3F4 | 1967 | Q9I3H8 | 3049 | Q9HWU5 | 4131 | Q9I1R9 | ||
| 886 | Q9I3G0 | 1968 | Q9I3H9 | 3050 | Q9HWU6 | 4132 | Q9I1S0 | ||
| 887 | Q9I3G2 | 1969 | Q9I3I0 | 3051 | Q9HWU7 | 4133 | Q9I1S1 | ||
| 888 | Q9I3G3 | 1970 | Q9I3I5 | 3052 | Q9HWU8 | 4134 | Q9I1S3 | ||
| 889 | Q9I3G5 | 1971 | Q9I3J2 | 3053 | Q9HWU9 | 4135 | Q9I1S4 | ||
| 890 | Q9I3H5 | 1972 | Q9I3J5 | 3054 | Q9HWV0 | 4136 | Q9I1S5 | ||
| 891 | Q9I3I1 | 1973 | Q9I3J7 | 3055 | Q9HWV1 | 4137 | Q9I1S6 | ||
| 892 | Q9I3I6 | 1974 | Q9I3J8 | 3056 | Q9HWV2 | 4138 | Q9I1S7 | ||
| 893 | Q9I3K1 | 1975 | Q9I3J9 | 3057 | Q9HWV3 | 4139 | Q9I1S8 | ||
| 894 | Q9I3K7 | 1976 | Q9I3K2 | 3058 | Q9HWV4 | 4140 | Q9I1S9 | ||
| 895 | Q9I3L4 | 1977 | Q9I3L0 | 3059 | Q9HWV5 | 4141 | Q9I1T0 | ||
| 896 | Q9I3N1 | 1978 | Q9I3L9 | 3060 | Q9HWV6 | 4142 | Q9I1T1 | ||
| 897 | Q9I3N2 | 1979 | Q9I3M7 | 3061 | Q9HWV7 | 4143 | Q9I1T2 | ||
| 898 | Q9I3N3 | 1980 | Q9I3M9 | 3062 | Q9HWV8 | 4144 | Q9I1T3 | ||
| 899 | Q9I3N7 | 1981 | Q9I3N0 | 3063 | Q9HWW0 | 4145 | Q9I1T5 | ||
| 900 | Q9I3P8 | 1982 | Q9I3N4 | 3064 | Q9HWW1 | 4146 | Q9I1T6 | ||
| 901 | Q9I3S1 | 1983 | Q9I3N6 | 3065 | Q9HWW2 | 4147 | Q9I1T7 | ||
| 902 | Q9I3W8 | 1984 | Q9I3P3 | 3066 | Q9HWW3 | 4148 | Q9I1T9 | ||
| 903 | Q9I3Y3 | 1985 | Q9I3P9 | 3067 | Q9HWW5 | 4149 | Q9I1U0 | ||
| 904 | Q9I407 | 1986 | Q9I3Q0 | 3068 | Q9HWW6 | 4150 | Q9I1U1 | ||
| 905 | Q9I418 | 1987 | Q9I3Q2 | 3069 | Q9HWW7 | 4151 | Q9I1U2 | ||
| 906 | Q9I423 | 1988 | Q9I3Q3 | 3070 | Q9HWW8 | 4152 | Q9I1U4 | ||
| 907 | Q9I424 | 1989 | Q9I3Q4 | 3071 | Q9HWX0 | 4153 | Q9I1U5 | ||
| 908 | Q9I425 | 1990 | Q9I3Q5 | 3072 | Q9HWX9 | 4154 | Q9I1U6 | ||
| 909 | Q9I427 | 1991 | Q9I3Q9 | 3073 | Q9HWY0 | 4155 | Q9I1U7 | ||
| 910 | Q9I441 | 1992 | Q9I3T6 | 3074 | Q9HWY2 | 4156 | Q9I1U8 | ||
| 911 | Q9I452 | 1993 | Q9I3T9 | 3075 | Q9HWY3 | 4157 | Q9I1U9 | ||
| 912 | Q9I463 | 1994 | Q9I3U8 | 3076 | Q9HWY4 | 4158 | Q9I1V3 | ||
| 913 | Q9I468 | 1995 | Q9I3X1 | 3077 | Q9HWY6 | 4159 | Q9I1V4 | ||
| 914 | Q9I471 | 1996 | Q9I3X8 | 3078 | Q9HWY7 | 4160 | Q9I1V5 | ||
| 915 | Q9I472 | 1997 | Q9I402 | 3079 | Q9HWY8 | 4161 | Q9I1V6 | ||
| 916 | Q9I496 | 1998 | Q9I403 | 3080 | Q9HWY9 | 4162 | Q9I1V7 | ||
| 917 | Q9I4E1 | 1999 | Q9I404 | 3081 | Q9HWZ2 | 4163 | Q9I1V8 | ||
| 918 | Q9I4E6 | 2000 | Q9I405 | 3082 | Q9HWZ4 | 4164 | Q9I1V9 | ||
| 919 | Q9I4F8 | 2001 | Q9I408 | 3083 | Q9HWZ5 | 4165 | Q9I1W1 | ||
| 920 | Q9I4F9 | 2002 | Q9I417 | 3084 | Q9HWZ7 | 4166 | Q9I1W6 | ||
| 921 | Q9I4G4 | 2003 | Q9I444 | 3085 | Q9HWZ8 | 4167 | Q9I1W7 | ||
| 922 | Q9I4H2 | 2004 | Q9I450 | 3086 | Q9HWZ9 | 4168 | Q9I1X0 | ||
| 923 | Q9I4H5 | 2005 | Q9I457 | 3087 | Q9HX00 | 4169 | Q9I1X2 | ||
| 924 | Q9I4I2 | 2006 | Q9I465 | 3088 | Q9HX01 | 4170 | Q9I1X3 | ||
| 925 | Q9I4L0 | 2007 | Q9I467 | 3089 | Q9HX05 | 4171 | Q9I1X4 | ||
| 926 | Q9I4N1 | 2008 | Q9I469 | 3090 | Q9HX06 | 4172 | Q9I1X5 | ||
| 927 | Q9I4N4 | 2009 | Q9I473 | 3091 | Q9HX09 | 4173 | Q9I1X6 | ||
| 928 | Q9I4P4 | 2010 | Q9I486 | 3092 | Q9HX10 | 4174 | Q9I1X8 | ||
| 929 | Q9I4S5 | 2011 | Q9I487 | 3093 | Q9HX12 | 4175 | Q9I1X9 | ||
| 930 | Q9I4S9 | 2012 | Q9I490 | 3094 | Q9HX13 | 4176 | Q9I1Y0 | ||
| 931 | Q9I4W5 | 2013 | Q9I495 | 3095 | Q9HX14 | 4177 | Q9I1Y1 | ||
| 932 | Q9I4W8 | 2014 | Q9I4B6 | 3096 | Q9HX15 | 4178 | Q9I1Y2 | ||
| 933 | Q9I4W9 | 2015 | Q9I4C1 | 3097 | Q9HX16 | 4179 | Q9I1Y4 | ||
| 934 | Q9I4X2 | 2016 | Q9I4C4 | 3098 | Q9HX18 | 4180 | Q9I1Y8 | ||
| 935 | Q9I4X4 | 2017 | Q9I4C8 | 3099 | Q9HX19 | 4181 | Q9I1Y9 | ||
| 936 | Q9I4Z2 | 2018 | Q9I4D1 | 3100 | Q9HX26 | 4182 | Q9I1Z0 | ||
| 937 | Q9I4Z3 | 2019 | Q9I4D3 | 3101 | Q9HX27 | 4183 | Q9I1Z1 | ||
| 938 | Q9I4Z4 | 2020 | Q9I4E9 | 3102 | Q9HX29 | 4184 | Q9I1Z2 | ||
| 939 | Q9I502 | 2021 | Q9I4G1 | 3103 | Q9HX30 | 4185 | Q9I1Z3 | ||
| 940 | Q9I514 | 2022 | Q9I4G2 | 3104 | Q9HX34 | 4186 | Q9I1Z4 | ||
| 941 | Q9I520 | 2023 | Q9I4G5 | 3105 | Q9HX35 | 4187 | Q9I1Z5 | ||
| 942 | Q9I524 | 2024 | Q9I4H9 | 3106 | Q9HX36 | 4188 | Q9I1Z6 | ||
| 943 | Q9I525 | 2025 | Q9I4I1 | 3107 | Q9HX38 | 4189 | Q9I1Z8 | ||
| 944 | Q9I526 | 2026 | Q9I4L1 | 3108 | Q9HX39 | 4190 | Q9I1Z9 | ||
| 945 | Q9I534 | 2027 | Q9I4L3 | 3109 | Q9HX43 | 4191 | Q9I200 | ||
| 946 | Q9I541 | 2028 | Q9I4M5 | 3110 | Q9HX44 | 4192 | Q9I201 | ||
| 947 | Q9I544 | 2029 | Q9I4M8 | 3111 | Q9HX47 | 4193 | Q9I202 | ||
| 948 | Q9I553 | 2030 | Q9I4N0 | 3112 | Q9HX49 | 4194 | Q9I204 | ||
| 949 | Q9I574 | 2031 | Q9I4N6 | 3113 | Q9HX50 | 4195 | Q9I205 | ||
| 950 | Q9I576 | 2032 | Q9I4P0 | 3114 | Q9HX51 | 4196 | Q9I206 | ||
| 951 | Q9I5A4 | 2033 | Q9I4P3 | 3115 | Q9HX52 | 4197 | Q9I207 | ||
| 952 | Q9I5E2 | 2034 | Q9I4P5 | 3116 | Q9HX53 | 4198 | Q9I208 | ||
| 953 | Q9I5E3 | 2035 | Q9I4P6 | 3117 | Q9HX54 | 4199 | Q9I212 | ||
| 954 | Q9I5F5 | 2036 | Q9I4P7 | 3118 | Q9HX55 | 4200 | Q9I213 | ||
| 955 | Q9I5G5 | 2037 | Q9I4P8 | 3119 | Q9HX56 | 4201 | Q9I214 | ||
| 956 | Q9I5G7 | 2038 | Q9I4P9 | 3120 | Q9HX57 | 4202 | Q9I215 | ||
| 957 | Q9I5G8 | 2039 | Q9I4Q0 | 3121 | Q9HX58 | 4203 | Q9I216 | ||
| 958 | Q9I5J6 | 2040 | Q9I4Q1 | 3122 | Q9HX59 | 4204 | Q9I217 | ||
| 959 | Q9I5Q3 | 2041 | Q9I4Q2 | 3123 | Q9HX60 | 4205 | Q9I218 | ||
| 960 | Q9I5Q9 | 2042 | Q9I4Q6 | 3124 | Q9HX61 | 4206 | Q9I219 | ||
| 961 | Q9I5R0 | 2043 | Q9I4R3 | 3125 | Q9HX62 | 4207 | Q9I220 | ||
| 962 | Q9I5R6 | 2044 | Q9I4R5 | 3126 | Q9HX63 | 4208 | Q9I221 | ||
| 963 | Q9I5R7 | 2045 | Q9I4R6 | 3127 | Q9HX64 | 4209 | Q9I223 | ||
| 964 | Q9I5T1 | 2046 | Q9I4R7 | 3128 | Q9HX65 | 4210 | Q9I224 | ||
| 965 | Q9I5U2 | 2047 | Q9I4R8 | 3129 | Q9HX67 | 4211 | Q9I225 | ||
| 966 | Q9I5U3 | 2048 | Q9I4R9 | 3130 | Q9HX68 | 4212 | Q9I226 | ||
| 967 | Q9I5U5 | 2049 | Q9I4U3 | 3131 | Q9HX71 | 4213 | Q9I227 | ||
| 968 | Q9I5V6 | 2050 | Q9I4W0 | 3132 | Q9HX73 | 4214 | Q9I228 | ||
| 969 | Q9I5V7 | 2051 | Q9I4W7 | 3133 | Q9HX74 | 4215 | Q9I229 | ||
| 970 | Q9I5W0 | 2052 | Q9I4Y4 | 3134 | Q9HX75 | 4216 | Q9I230 | ||
| 971 | Q9I5Y1 | 2053 | Q9I504 | 3135 | Q9HX76 | 4217 | Q9I232 | ||
| 972 | Q9I5Y4 | 2054 | Q9I516 | 3136 | Q9HX77 | 4218 | Q9I233 | ||
| 973 | Q9I5Y8 | 2055 | Q9I523 | 3137 | Q9HX78 | 4219 | Q9I236 | ||
| 974 | Q9I614 | 2056 | Q9I528 | 3138 | Q9HX80 | 4220 | Q9I239 | ||
| 975 | Q9I617 | 2057 | Q9I529 | 3139 | Q9HX81 | 4221 | Q9I240 | ||
| 976 | Q9I618 | 2058 | Q9I537 | 3140 | Q9HX82 | 4222 | Q9I241 | ||
| 977 | Q9I632 | 2059 | Q9I539 | 3141 | Q9HX84 | 4223 | Q9I242 | ||
| 978 | Q9I636 | 2060 | Q9I540 | 3142 | Q9HX85 | 4224 | Q9I243 | ||
| 979 | Q9I648 | 2061 | Q9I548 | 3143 | Q9HX86 | 4225 | Q9I246 | ||
| 980 | Q9I689 | 2062 | Q9I549 | 3144 | Q9HX87 | 4226 | Q9I247 | ||
| 981 | Q9I693 | 2063 | Q9I557 | 3145 | Q9HX88 | 4227 | Q9I248 | ||
| 982 | Q9I697 | 2064 | Q9I559 | 3146 | Q9HX89 | 4228 | Q9I249 | ||
| 983 | Q9I6B3 | 2065 | Q9I560 | 3147 | Q9HX90 | 4229 | Q9I250 | ||
| 984 | Q9I6B4 | 2066 | Q9I572 | 3148 | Q9HX91 | 4230 | Q9I251 | ||
| 985 | Q9I6B7 | 2067 | Q9I573 | 3149 | Q9HX92 | 4231 | Q9I252 | ||
| 986 | Q9I6C1 | 2068 | Q9I575 | 3150 | Q9HX94 | 4232 | Q9I253 | ||
| 987 | Q9I6D1 | 2069 | Q9I583 | 3151 | Q9HX96 | 4233 | Q9I255 | ||
| 988 | Q9I6D3 | 2070 | Q9I590 | 3152 | Q9HXA3 | 4234 | Q9I256 | ||
| 989 | Q9I6E0 | 2071 | Q9I592 | 3153 | Q9HXA4 | 4235 | Q9I257 | ||
| 990 | Q9I6E3 | 2072 | Q9I594 | 3154 | Q9HXA5 | 4236 | Q9I258 | ||
| 991 | Q9I6F6 | 2073 | Q9I595 | 3155 | Q9HXA6 | 4237 | Q9I259 | ||
| 992 | Q9I6G0 | 2074 | Q9I5A3 | 3156 | Q9HXA7 | 4238 | Q9I260 | ||
| 993 | Q9I6G1 | 2075 | Q9I5C9 | 3157 | Q9HXA8 | 4239 | Q9I261 | ||
| 994 | Q9I6I9 | 2076 | Q9I5D1 | 3158 | Q9HXA9 | 4240 | Q9I264 | ||
| 995 | Q9I6L0 | 2077 | Q9I5E4 | 3159 | Q9HXB3 | 4241 | Q9I266 | ||
| 996 | Q9I6M4 | 2078 | Q9I5E6 | 3160 | Q9HXB4 | 4242 | Q9I267 | ||
| 997 | Q9I6P3 | 2079 | Q9I5F0 | 3161 | Q9HXB5 | 4243 | Q9I268 | ||
| 998 | Q9I6P4 | 2080 | Q9I5F2 | 3162 | Q9HXB6 | 4244 | Q9I269 | ||
| 999 | Q9I6R0 | 2081 | Q9I5F7 | 3163 | Q9HXB7 | 4245 | Q9I270 | ||
| 1000 | Q9I6V2 | 2082 | Q9I5G3 | 3164 | Q9HXB8 | 4246 | Q9I271 | ||
| 1001 | Q9I6V7 | 2083 | Q9I5G4 | 3165 | Q9HXC0 | 4247 | Q9I272 | ||
| 1002 | Q9I6Y5 | 2084 | Q9I5G9 | 3166 | Q9HXC1 | 4248 | Q9I274 | ||
| 1003 | Q9I6Z0 | 2085 | Q9I5H2 | 3167 | Q9HXC6 | 4249 | Q9I275 | ||
| 1004 | Q9I6Z2 | 2086 | Q9I5J7 | 3168 | Q9HXC8 | 4250 | Q9I276 | ||
| 1005 | Q9I6Z3 | 2087 | Q9I5L3 | 3169 | Q9HXC9 | 4251 | Q9I277 | ||
| 1006 | Q9I6Z9 | 2088 | Q9I5L8 | 3170 | Q9HXD0 | 4252 | Q9I278 | ||
| 1007 | Q9I703 | 2089 | Q9I5M1 | 3171 | Q9HXD1 | 4253 | Q9I279 | ||
| 1008 | Q9I719 | 2090 | Q9I5M4 | 3172 | Q9HXD5 | 4254 | Q9I280 | ||
| 1009 | Q9I726 | 2091 | Q9I5N4 | 3173 | Q9HXD7 | 4255 | Q9I281 | ||
| 1010 | Q9I727 | 2092 | Q9I5N6 | 3174 | Q9HXD8 | 4256 | Q9I283 | ||
| 1011 | Q9I733 | 2093 | Q9I5N7 | 3175 | Q9HXE1 | 4257 | Q9I284 | ||
| 1012 | Q9I737 | 2094 | Q9I5P0 | 3176 | Q9HXE6 | 4258 | Q9I285 | ||
| 1013 | Q9I741 | 2095 | Q9I5P4 | 3177 | Q9HXE7 | 4259 | Q9I286 | ||
| 1014 | Q9I742 | 2096 | Q9I5P5 | 3178 | Q9HXE8 | 4260 | Q9I287 | ||
| 1015 | Q9I745 | 2097 | Q9I5P7 | 3179 | Q9HXF0 | 4261 | Q9I288 | ||
| 1016 | Q9I747 | 2098 | Q9I5P9 | 3180 | Q9HXF1 | 4262 | Q9I293 | ||
| 1017 | Q9I748 | 2099 | Q9I5Q2 | 3181 | Q9HXF2 | 4263 | Q9I294 | ||
| 1018 | Q9I749 | 2100 | Q9I5Q4 | 3182 | Q9HXF4 | 4264 | Q9I295 | ||
| 1019 | Q9I750 | 2101 | Q9I5Q6 | 3183 | Q9HXF6 | 4265 | Q9I2A1 | ||
| 1020 | Q9I758 | 2102 | Q9I5T5 | 3184 | Q9HXF8 | 4266 | Q9I2A3 | ||
| 1021 | Q9I767 | 2103 | Q9I5T8 | 3185 | Q9HXF9 | 4267 | Q9I2A4 | ||
| 1022 | Q9I7A5 | 2104 | Q9I5U7 | 3186 | Q9HXG0 | 4268 | Q9I2A5 | ||
| 1023 | Q9I7A8 | 2105 | Q9I5U8 | 3187 | Q9HXG1 | 4269 | Q9I2A6 | ||
| 1024 | Q9I7A9 | 2106 | Q9I5V4 | 3188 | Q9HXG2 | 4270 | Q9I2A7 | ||
| 1025 | Q9I7C0 | 2107 | Q9I5V5 | 3189 | Q9HXG3 | 4271 | Q9I2B2 | ||
| 1026 | Q9I7C2 | 2108 | Q9I5V8 | 3190 | Q9HXG5 | 4272 | Q9I2B4 | ||
| 1027 | Q9I7C4 | 2109 | Q9I5W1 | 3191 | Q9HXG7 | 4273 | Q9I2B5 | ||
| 1028 | Q9K3C5 | 2110 | Q9I5Y5 | 3192 | Q9HXG8 | 4274 | Q9I2B6 | ||
| 1029 | Q9KGU6 | 2111 | Q9I5Z5 | 3193 | Q9HXG9 | 4275 | Q9I2B8 | ||
| 1030 | Q9KGU7 | 2112 | Q9I615 | 3194 | Q9HXH1 | 4276 | Q9I2B9 | ||
| 1031 | Q9LCT3 | 2113 | Q9I626 | 3195 | Q9HXH2 | 4277 | Q9I2C4 | ||
| 1032 | Q9LCT6 | 2114 | Q9I633 | 3196 | Q9HXH3 | 4278 | Q9I2C6 | ||
| 1033 | Q9RMT3 | 2115 | Q9I646 | 3197 | Q9HXH6 | 4279 | Q9I2C7 | ||
| 1034 | Q9RPT1 | 2116 | Q9I650 | 3198 | Q9HXI0 | 4280 | Q9I2C8 | ||
| 1035 | Q9S586 | 2117 | Q9I654 | 3199 | Q9HXI3 | 4281 | Q9I2D0 | ||
| 1036 | Q9X2N2 | 2118 | Q9I655 | 3200 | Q9HXI7 | 4282 | Q9I2D1 | ||
| 1037 | Q9X6P4 | 2119 | Q9I659 | 3201 | Q9HXJ6 | 4283 | Q9I2D3 | ||
| 1038 | Q9X6R0 | 2120 | Q9I668 | 3202 | Q9HXJ9 | 4284 | Q9I2D4 | ||
| 1039 | Q9X6V6 | 2121 | Q9I669 | 3203 | Q9HXK0 | 4285 | Q9I2D5 | ||
| 1040 | Q9X6V9 | 2122 | Q9I678 | 3204 | Q9HXK1 | 4286 | Q9I2D6 | ||
| 1041 | Q9XCL6 | 2123 | Q9I683 | 3205 | Q9HXK2 | 4287 | Q9I2D7 | ||
| 1042 | Q9XCX8 | 2124 | Q9I687 | 3206 | Q9HXK3 | 4288 | Q9I2D8 | ||
| 1043 | Q9XCX9 | 2125 | Q9I690 | 3207 | Q9HXK4 | 4289 | Q9I2D9 | ||
| 1044 | Q9Z9H0 | 2126 | Q9I691 | 3208 | Q9HXK6 | 4290 | Q9I2E0 | ||
| 1045 | Q9ZFE4 | 2127 | Q9I692 | 3209 | Q9HXK7 | 4291 | Q9I2E1 | ||
| 1046 | Q9ZFK4 | 2128 | Q9I694 | 3210 | Q9HXK8 | 4292 | Q9I2E3 | ||
| 1047 | Q9ZI86 | 2129 | Q9I696 | 3211 | Q9HXK9 | 4293 | Q9I2E7 | ||
| 1048 | E1JGJ8 | 2130 | Q9I698 | 3212 | Q9HXL0 | 4294 | Q9I2E8 | ||
| 1049 | G3XCT6 | 2131 | Q9I699 | 3213 | Q9HXL1 | 4295 | Q9I2E9 | ||
| 1050 | G3XCT7 | 2132 | Q9I6A1 | 3214 | Q9HXL2 | 4296 | Q9I2F0 | ||
| 1051 | G3XCU6 | 2133 | Q9I6A9 | 3215 | Q9HXL3 | 4297 | Q9I2F2 | ||
| 1052 | G3XCU9 | 2134 | Q9I6B9 | 3216 | Q9HXL7 | 4298 | Q9I2F3 | ||
| 1053 | G3XCV4 | 2135 | Q9I6C0 | 3217 | Q9HXL9 | 4299 | Q9I2F5 | ||
| 1054 | G3XCV6 | 2136 | Q9I6C2 | 3218 | Q9HXM0 | 4300 | Q9I2F7 | ||
| 1055 | G3XCW2 | 2137 | Q9I6C4 | 3219 | Q9HXM2 | 4301 | Q9I2F9 | ||
| 1056 | G3XCW6 | 2138 | Q9I6C8 | 3220 | Q9HXM3 | 4302 | Q9I2G0 | ||
| 1057 | G3XCW8 | 2139 | Q9I6D8 | 3221 | Q9HXM4 | 4303 | Q9I2G1 | ||
| 1058 | G3XCX6 | 2140 | Q9I6F2 | 3222 | Q9HXM7 | 4304 | Q9I2G2 | ||
| 1059 | G3XCY2 | 2141 | Q9I6G3 | 3223 | Q9HXM9 | 4305 | Q9I2G4 | ||
| 1060 | G3XCY5 | 2142 | Q9I6G5 | 3224 | Q9HXN0 | 4306 | Q9I2G5 | ||
| 1061 | G3XCY7 | 2143 | Q9I6G7 | 3225 | Q9HXN3 | 4307 | Q9I2G6 | ||
| 1062 | G3XCZ2 | 2144 | Q9I6H4 | 3226 | Q9HXN6 | 4308 | Q9I2G7 | ||
| 1063 | G3XCZ5 | 2145 | Q9I6H5 | 3227 | Q9HXN8 | 4309 | Q9I2G8 | ||
| 1064 | G3XCZ6 | 2146 | Q9I6I6 | 3228 | Q9HXP1 | 4310 | Q9I2G9 | ||
| 1065 | G3XD00 | 2147 | Q9I6I7 | 3229 | Q9HXP2 | 4311 | Q9I2H0 | ||
| 1066 | G3XD05 | 2148 | Q9I6I8 | 3230 | Q9HXP4 | 4312 | Q9I2H1 | ||
| 1067 | G3XD11 | 2149 | Q9I6J4 | 3231 | Q9HXP5 | 4313 | Q9I2H2 | ||
| 1068 | G3XD13 | 2150 | Q9I6J5 | 3232 | Q9HXP6 | 4314 | Q9I2H3 | ||
| 1069 | G3XD15 | 2151 | Q9I6K3 | 3233 | Q9HXP7 | 4315 | Q9I2H4 | ||
| 1070 | G3XD16 | 2152 | Q9I6K8 | 3234 | Q9HXQ4 | 4316 | Q9I2H5 | ||
| 1071 | G3XD17 | 2153 | Q9I6L3 | 3235 | Q9HXQ5 | 4317 | Q9I2H8 | ||
| 1072 | G3XD18 | 2154 | Q9I6M3 | 3236 | Q9HXQ8 | 4318 | Q9I2H9 | ||
| 1073 | G3XD21 | 2155 | Q9I6N0 | 3237 | Q9HXQ9 | 4319 | Q9I2I0 | ||
| 1074 | G3XD35 | 2156 | Q9I6Q3 | 3238 | Q9HXR0 | 4320 | Q9I2I1 | ||
| 1075 | G3XD36 | 2157 | Q9I6Q8 | 3239 | Q9HXR1 | 4321 | Q9I2I3 | ||
| 1076 | G3XD49 | 2158 | Q9I6R1 | 3240 | Q9HXR2 | 4322 | Q9I2I4 | ||
| 1077 | G3XD50 | 2159 | Q9I6R2 | 3241 | Q9HXR3 | 4323 | Q9I2I5 | ||
| 1078 | G3XD53 | 2160 | Q9I6R6 | 3242 | Q9HXR4 | 4324 | Q9I2I6 | ||
| 1079 | G3XD54 | 2161 | Q9I6S4 | 3243 | Q9HXR5 | 4325 | Q9I2I7 | ||
| 1080 | G3XD58 | 2162 | Q9I6S5 | 3244 | Q9HXR6 | 4326 | Q9I2I8 | ||
| 1081 | G3XD63 | 2163 | Q9I6S8 | 3245 | Q9HXR8 | 4327 | Q9I2I9 | ||
| 1082 | G3XD66 | 2164 | Q9I6S9 | 3246 | Q9HXR9 | 4328 | Q9I2J0 |
3.2.2. Identification of essential proteins
The dataset of 5155 non-homologous proteins was subjected to essentiality analysis using the DEG-15. This analysis identified 149 proteins strongly associated with essential bacterial genes cataloged in DEG-15. These essential proteins are critical for the survival of P. aeruginosa and are therefore considered potential therapeutic targets. The results of this analysis, including protein annotations and associated essential gene identifiers, are provided in Table 2.
Table 2.
Non-human homologous essential proteins of P. aeruginosa PAO1 identified through comparative proteomics analysis.
| S.NO | DEG ACCESSION NO | NAME | UNIPROT ID | Homology with human proteins |
|---|---|---|---|---|
| 1 | DEG10360001 | chromosome replication initiator DnaA | Q9I7C5 | – |
| 2 | DEG10360002 | DNA polymerase III subunit beta | Q9I7C4 | – |
| 3 | DEG10360004 | D,D-heptose 1,7-bisphosphate phosphatase | Q9I7C0 | – |
| 4 | DEG10360005 | glycyl-tRNA synthetase subunit beta | Q9I7B8 | – |
| 5 | DEG10360006 | glycyl-tRNA synthetase subunit alpha | Q9I7B7 | – |
| 6 | DEG10360011 | transcriptional regulator | Q9I709 | – |
| 7 | DEG10360013 | prolipoprotein diacylglyceryl transferase | Q9I6F2 | – |
| 8 | DEG10360016 | phosphopantetheine adenylyltransferase | Q9I6D1 | – |
| 9 | DEG10360017 | RNA polymerase factor sigma-32 | P42378 | – |
| 10 | DEG10360018 | Holliday junction resolvase-like protein | Q9I699 | – |
| 11 | DEG10360019 | cytosine permease | Q9I679 | – |
| 12 | DEG10360022 | fructose-1,6-bisphosphate aldolase | Q9I5Y1 | – |
| 13 | DEG10360023 | RNA polymerase sigma factor RpoD | P26480 | – |
| 14 | DEG10360024 | DNA primase | Q9I5W0 | – |
| 15 | DEG10360026 | dihydroneopterin aldolase | Q9I5V5 | – |
| 16 | DEG10360027 | 4-hydroxythreonine-4-phosphate dehydrogenase | Q9I5U4 | – |
| 17 | DEG10360028 | peptidyl-prolyl cis-trans isomerase SurA | Q9I5U3 | – |
| 18 | DEG10360030 | transcriptional regulator PrtR | Q06553 | – |
| 19 | DEG10360033 | HxcU pseudopilin | Q9I5P7 | – |
| 20 | DEG10360037 | pyridoxine 5′-phosphate synthase | Q9I5G5 | – |
| 21 | DEG10360039 | aspartate kinase | O69077 | – |
| 22 | DEG10360040 | transcriptional regulator | Q9I551 | – |
| 23 | DEG10360041 | DNA replication initiation factor | Q9I511 | – |
| 24 | DEG10360044 | TolQ protein | P50598 | – |
| 25 | DEG10360045 | TolR protein | P50599 | – |
| 26 | DEG10360046 | TolA protein | P50600 | – |
| 27 | DEG10360047 | translocation protein TolB | P50601 | – |
| 28 | DEG10360050 | monovalent cationH + antiporter subunit G | Q9I4R5 | – |
| 29 | DEG10360054 | erythronate-4-phosphate dehydrogenase | Q9I3W9 | – |
| 30 | DEG10360056 | cell division protein ZipA | Q9I3I5 | – |
| 31 | DEG10360057 | NAD-dependent DNA ligase LigA | Q9I3I4 | – |
| 32 | DEG10360059 | succinate dehydrogenase subunit C | Q9I3D7 | – |
| 33 | DEG10360060 | succinate dehydrogenase subunit D | Q9I3D6 | – |
| 34 | DEG10360069 | 3-hydroxydecanoyl-ACP dehydratase | O33877 | – |
| 35 | DEG10360070 | chorismate synthase | Q9I344 | – |
| 36 | DEG10360071 | RNA polymerase sigma factor SigX | Q9I2W5 | – |
| 37 | DEG10360072 | bifunctional aconitate hydratase 22-methylisocitrate dehydratase | Q9I2V5 | – |
| 38 | DEG10360073 | UDP-2,3-diacylglucosamine hydrolase | Q9I2V0 | – |
| 39 | DEG10360079 | hypothetical protein | Q9I2D5 | – |
| 40 | DEG10360080 | ring-cleaving dioxygenase | P23205 | – |
| 41 | DEG10360081 | hypothetical protein | Q9I1V9 | – |
| 42 | DEG10360083 | sulfur transfer complex subunit TusD | Q9I0N3 | – |
| 43 | DEG10360085 | outer-membrane lipoprotein carrier protein | Q9I0M4 | – |
| 44 | DEG10360086 | DNA translocase FtsK | Q9I0M3 | – |
| 45 | DEG10360089 | radical activating enzyme | Q9I0B6 | – |
| 46 | DEG10360093 | translation initiation factor IF-3 | Q9I0A0 | – |
| 47 | DEG10360095 | two-component response regulator | Q9I045 | – |
| 48 | DEG10360096 | trans-2-enoyl-CoA reductase | Q9HZP8 | – |
| 49 | DEG10360097 | electron transfer flavoprotein subunit alpha | Q9HZP7 | – |
| 50 | DEG10360100 | thymidylate kinase | Q9HZN8 | – |
| 51 | DEG10360102 | UDP-N-acetylenolpyruvoylglucosamine reductase | Q9HZM7 | – |
| 52 | DEG10360103 | 3-deoxy-manno-octulosonate cytidylyltransferase | Q9HZM5 | – |
| 53 | DEG10360104 | tetraacyldisaccharide 4′-kinase | Q9HZM3 | – |
| 54 | DEG10360105 | hypothetical protein | Q9HZL8 | – |
| 55 | DEG10360107 | hypothetical protein | Q9HZL6 | – |
| 56 | DEG10360111 | aspartate-semialdehyde dehydrogenase | Q51344 | – |
| 57 | DEG10360113 | nucleotide sugar epimerasedehydratase WbpM | Q9HZ86 | – |
| 58 | DEG10360114 | glycosyltransferase WbpL | G3XD50 | – |
| 59 | DEG10360115 | glycosyl transferase WbpJ | Q9HZ80 | – |
| 60 | DEG10360116 | UDP-N-acetylglucosamine 2-epimerase | G3XD61 | – |
| 61 | DEG10360118 | LPS biosynthesis protein WbpG | Q9HZ78 | – |
| 62 | DEG10360119 | imidazole glycerol phosphate synthase subunit HisF2 | P72139 | – |
| 63 | DEG10360121 | B-band O-antigen polymerase | G3XCW3 | – |
| 64 | DEG10360123 | UDP-2-acetamido-3-amino-23-dideoxy-d-glucuronic acid N-acetyltransferase WbpD | G3XD01 | – |
| 65 | DEG10360124 | UDP-2-acetamido-2-deoxy-d-glucuronic acid 3-dehydrogenase WbpB | G3XD23 | – |
| 66 | DEG10360128 | DNA gyrase subunit A | P48372 | – |
| 67 | DEG10360130 | ferredoxin-NADP reductase | Q9HYK7 | – |
| 68 | DEG10360132 | 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase | P57708 | – |
| 69 | DEG10360133 | 2-dehydro-3-deoxyphosphooctonate aldolase | Q9ZFK4 | – |
| 70 | DEG10360135 | hypothetical protein | Q9HXZ3 | – |
| 71 | DEG10360138 | lipid-A-disaccharide synthase | Q9HXY8 | – |
| 72 | DEG10360139 | UDP-N-acetylglucosamine acyltransferase | Q9X6P4 | – |
| 73 | DEG10360140 | (3R)-hydroxymyristoyl-ACP dehydratase | Q9HXY7 | – |
| 74 | DEG10360141 | UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase | Q9HXY6 | – |
| 75 | DEG10360143 | 1-deoxy-D-xylulose 5-phosphate reductoisomerase | Q9KGU6 | – |
| 76 | DEG10360146 | ribosome recycling factor | O82853 | – |
| 77 | DEG10360147 | uridylate kinase | O82852 | – |
| 78 | DEG10360149 | tetrahydrodipicolinate succinylase | G3XD76 | – |
| 79 | DEG10360150 | hypothetical protein | Q9HXV5 | – |
| 80 | DEG10360153 | chemotaxis-specific methylesterase | Q9HXT8 | – |
| 81 | DEG10360154 | NalC protein | Q9HXS0 | – |
| 82 | DEG10360155 | 16S rRNA-processing protein RimM | Q9HXQ0 | – |
| 83 | DEG10360160 | histidyl-tRNA synthetase | Q9HXJ5 | – |
| 84 | DEG10360161 | 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase | Q9HXJ4 | – |
| 85 | DEG10360166 | preprotein translocase subunit SecD | Q9HXI1 | – |
| 86 | DEG10360167 | hypothetical protein | Q9HXH5 | – |
| 87 | DEG10360168 | hypothetical protein | Q9HXH4 | – |
| 88 | DEG10360171 | Metalloprotease | Q9HX37 | – |
| 89 | DEG10360172 | apolipoprotein N-acyltransferase | Q9ZI86 | – |
| 90 | DEG10360174 | DNA polymerase III subunit delta | Q9HX31 | – |
| 91 | DEG10360178 | aromatic acid decarboxylase | Q9HX08 | – |
| 92 | DEG10360179 | inorganic pyrophosphatase | Q9HWZ6 | – |
| 93 | DEG10360181 | 1-deoxy-D-xylulose-5-phosphate synthase | Q9KGU7 | – |
| 94 | DEG10360182 | thiamine monophosphate kinase | Q9HWX7 | – |
| 95 | DEG10360183 | transcription antitermination protein NusB | Q9HWX6 | – |
| 96 | DEG10360185 | hypothetical protein | Q9HWT5 | – |
| 97 | DEG10360186 | 50S ribosomal protein L17 | O52761 | – |
| 98 | DEG10360187 | DNA-directed RNA polymerase subunit alpha | O52760 | – |
| 99 | DEG10360191 | preprotein translocase subunit SecY | Q9HWF5 | – |
| 100 | DEG10360192 | 50S ribosomal protein L15 | Q9HWF4 | – |
| 101 | DEG10360193 | 30S ribosomal protein S5 | Q9HWF2 | – |
| 102 | DEG10360194 | 50S ribosomal protein L6 | Q9HWF0 | – |
| 103 | DEG10360195 | 30S ribosomal protein S8 | Q9HWE9 | – |
| 104 | DEG10360196 | 50S ribosomal protein L5 | Q9HWE7 | – |
| 105 | DEG10360198 | 30S ribosomal protein S3 | Q9HWE1 | – |
| 106 | DEG10360207 | 50S ribosomal protein L7L12 | Q9HWC8 | – |
| 107 | DEG10360208 | 50S ribosomal protein L10 | Q9HWC7 | – |
| 108 | DEG10360209 | 50S ribosomal protein L1 | Q9HWC6 | – |
| 109 | DEG10360211 | transcription antitermination protein NusG | Q9HWC4 | – |
| 110 | DEG10360212 | preprotein translocase subunit SecE | Q9HWC3 | – |
| 111 | DEG10360213 | pantothenate kinase | Q9HWC1 | – |
| 112 | DEG10360217 | preprotein translocase subunit SecA | Q9LCT3 | – |
| 113 | DEG10360218 | hypothetical protein | Q9HW03 | – |
| 114 | DEG10360219 | UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase | P47205 | – |
| 115 | DEG10360220 | cell division protein FtsZ | P47204 | – |
| 116 | DEG10360221 | cell division protein FtsA | P47203 | – |
| 117 | DEG10360222 | UDP-N-acetylmuramate--L-alanine ligase | Q9HW02 | – |
| 118 | DEG10360223 | undecaprenyldiphospho-muramoylpentapeptide beta-N- acetylglucosaminyltransferase | Q9HW01 | – |
| 119 | DEG10360224 | cell division protein FtsW | Q9HW00 | – |
| 120 | DEG10360225 | UDP-N-acetylmuramoyl-L-alanyl-D-glutamate synthetase | Q9HVZ9 | – |
| 121 | DEG10360226 | phospho-N-acetylmuramoyl-pentapeptide- transferase | Q9HVZ8 | – |
| 122 | DEG10360227 | UDP-N-acetylmuramoyl-tripeptide--D-alanyl-D- alanine ligase | Q9HVZ7 | – |
| 123 | DEG10360228 | UDP-N-acetylmuramoylalanyl-D-glutamate--2, 6-diaminopimelate ligase | Q59650 | – |
| 124 | DEG10360231 | phosphoheptose isomerase | Q9HVZ0 | – |
| 125 | DEG10360232 | cytochrome C1 | Q9HVY6 | – |
| 126 | DEG10360236 | UDP-N-acetylglucosamine 1-carboxyvinyltransferase | Q9HVW7 | – |
| 127 | DEG10360237 | arabinose-5-phosphate isomerase KdsD | Q9HVW0 | – |
| 128 | DEG10360238 | hypothetical protein | Q9HVV8 | – |
| 129 | DEG10360239 | hypothetical protein | Q9HVV7 | – |
| 130 | DEG10360241 | rod shape-determining protein MreD | Q9HVU2 | – |
| 131 | DEG10360242 | rod shape-determining protein MreC | Q9HVU1 | – |
| 132 | DEG10360251 | hypothetical protein | Q9HVM2 | – |
| 133 | DEG10360254 | hypothetical protein | Q9HVF5 | – |
| 134 | DEG10360262 | hypothetical protein | Q9HVB6 | – |
| 135 | DEG10360263 | hypothetical protein | Q9HVB0 | – |
| 136 | DEG10360264 | 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase | Q9HV71 | – |
| 137 | DEG10360271 | dihydropteroate synthase | Q9HV49 | – |
| 138 | DEG10360279 | acetyl-CoA carboxylase biotin carboxyl carrier protein subunit | P37799 | – |
| 139 | DEG10360281 | NAD synthetase | Q9HUP3 | – |
| 140 | DEG10360288 | cAMP phosphodiesterase | Q9HUJ6 | – |
| 141 | DEG10360289 | 3-deoxy-D-manno-octulosonic-acid transferase | Q9HUH7 | – |
| 142 | DEG10360290 | hypothetical protein | Q9HUH4 | – |
| 143 | DEG10360298 | lipopolysaccharide kinase WaaP | Q9HUF7 | – |
| 144 | DEG10360299 | UDP-glucose:(heptosyl) LPS alpha 1,3-glucosyltransferase WaaG | Q9HUF6 | – |
| 145 | DEG10360315 | hypothetical protein | Q9HTV3 | – |
| 146 | DEG10360318 | uroporphyrinogen-III synthase | P48246 | – |
| 147 | DEG10360320 | diaminopimelate epimerase | Q51564 | – |
| 148 | DEG10360328 | bifunctional glucosamine-1-phosphate acetyltransferaseN-acetylglucosamine-1-phosphate uridyltransferase | Q9HT22 | – |
| 149 | DEG10360332 | ATP synthase F0F1 subunit delta | Q9HT17 | – |
3.3. Metabolic pathway analysis and subcellular localization
3.3.1. Metabolic pathway mapping
Non-homologous essential proteins were mapped to known metabolic pathways using the KEGG pathway database. Upon comparing the host and pathogen pathways, of the 149 non-homologous essential proteins, 87 proteins were found to be unique to P. aeruginosa. These proteins were prioritized for drug development, as they are involved in pathways not present in humans, reducing the risk of off-target effects. Eight proteins shared common pathways with the human proteome, potentially leading to off-target effects on human cells. The remaining 54 proteins had unknown pathways, with 33 proteins assigned a KEGG Orthology (KO) identifier, while the KO for the remaining 21 proteins was unassigned (Fig. 2; Table 3).
Fig. 2.
Distribution of essential and non-homologous proteins involved in the unique metabolic pathways of P. aeruginosa.
Table 3.
KEGG Pathway analysis of Non-host homologous proteins of P. aeruginosa.
| S.NO | DEG Accession No | KO Entry | Essential Protein | Unique pathway | Common pathway |
|---|---|---|---|---|---|
| 1 | DEG10360001 | K02313 | chromosome replication initiator DnaA | Two-component system | |
| 2 | DEG10360002 | K02338 | DNA polymerase III subunit beta | DNA replication, | |
| Mismatch repair, | |||||
| Homologous recombination | |||||
| 3 | DEG10360004 | K03273 | D,D-heptose 1,7-bisphosphate phosphatase | Lipopolysaccharide biosynthesis, | |
| Biosynthesis of nucleotide sugars | |||||
| 4 | DEG10360005 | K01879 | glycyl-tRNA synthetase subunit beta | Aminoacyl-tRNA biosynthesis | |
| 5 | DEG10360006 | K01878 | glycyl-tRNA synthetase subunit alpha | Aminoacyl-tRNA biosynthesis | |
| 6 | DEG10360011 | – | transcriptional regulator | – | – |
| 7 | DEG10360013 | K13292 | prolipoprotein diacylglyceryl transferase | unclasssified | – |
| 8 | DEG10360016 | K00954 | phosphopantetheine adenylyltransferase | Pantothenate and CoA biosynthesis, | Pantothenate and CoA biosynthesis, |
| Metabolic pathways, | Metabolic pathways, | ||||
| Biosynthesis of cofactors | Biosynthesis of cofactors | ||||
| 9 | DEG10360017 | K03089 | RNA polymerase factor sigma-32 | – | – |
| 10 | DEG10360018 | – | Holliday junction resolvase-like protein | – | – |
| 11 | DEG10360019 | K10974 | cytosine permease | – | – |
| 12 | DEG10360022 | K01624 | fructose-1,6-bisphosphate aldolase | Glycolysis/Gluconeogenesis, | Glycolysis/Gluconeogenesis, Fructose and mannose metabolism, Galactose metabolism, Tricarboxylic acid cycle and glyoxylate/dicarboxylate metabolism |
| Pentose phosphate pathway, | |||||
| Fructose and mannose metabolism, | |||||
| Methane metabolism, | |||||
| Biosynthesis of secondary metabolites, | |||||
| Microbial metabolism in diverse environments, | |||||
| Carbon metabolism, | |||||
| Biosynthesis of amino acids | |||||
| 13 | DEG10360023 | K03086 | RNA polymerase sigma factor RpoD | Flagellar assembly | – |
| 14 | DEG10360024 | K02316 | DNA primase | DNA replication | – |
| 15 | DEG10360026 | K01633 | dihydroneopterin aldolase | Folate biosynthesis, | – |
| Metabolic pathways, | |||||
| Biosynthesis of cofactors | |||||
| 16 | DEG10360027 | K00097 | 4-hydroxythreonine-4-phosphate dehydrogenase | Vitamin B6 metabolism, | – |
| Biosynthesis of cofactors | |||||
| 17 | DEG10360028 | K03771 | peptidyl-prolyl cis-trans isomerase SurA | – | – |
| 18 | DEG10360030 | – | transcriptional regulator PrtR | – | – |
| 19 | DEG10360033 | K02457 | HxcU pseudopilin | Bacterial secretion system | – |
| 20 | DEG10360037 | K03474 | pyridoxine 5′-phosphate synthase | Vitamin B6 metabolism, | |
| Biosynthesis of cofactors | |||||
| 21 | DEG10360039 | K00928 | aspartate kinase | Glycine, serine and threonine metabolism, | – |
| Monobactam biosynthesis, | |||||
| Cysteine and methionine metabolism, | |||||
| Lysine biosynthesis, | |||||
| Metabolic pathways, | |||||
| Biosynthesis of secondary metabolites, | |||||
| Microbial metabolism in diverse environments, | |||||
| 2-Oxocarboxylic acid metabolism, | |||||
| Biosynthesis of amino acids | |||||
| 22 | DEG10360040 | – | transcriptional regulator | – | – |
| 23 | DEG10360041 | – | DNA replication initiation factor | – | – |
| 24 | DEG10360044 | K03562 | TolQ protein | – | – |
| 25 | DEG10360045 | K03560 | TolR protein | – | – |
| 26 | DEG10360046 | K00615 | TolA protein | Pentose phosphate pathway, | Pentose phosphate pathway, Metabolic pathways, Carbon metabolism, Biosynthesis of amino acids |
| Metabolic pathways, | |||||
| Biosynthesis of secondary metabolites, | |||||
| Microbial metabolism in diverse environments, | |||||
| Carbon metabolism, | |||||
| Biosynthesis of amino acids | |||||
| 27 | DEG10360047 | K03641 | TolB protein | – | – |
| 28 | DEG10360050 | K05564 | monovalent cationH + antiporter subunit G | – | – |
| 29 | DEG10360054 | K03473 | erythronate-4-phosphate dehydrogenase | Vitamin B6 metabolism, | |
| Biosynthesis of cofactors | |||||
| 30 | DEG10360056 | K03528 | cell division protein ZipA | – | – |
| 31 | DEG10360057 | K01972 | NAD-dependent DNA ligase LigA | DNA replication, | Cell adhesion molecules |
| Base excision repair, | |||||
| Nucleotide excision repair, | |||||
| Mismatch repair | |||||
| 32 | DEG10360059 | K00241 | succinate dehydrogenase subunit C | Citrate cycle (TCA cycle), | Citrate cycle (TCA cycle), Oxidative phosphorylation, Metabolic pathways, Carbon metabolism, Thermogenesis, Non-alcoholic fatty liver disease, Alzheimer disease, Parkinson disease, Amyotrophic lateral sclerosis, Huntington disease, Prion disease, Pathways of neurodegeneration - multiple diseases, Chemical carcinogenesis - reactive oxygen species, Diabetic cardiomyopathy |
| Oxidative phosphorylation, | |||||
| Butanoate metabolism, | |||||
| Metabolic pathways, | |||||
| Biosynthesis of secondary metabolites, | |||||
| Microbial metabolism in diverse environments, | |||||
| Carbon metabolism | |||||
| 33 | DEG10360060 | K00242 | succinate dehydrogenase subunit D | Citrate cycle (TCA cycle), | Citrate cycle (TCA cycle), Oxidative phosphorylation, Metabolic pathways, Carbon metabolism, Thermogenesis, Non-alcoholic fatty liver disease, Alzheimer disease, Parkinson disease, Amyotrophic lateral sclerosis, Huntington disease, Prion disease, Pathways of neurodegeneration - multiple diseases, Chemical carcinogenesis - reactive oxygen species, Diabetic cardiomyopathy |
| Oxidative phosphorylation, | |||||
| Butanoate metabolism, | |||||
| Metabolic pathways, | |||||
| Biosynthesis of secondary metabolites, | |||||
| Microbial metabolism in diverse environments, | |||||
| Carbon metabolism | |||||
| 34 | DEG10360069 | K01716 | 3-hydroxydecanoyl-ACP dehydratase | Fatty acid biosynthesis, | – |
| Fatty acid metabolism | |||||
| 35 | DEG10360070 | K01736 | chorismate synthase | Phenylalanine, tyrosine and tryptophan biosynthesis, | – |
| Metabolic pathways, | |||||
| Biosynthesis of secondary metabolites, | |||||
| Biosynthesis of amino acids | |||||
| 36 | DEG10360071 | K03088 | RNA polymerase sigma factor SigX | – | – |
| 37 | DEG10360072 | K01682 | bifunctional aconitate hydratase 22-methylisocitrate dehydratase | Citrate cycle (TCA cycle), | |
| Glyoxylate and dicarboxylate metabolism, | |||||
| Propanoate metabolism, | |||||
| Metabolic pathways, | |||||
| Biosynthesis of secondary metabolites, | |||||
| Microbial metabolism in diverse environments, | |||||
| Carbon metabolism, | |||||
| 2-Oxocarboxylic acid metabolism, | |||||
| Biosynthesis of amino acids | |||||
| 38 | DEG10360073 | K03269 | UDP-2,3-diacylglucosamine hydrolase | Lipopolysaccharide biosynthesis | – |
| 39 | DEG10360079 | – | hypothetical protein | – | – |
| 40 | DEG10360080 | – | ring-cleaving dioxygenase | – | – |
| 41 | DEG10360081 | – | hypothetical protein | – | – |
| 42 | DEG10360083 | K07235 | sulfur transfer complex subunit TusD | Sulfur relay system | – |
| 43 | DEG10360085 | K03634 | outer-membrane lipoprotein carrier protein | – | – |
| 44 | DEG10360086 | K03466 | DNA translocase FtsK | – | – |
| 45 | DEG10360089 | K10026 | Probable radical activating enzyme | – | – |
| 46 | DEG10360093 | K02520 | translation initiation factor IF-3 | – | – |
| 47 | DEG10360095 | K07315 | two-component response regulator | – | – |
| 48 | DEG10360096 | K00209 | trans-2-enoyl-CoA reductase | Fatty acid biosynthesis, | |
| Butanoate metabolism, | |||||
| Metabolic pathways, | |||||
| Biosynthesis of secondary metabolites, | |||||
| Microbial metabolism in diverse environments, | |||||
| Carbon metabolism, | |||||
| Fatty acid metabolism | |||||
| 49 | DEG10360097 | K03522 | electron transfer flavoprotein subunit alpha | – | – |
| 50 | DEG10360100 | K00943 | thymidylate kinase | Pyrimidine metabolism, | – |
| Metabolic pathways, | |||||
| Nucleotide metabolism | |||||
| 51 | DEG10360102 | K00075 | UDP-N-acetylenolpyruvoylglucosamine reductase | Amino sugar and nucleotide sugar metabolism, | – |
| Peptidoglycan biosynthesis, | |||||
| Biosynthesis of nucleotide sugars | |||||
| 52 | DEG10360103 | K00979 | 3-deoxy-manno-octulosonate cytidylyltransferase | Lipopolysaccharide biosynthesis, | – |
| Metabolic pathways, | |||||
| Biosynthesis of nucleotide sugars | |||||
| 53 | DEG10360104 | K00912 | tetraacyldisaccharide 4′-kinase | Lipopolysaccharide biosynthesis | – |
| 54 | DEG10360105 | K09808 | hypothetical protein | ABC transporters | – |
| 55 | DEG10360107 | K09808 | hypothetical protein | ABC transporters | – |
| 56 | DEG10360111 | K11906 | aspartate-semialdehyde dehydrogenase | Bacterial secretion system | – |
| 57 | DEG10360113 | K24300 | nucleotide sugar epimerasedehydratase WbpM | O-Antigen nucleotide sugar biosynthesis, | – |
| Teichoic acid biosynthesis, | |||||
| Metabolic pathways, | |||||
| Biosynthesis of nucleotide sugars | |||||
| 58 | DEG10360114 | K13007 | glycosyltransferase WbpL | – | – |
| 59 | DEG10360115 | – | glycosyl transferase WbpJ | – | – |
| 60 | DEG10360116 | K13019 | UDP-N-acetylglucosamine 2-epimerase |
O-Antigen nucleotide sugar biosynthesis |
Amino sugar and nucleotide sugar metabolism, Biosynthesis of nucleotide sugars |
| 61 | DEG10360118 | K24326 | LPS biosynthesis protein WbpG | O-Antigen nucleotide sugar biosynthesis, Biosynthesis of nucleotide sugars |
– |
| 62 | DEG10360119 | K02500 | imidazole glycerol phosphate synthase subunit HisF2 | Histidine metabolism, | – |
| Biosynthesis of secondary metabolites, | |||||
| Biosynthesis of amino acids | |||||
| 63 | DEG10360121 | – | B-band O-antigen polymerase | – | – |
| 64 | DEG10360123 | K13018 | UDP-2-acetamido-3-amino-23-dideoxy-d-glucuronic acid N-acetyltransferase WbpD | Amino sugar and nucleotide sugar metabolism, | – |
| O-Antigen nucleotide sugar biosynthesis, | |||||
| Metabolic pathways, | |||||
| Biosynthesis of nucleotide sugars | |||||
| 65 | DEG10360124 | K13016 | UDP-2-acetamido-2-deoxy-d-glucuronic acid 3-dehydrogenase WbpB | Amino sugar and nucleotide sugar metabolism, | – |
| O-Antigen nucleotide sugar biosynthesis, | |||||
| Metabolic pathways, | |||||
| Biosynthesis of nucleotide sugars | |||||
| 66 | DEG10360128 | K02469 | DNA gyrase subunit A | – | – |
| 67 | DEG10360130 | K00528 | ferredoxin-NADP reductase | – | – |
| 68 | DEG10360132 | K01770 | 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase | Terpenoid backbone biosynthesis, | – |
| Biosynthesis of secondary metabolites | |||||
| 69 | DEG10360133 | K01627 | 2-dehydro-3-deoxyphosphooctonate aldolase | Lipopolysaccharide biosynthesis, | – |
| Metabolic pathways, | |||||
| Biosynthesis of nucleotide sugars | |||||
| 70 | DEG10360135 | – | hypothetical protein | – | – |
| 71 | DEG10360138 | K00748 | lipid-A-disaccharide synthase | Lipopolysaccharide biosynthesis | – |
| 72 | DEG10360139 | K00677 | UDP-N-acetylglucosamine acyltransferase | Lipopolysaccharide biosynthesis, | |
| Cationic antimicrobial peptide (CAMP) resistance | |||||
| 73 | DEG10360140 | K02372 | (3R)-hydroxymyristoyl-ACP dehydratase | Fatty acid biosynthesis, | |
| Biotin metabolism, | |||||
| Fatty acid metabolism, | |||||
| Biosynthesis of cofactors | |||||
| 74 | DEG10360141 | K02536 | UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase | Lipopolysaccharide biosynthesis | |
| 75 | DEG10360143 | K00099 | 1-deoxy-D-xylulose 5-phosphate reductoisomerase | Terpenoid backbone biosynthesis, | – |
| Metabolic pathways, | |||||
| Biosynthesis of secondary metabolites | |||||
| 76 | DEG10360146 | K02838 | ribosome recycling factor | – | – |
| 77 | DEG10360147 | K09903 | uridylate kinase | Pyrimidine metabolism, | – |
| Metabolic pathways, | |||||
| Nucleotide metabolism, | |||||
| Biosynthesis of cofactors | |||||
| 78 | DEG10360149 | K00674 | tetrahydrodipicolinate succinylase | Lysine biosynthesis, | – |
| Microbial metabolism in diverse environments, | |||||
| Biosynthesis of amino acids | |||||
| 79 | DEG10360150 | K14742 | hypothetical protein (tRNA threonylcarbamoyladenosine biosynthesis protein TsaB) | – | – |
| 80 | DEG10360153 | K03412 | chemotaxis-specific methylesterase | Two-component system, | – |
| Bacterial chemotaxis | |||||
| 81 | DEG10360154 | K18130 | NalC protein | beta-Lactam resistance | – |
| 82 | DEG10360155 | K02860 | 16S rRNA-processing protein RimM | – | – |
| 83 | DEG10360160 | K01892 | histidyl-tRNA synthetase | Aminoacyl-tRNA biosynthesis | – |
| 84 | DEG10360161 | K03526 | 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase | Terpenoid backbone biosynthesis, | – |
| Biosynthesis of secondary metabolites | |||||
| 85 | DEG10360166 | K03072 | preprotein translocase subunit SecD | Protein export, | – |
| Bacterial secretion system | |||||
| 86 | DEG10360167 | K11720 | hypothetical protein (lipopolysaccharide export system permease protein LptG) | ABC transporters | – |
| 87 | DEG10360168 | K07091 | hypothetical protein (Lipopolysaccharide export system permease protein LptF) | ABC transporters | – |
| 88 | DEG10360171 | – | metalloprotease | – | – |
| 89 | DEG10360172 | K03820 | apolipoprotein N-acyltransferase | – | – |
| 90 | DEG10360174 | K02340 | DNA polymerase III subunit delta | DNA replication, | – |
| Mismatch repair, | |||||
| Homologous recombination | |||||
| 91 | DEG10360178 | – | aromatic acid decarboxylase | Ubiquinone and other terpenoid-quinone biosynthesis, | – |
| Aminobenzoate degradation, | |||||
| Riboflavin metabolism, | |||||
| Terpenoid backbone biosynthesis, | |||||
| Metabolic pathways, | |||||
| Biosynthesis of secondary metabolites, | |||||
| Microbial metabolism in diverse environments, | |||||
| Degradation of aromatic compounds, | |||||
| Biosynthesis of cofactor | |||||
| 92 | DEG10360179 | K01507 | inorganic pyrophosphatase | Oxidative phosphorylation | Oxidative phosphorylation |
| 93 | DEG10360181 | K01662 | 1-deoxy-D-xylulose-5-phosphate synthase | Thiamine metabolism, | – |
| Terpenoid backbone biosynthesis, | |||||
| Metabolic pathways, | |||||
| Biosynthesis of secondary metabolites | |||||
| 94 | DEG10360182 | K00946 | thiamine monophosphate kinase | Thiamine metabolism, | – |
| Biosynthesis of cofactors | |||||
| 95 | DEG10360183 | K03625 | transcription antitermination protein NusB | – | – |
| 96 | DEG10360185 | – | hypothetical protein (Thioesterase domain-containing protein) | – | – |
| 97 | DEG10360186 | K02879 | 50S ribosomal protein L17 | Ribosome | – |
| 98 | DEG10360187 | K03040 | DNA-directed RNA polymerase subunit alpha | RNA polymerase | – |
| 99 | DEG10360191 | K03076 | preprotein translocase subunit SecY | Quorum sensing, | – |
| Protein export, | |||||
| Bacterial secretion system | |||||
| 100 | DEG10360192 | K02876 | 50S ribosomal protein L15 | Ribosome | – |
| 101 | DEG10360193 | K02988 | 30S ribosomal protein S5 | Ribosome | – |
| 102 | DEG10360194 | K02933 | 50S ribosomal protein L6 | Ribosome | – |
| 103 | DEG10360195 | K02994 | 30S ribosomal protein S8 | Ribosome | – |
| 104 | DEG10360196 | K02931 | 50S ribosomal protein L5 | Ribosome | – |
| 105 | DEG10360198 | K02982 | 30S ribosomal protein S3 | Ribosome | – |
| 106 | DEG10360207 | K02935 | 50S ribosomal protein L7L12 | Ribosome | – |
| 107 | DEG10360208 | K02863 | 50S ribosomal protein L10 | Ribosome | – |
| 108 | DEG10360209 | K02867 | 50S ribosomal protein L1 | Ribosome | – |
| 109 | DEG10360211 | K02601 | transcription antitermination protein NusG | – | – |
| 110 | DEG10360212 | K03073 | preprotein translocase subunit SecE | Quorum sensing, | – |
| Protein export, | |||||
| Bacterial secretion system | |||||
| 111 | DEG10360213 | – | pantothenate kinase | – | – |
| 112 | DEG10360217 | K03070 | preprotein translocase subunit SecA | Quorum sensing, | – |
| Protein export, | |||||
| Bacterial secretion system | |||||
| 113 | DEG10360218 | – | hypothetical protein (DUF721 domain-containing protein) | – | – |
| 114 | DEG10360219 | K02535 | UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase | Lipopolysaccharide biosynthesis | – |
| 115 | DEG10360220 | K03531 | cell division protein FtsZ | – | – |
| 116 | DEG10360221 | K03590 | cell division protein FtsA | – | – |
| 117 | DEG10360222 | K01924 | UDP-N-acetylmuramate--L-alanine ligase | Peptidoglycan biosynthesis | – |
| 118 | DEG10360223 | K02563 | undecaprenyldiphospho-muramoylpentapeptide beta-N- acetylglucosaminyltransferase | Peptidoglycan biosynthesis, | |
| Vancomycin resistance | |||||
| 119 | DEG10360224 | K03588 | cell division protein FtsW | – | – |
| 120 | DEG10360225 | K01925 | UDP-N-acetylmuramoyl-L-alanyl-D-glutamate synthetase | D-Amino acid metabolism, | – |
| Peptidoglycan biosynthesis | |||||
| 121 | DEG10360226 | K01000 | phospho-N-acetylmuramoyl-pentapeptide- transferase | Peptidoglycan biosynthesis, | – |
| Vancomycin resistance | |||||
| 122 | DEG10360227 | K01929 | UDP-N-acetylmuramoyl-tripeptide--D-alanyl-D- alanine ligase | Lysine biosynthesis, | – |
| Peptidoglycan biosynthesis, | |||||
| Vancomycin resistance | |||||
| 123 | DEG10360228 | K01928 | UDP-N-acetylmuramoylalanyl-D-glutamate--2, 6-diaminopimelate ligase | Lysine biosynthesis, | – |
| Peptidoglycan biosynthesis | |||||
| 124 | DEG10360231 | K03271 | phosphoheptose isomerase | Lipopolysaccharide biosynthesis, | – |
| Biosynthesis of nucleotide sugars | |||||
| 125 | DEG10360232 | K00413 | cytochrome C1 | Oxidative phosphorylation, | – |
| Metabolic pathways, | |||||
| Two-component system | |||||
| 126 | DEG10360236 | K00790 | UDP-N-acetylglucosamine 1-carboxyvinyltransferase | Amino sugar and nucleotide sugar metabolism, | – |
| Peptidoglycan biosynthesis, | |||||
| Biosynthesis of nucleotide sugars | |||||
| 127 | DEG10360237 | K06041 | arabinose-5-phosphate isomerase KdsD | Lipopolysaccharide biosynthesis, | – |
| Biosynthesis of nucleotide sugars | |||||
| 128 | DEG10360238 | K11719 | hypothetical protein (Lipopolysaccharide export system protein LptC) | – | – |
| 129 | DEG10360239 | K09774 | hypothetical protein (Lipopolysaccharide export system protein LptH) | – | – |
| 130 | DEG10360241 | K03571 | rod shape-determining protein MreD | – | – |
| 131 | DEG10360242 | K03570 | rod shape-determining protein MreC | – | – |
| 132 | DEG10360251 | – | hypothetical protein (Probable lipid II flippase MurJ) | – | – |
| 133 | DEG10360254 | – | hypothetical protein (Phospholipid/glycerol acyltransferase domain-containing protein) | – | – |
| 134 | DEG10360262 | – | hypothetical protein (Protein TonB) | – | – |
| 135 | DEG10360263 | – | hypothetical protein (Chromosome partitioning protein) | – | – |
| 136 | DEG10360264 | K00950 | 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase | Folate biosynthesis, | – |
| Metabolic pathways, | |||||
| Biosynthesis of cofactors | |||||
| 137 | DEG10360271 | K18974 | dihydropteroate synthase | Folate biosynthesis, | – |
| Biosynthesis of cofactors | |||||
| 138 | DEG10360279 | K02160 | acetyl-CoA carboxylase biotin carboxyl carrier protein subunit | Fatty acid biosynthesis, | Fatty acid biosynthesis, Pyruvate metabolism, Propanoate metabolism, Metabolic pathways, AMPK signaling pathway, Insulin signaling pathway, Adipocytokine signaling pathway, Glucagon signaling pathway Insulin resistance, Alcoholic liver disease |
| Pyruvate metabolism, | |||||
| Propanoate metabolism, | |||||
| Biosynthesis of secondary metabolites, | |||||
| Microbial metabolism in diverse environments, | |||||
| Carbon metabolism, | |||||
| Fatty acid metabolism | |||||
| 139 | DEG10360281 | K01916 | NAD synthetase | Nicotinate and nicotinamide metabolism, | – |
| Metabolic pathways, | |||||
| Biosynthesis of cofactors | |||||
| 140 | DEG10360288 | K03651 | cAMP phosphodiesterase | Purine metabolism, | – |
| Metabolic pathways, | |||||
| Biofilm formation - Pseudomonas aeruginosa | |||||
| 141 | DEG10360289 | K02527 | 3-deoxy-D-manno-octulosonic-acid transferase | Lipopolysaccharide biosynthesis, | – |
| Metabolic pathways | |||||
| 142 | DEG10360290 | – | hypothetical protein (Probable FAD-dependent oxidoreductase PA4991) | – | – |
| 143 | DEG10360298 | K02848 | lipopolysaccharide kinase WaaP | Lipopolysaccharide biosynthesis | – |
| 144 | DEG10360299 | K02844 | UDP-glucose:(heptosyl) LPS alpha 1,3-glucosyltransferase WaaG | Lipopolysaccharide biosynthesis | – |
| 145 | DEG10360315 | – | hypothetical protein (3-octaprenyl-4-hydroxybenzoate carboxy-lyase) | – | – |
| 146 | DEG10360318 | K01719 | uroporphyrinogen-III synthase | Porphyrin metabolism, | – |
| Metabolic pathways, | |||||
| Biosynthesis of secondary metabolites, | |||||
| Microbial metabolism in diverse environments, | |||||
| Biosynthesis of cofactors | |||||
| 147 | DEG10360320 | K01778 | diaminopimelate epimerase | Lysine biosynthesis, | – |
| D-Amino acid metabolism, | |||||
| Biosynthesis of secondary metabolites, | |||||
| Microbial metabolism in diverse environments, | |||||
| Biosynthesis of amino acids | |||||
| 148 | DEG10360328 | K04042 | bifunctional glucosamine-1-phosphate acetyltransferaseN-acetylglucosamine-1-phosphate uridyltransferase | Amino sugar and nucleotide sugar metabolism, | – |
| O-Antigen nucleotide sugar biosynthesis, | |||||
| Biosynthesis of nucleotide sugars | |||||
| 149 | DEG10360332 | K02113 | ATP synthase F0F1 subunit delta | Oxidative phosphorylation | – |
3.3.2. Subcellular localization
Protein localization is crucial for identifying effective drug targets. The 87 proteins with unique pathways were selected for subcellular localization prediction. Of these, 77 proteins were localized to the cytoplasm, and 10 proteins were found to be associated with the cell membrane (Table 4). Cytoplasmic proteins are considered attractive drug targets, while membrane proteins are potential candidates for vaccine development [54].
Table 4.
Subcellular localization prediction of proteins using Psortb
| S.NO | DEG ACCESSION NO | Protein Name | Cellular localization |
|---|---|---|---|
| 1 | DEG10360001 | Chromosome replication initiator DnaA | Cytoplasmic |
| 2 | DEG10360002 | DNA polymerase III subunit beta | Cytoplasmic |
| 3 | DEG10360004 | D,D-heptose 1,7-bisphosphate phosphatase | Cytoplasmic |
| 4 | DEG10360005 | Glycyl-tRNA synthetase subunit beta | Cytoplasmic |
| 5 | DEG10360006 | Glycyl-tRNA synthetase subunit alpha | Cytoplasmic |
| 6 | DEG10360023 | RNA polymerase sigma factor RpoD | Cytoplasmic |
| 7 | DEG10360024 | DNA primase | Cytoplasmic |
| 8 | DEG10360026 | Dihydroneopterin aldolase | Cytoplasmic |
| 9 | DEG10360027 | 4-hydroxythreonine-4-phosphate dehydrogenase | Cytoplasmic |
| 10 | DEG10360033 | HxcU pseudopilin | Cytoplasmic membrane |
| 11 | DEG10360037 | Pyridoxine 5′-phosphate synthase | Cytoplasmic |
| 12 | DEG10360039 | Aspartate kinase | Cytoplasmic |
| 13 | DEG10360054 | Erythronate-4-phosphate dehydrogenase | Cytoplasmic |
| 14 | DEG10360069 | 3-hydroxydecanoyl-ACP dehydratase | Cytoplasmic |
| 15 | DEG10360070 | Chorismate synthase | Cytoplasmic |
| 16 | DEG10360072 | Bifunctional aconitate hydratase 22-methylisocitrate dehydratase | Cytoplasmic |
| 17 | DEG10360073 | UDP-2,3-diacylglucosamine hydrolase | Cytoplasmic |
| 18 | DEG10360083 | Sulfur transfer complex subunit TusD | Cytoplasmic |
| 19 | DEG10360096 | Trans-2-enoyl-CoA reductase | Cytoplasmic |
| 20 | DEG10360100 | Thymidylate kinase | Cytoplasmic |
| 21 | DEG10360102 | UDP-N-acetylenolpyruvoylglucosamine reductase | Cytoplasmic |
| 22 | DEG10360103 | 3-deoxy-manno-octulosonate cytidylyltransferase | Cytoplasmic |
| 23 | DEG10360104 | Tetraacyldisaccharide 4′-kinase | Cytoplasmic |
| 24 | DEG10360105 | Hypothetical protein (Lipoprotein-releasing ABC transporter permease subunit) | Cytoplasmic Membrane |
| 25 | DEG10360107 | Hypothetical protein | Cytoplasmic Membrane |
| 26 | DEG10360111 | Aspartate-semialdehyde dehydrogenase | Cytoplasmic |
| 27 | DEG10360113 | Nucleotide sugar epimerasedehydratase WbpM | Cytoplasmic Membrane |
| 28 | DEG10360118 | LPS biosynthesis protein WbpG | Cytoplasmic |
| 29 | DEG10360119 | Imidazole glycerol phosphate synthase subunit HisF2 | Cytoplasmic |
| 30 | DEG10360123 | UDP-2-acetamido-3-amino-23-dideoxy-d-glucuronic acid N-acetyltransferase WbpD | Cytoplasmic |
| 31 | DEG10360124 | UDP-2-acetamido-2-deoxy-d-glucuronic acid 3-dehydrogenase WbpB | Cytoplasmic |
| 32 | DEG10360132 | 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase | Cytoplasmic |
| 33 | DEG10360133 | 2-dehydro-3-deoxyphosphooctonate aldolase | Cytoplasmic |
| 34 | DEG10360138 | Lipid-A-disaccharide synthase | Cytoplasmic |
| 35 | DEG10360139 | UDP-N-acetylglucosamine acyltransferase | Cytoplasmic |
| 36 | DEG10360140 | (3R)-hydroxymyristoyl-ACP dehydratase | Cytoplasmic |
| 37 | DEG10360141 | UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase | Cytoplasmic |
| 38 | DEG10360143 | 1-deoxy-D-xylulose 5-phosphate reductoisomerase | Cytoplasmic |
| 39 | DEG10360147 | Uridylate kinase | Cytoplasmic |
| 40 | DEG10360149 | tetrahydrodipicolinate succinylase | Cytoplasmic |
| 41 | DEG10360153 | chemotaxis-specific methylesterase | Cytoplasmic |
| 42 | DEG10360154 | NalC protein | Cytoplasmic |
| 43 | DEG10360160 | Histidyl-tRNA synthetase | Cytoplasmic |
| 44 | DEG10360161 | 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase | Cytoplasmic |
| 45 | DEG10360166 | Preprotein translocase subunit SecD | Cytoplasmic Membrane |
| 46 | DEG10360167 | Hypothetical protein (lipopolysaccharide export system permease protein LptG) | Cytoplasmic Membrane |
| 47 | DEG10360168 | Hypothetical protein (Lipopolysaccharide export system permease protein LptF) | Cytoplasmic Membrane |
| 48 | DEG10360174 | DNA polymerase III subunit delta | Cytoplasmic |
| 49 | DEG10360178 | Aromatic acid decarboxylase | Cytoplasmic |
| 50 | DEG10360181 | 1-deoxy-D-xylulose-5-phosphate synthase | Cytoplasmic |
| 51 | DEG10360182 | Thiamine monophosphate kinase | Cytoplasmic |
| 52 | DEG10360186 | 50S ribosomal protein L17 | Cytoplasmic |
| 53 | DEG10360187 | DNA-directed RNA polymerase subunit alpha | Cytoplasmic |
| 54 | DEG10360191 | Preprotein translocase subunit SecY | Cytoplasmic Membrane |
| 55 | DEG10360192 | 50S ribosomal protein L15 | Cytoplasmic |
| 56 | DEG10360193 | 30S ribosomal protein S5 | Cytoplasmic |
| 57 | DEG10360194 | 50S ribosomal protein L6 | Cytoplasmic |
| 58 | DEG10360195 | 30S ribosomal protein S8 | Cytoplasmic |
| 59 | DEG10360196 | 50S ribosomal protein L5 | Cytoplasmic |
| 60 | DEG10360198 | 30S ribosomal protein S3 | Cytoplasmic |
| 61 | DEG10360207 | 50S ribosomal protein L7L12 | Cytoplasmic |
| 62 | DEG10360208 | 50S ribosomal protein L10 | Cytoplasmic |
| 63 | DEG10360209 | 50S ribosomal protein L1 | Cytoplasmic |
| 64 | DEG10360212 | Preprotein translocase subunit SecE | Cytoplasmic Membrane |
| 65 | DEG10360217 | Preprotein translocase subunit SecA | Cytoplasmic |
| 66 | DEG10360219 | UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase | Cytoplasmic |
| 67 | DEG10360222 | UDP-N-acetylmuramate--L-alanine ligase | Cytoplasmic |
| 68 | DEG10360223 | Undecaprenyldiphospho-muramoylpentapeptide beta-N- acetylglucosaminyltransferase | Cytoplasmic |
| 69 | DEG10360225 | UDP-N-acetylmuramoyl-L-alanyl-D-glutamate synthetase | Cytoplasmic |
| 70 | DEG10360226 | Phospho-N-acetylmuramoyl-pentapeptide- transferase | Cytoplasmic Membrane |
| 71 | DEG10360227 | UDP-N-acetylmuramoyl-tripeptide--D-alanyl-D- alanine ligase | Cytoplasmic |
| 72 | DEG10360228 | UDP-N-acetylmuramoylalanyl-D-glutamate--2, 6-diaminopimelate ligase | Cytoplasmic |
| 73 | DEG10360231 | Phosphoheptose isomerase | Cytoplasmic |
| 74 | DEG10360232 | Cytochrome C1 | Cytoplasmic |
| 75 | DEG10360236 | UDP-N-acetylglucosamine 1-carboxyvinyltransferase | Cytoplasmic |
| 76 | DEG10360237 | arabinose-5-phosphate isomerase KdsD | Cytoplasmic |
| 77 | DEG10360264 | 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase | Cytoplasmic |
| 78 | DEG10360271 | Dihydropteroate synthase | Cytoplasmic |
| 79 | DEG10360281 | NAD synthetase | Cytoplasmic |
| 80 | DEG10360288 | cAMP phosphodiesterase | Cytoplasmic |
| 81 | DEG10360289 | 3-deoxy-D-manno-octulosonic-acid transferase | Cytoplasmic |
| 82 | DEG10360298 | Lipopolysaccharide kinase WaaP | Cytoplasmic |
| 83 | DEG10360299 | UDP-glucose:(heptosyl) LPS alpha 1,3-glucosyltransferase WaaG | Cytoplasmic |
| 84 | DEG10360318 | Uroporphyrinogen-III synthase | Cytoplasmic |
| 85 | DEG10360320 | Diaminopimelate epimerase | Cytoplasmic |
| 86 | DEG10360328 | Bifunctional glucosamine-1-phosphate acetyltransferaseN-acetylglucosamine-1-phosphate uridyltransferase | Cytoplasmic |
| 87 | DEG10360332 | ATP synthase F0F1 subunit delta | Cytoplasmic |
3.4. Druggability assessment
3.4.1. Drug target identification and prioritization
The evaluation of a protein's druggability is a crucial step in the identification of drug targets and was performed with the assumption that druggable proteins should bind with drug-like compounds. Consequently, potential drug targets were identified using the DrugBank database. The DrugBank database was systematically analyzed using BLASTp to assess the sequence homology of 87 essential non-homologous proteins, identified during the subcellular localization prediction phase, with annotated drug targets within DrugBank. The cut-off values for the search were: E-value = 0.0001, bit score >100, and identity >10 %. The DrugBank hits were classified into common targets or those with druggability potential. The remaining hits were regarded as distinctive drug targets and subjected to further experimental validation. Out of the 87 proteins, 45 non-homologous essential proteins were identified as unique targets (Table 5).
Table 5.
Unique Drug Targets Prioritization using DrugBank Database Analysis.
| S.NO | DEG ACCESSION NO | Definition | Similarity with Drug bank targets |
|---|---|---|---|
| 1 | DEG10360001 | Chromosome replication initiator DnaA | – |
| 2 | DEG10360002 | DNA polymerase III subunit beta | Yes |
| 3 | DEG10360004 | D,D-heptose 1,7-bisphosphate phosphatase | – |
| 4 | DEG10360005 | Glycyl-tRNA synthetase subunit beta | – |
| 5 | DEG10360006 | Glycyl-tRNA synthetase subunit alpha | – |
| 6 | DEG10360023 | RNA polymerase sigma factor RpoD | Yes |
| 7 | DEG10360024 | DNA primase | – |
| 8 | DEG10360026 | Dihydroneopterin aldolase | – |
| 9 | DEG10360027 | 4-hydroxythreonine-4-phosphate dehydrogenase | Yes |
| 10 | DEG10360033 | HxcU pseudopilin | – |
| 11 | DEG10360037 | Pyridoxine 5′-phosphate synthase | Yes |
| 12 | DEG10360039 | Aspartate kinase | – |
| 13 | DEG10360054 | Erythronate-4-phosphate dehydrogenase | Yes |
| 14 | DEG10360069 | 3-hydroxydecanoyl-ACP dehydratase | Yes |
| 15 | DEG10360070 | Chorismate synthase | Yes |
| 16 | DEG10360072 | Bifunctional aconitate hydratase 22-methylisocitrate dehydratase | Yes |
| 17 | DEG10360073 | UDP-2,3-diacylglucosamine hydrolase | – |
| 18 | DEG10360083 | Sulfur transfer complex subunit TusD | – |
| 19 | DEG10360096 | Trans-2-enoyl-CoA reductase | – |
| 20 | DEG10360100 | Thymidylate kinase | Yes |
| 21 | DEG10360102 | UDP-N-acetylenolpyruvoylglucosamine reductase | Yes |
| 22 | DEG10360103 | 3-deoxy-manno-octulosonate cytidylyltransferase | Yes |
| 23 | DEG10360104 | Tetraacyldisaccharide 4′-kinase | – |
| 24 | DEG10360105 | Hypothetical protein (Lipoprotein-releasing ABC transporter permease subunit) | – |
| 25 | DEG10360107 | Hypothetical protein | – |
| 26 | DEG10360111 | Aspartate-semialdehyde dehydrogenase | Yes |
| 27 | DEG10360113 | Nucleotide sugar epimerasedehydratase WbpM | – |
| 28 | DEG10360118 | LPS biosynthesis protein WbpG | – |
| 29 | DEG10360119 | Imidazole glycerol phosphate synthase subunit HisF2 | – |
| 30 | DEG10360123 | UDP-2-acetamido-3-amino-23-dideoxy-d-glucuronic acid N-acetyltransferase WbpD | Yes |
| 31 | DEG10360124 | UDP-2-acetamido-2-deoxy-d-glucuronic acid 3-dehydrogenase WbpB | – |
| 32 | DEG10360132 | 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase | Yes |
| 33 | DEG10360133 | 2-dehydro-3-deoxyphosphooctonate aldolase | Yes |
| 34 | DEG10360138 | Lipid-A-disaccharide synthase | – |
| 35 | DEG10360139 | UDP-N-acetylglucosamine acyltransferase | Yes |
| 36 | DEG10360140 | (3R)-hydroxymyristoyl-ACP dehydratase | Yes |
| 37 | DEG10360141 | UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase | Yes |
| 38 | DEG10360143 | 1-deoxy-D-xylulose 5-phosphate reductoisomerase | Yes |
| 39 | DEG10360147 | Uridylate kinase | – |
| 40 | DEG10360149 | tetrahydrodipicolinate succinylase | – |
| 41 | DEG10360153 | chemotaxis-specific methylesterase | – |
| 42 | DEG10360154 | NalC protein | – |
| 43 | DEG10360160 | Histidyl-tRNA synthetase | Yes |
| 44 | DEG10360161 | 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase | – |
| 45 | DEG10360166 | Preprotein translocase subunit SecD | – |
| 46 | DEG10360167 | Hypothetical protein (lipopolysaccharide export system permease protein LptG) | – |
| 47 | DEG10360168 | Hypothetical protein (Lipopolysaccharide export system permease protein LptF) | – |
| 48 | DEG10360174 | DNA polymerase III subunit delta | – |
| 49 | DEG10360178 | Aromatic acid decarboxylase | Yes |
| 50 | DEG10360181 | 1-deoxy-D-xylulose-5-phosphate synthase | Yes |
| 51 | DEG10360182 | Thiamine monophosphate kinase | – |
| 52 | DEG10360186 | 50S ribosomal protein L17 | – |
| 53 | DEG10360187 | DNA-directed RNA polymerase subunit alpha | Yes |
| 54 | DEG10360191 | Preprotein translocase subunit SecY | – |
| 55 | DEG10360192 | 50S ribosomal protein L15 | – |
| 56 | DEG10360193 | 30S ribosomal protein S5 | Yes |
| 57 | DEG10360194 | 50S ribosomal protein L6 | – |
| 58 | DEG10360195 | 30S ribosomal protein S8 | Yes |
| 59 | DEG10360196 | 50S ribosomal protein L5 | Yes |
| 60 | DEG10360198 | 30S ribosomal protein S3 | Yes |
| 61 | DEG10360207 | 50S ribosomal protein L7L12 | – |
| 62 | DEG10360208 | 50S ribosomal protein L10 | Yes |
| 63 | DEG10360209 | 50S ribosomal protein L1 | Yes |
| 64 | DEG10360212 | Preprotein translocase subunit SecE | – |
| 65 | DEG10360217 | Preprotein translocase subunit SecA | – |
| 66 | DEG10360219 | UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase | Yes |
| 67 | DEG10360222 | UDP-N-acetylmuramate--L-alanine ligase | Yes |
| 68 | DEG10360223 | Undecaprenyldiphospho-muramoylpentapeptide beta-N- acetylglucosaminyltransferase | Yes |
| 69 | DEG10360225 | UDP-N-acetylmuramoyl-L-alanyl-D-glutamate synthetase | Yes |
| 70 | DEG10360226 | Phospho-N-acetylmuramoyl-pentapeptide- transferase | – |
| 71 | DEG10360227 | UDP-N-acetylmuramoyl-tripeptide--D-alanyl-D- alanine ligase | Yes |
| 72 | DEG10360228 | UDP-N-acetylmuramoylalanyl-D-glutamate--2, 6-diaminopimelate ligase | Yes |
| 73 | DEG10360231 | Phosphoheptose isomerase | Yes |
| 74 | DEG10360232 | Cytochrome C1 | – |
| 75 | DEG10360236 | UDP-N-acetylglucosamine 1-carboxyvinyltransferase | Yes |
| 76 | DEG10360237 | arabinose-5-phosphate isomerase KdsD | – |
| 77 | DEG10360264 | 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase | Yes |
| 78 | DEG10360271 | Dihydropteroate synthase | Yes |
| 79 | DEG10360281 | NAD synthetase | Yes |
| 80 | DEG10360288 | cAMP phosphodiesterase | – |
| 81 | DEG10360289 | 3-deoxy-D-manno-octulosonic-acid transferase | – |
| 82 | DEG10360298 | Lipopolysaccharide kinase WaaP | – |
| 83 | DEG10360299 | UDP-glucose:(heptosyl) LPS alpha 1,3-glucosyltransferase WaaG | – |
| 84 | DEG10360318 | Uroporphyrinogen-III synthase | – |
| 85 | DEG10360320 | Diaminopimelate epimerase | – |
| 86 | DEG10360328 | Bifunctional glucosamine-1-phosphate acetyltransferaseN-acetylglucosamine-1-phosphate uridyltransferase | Yes |
| 87 | DEG10360332 | ATP synthase F0F1 subunit delta | Yes |
3.5. Virulence factor and antibiotic resistance analysis
3.5.1. Virulence factor identification
Pathogenic bacteria synthesize essential proteins, known as virulence factors, which play a key role in host invasion, disease development, and immune system evasion. These factors serve as a defensive shield for the pathogen against the host's immune responses [55]. To identify potential virulence factors from the 45 unique targets, the VFDB was employed, revealing 10 proteins with significant sequence similarity to well-characterized virulence factors such as lipopolysaccharide, pili, flagella, and alginate (Table 6).
Table 6.
Identification of Proteins involved in Pseudomonas sp. virulence using the Virulence Factor Database (VFDB).
| S.NO | Protein Name | VFDB sequence identity | Hit ID | Related Virulence factor |
|---|---|---|---|---|
| 1 | HxcU pseudopilin | 54 % | VFG000180 (xcpT) general secretion pathway protein G | Xcp secretion system |
| 2 | Nucleotide sugar epimerasedehydratase WbpM | 98 % | VFG014117 (wbpM) nucleotide sugar epimerase/dehydratase WbpM | LPS |
| 3 | LPS biosynthesis protein WbpG | 100 % | VFG014108 (wbpG) LPS biosynthesis protein WbpG | LPS |
| 4 | Imidazole glycerol phosphate synthase subunit HisF2 | 100 % | VFG014107 (hisF2) imidazole glycerol phosphate synthase subunit HisF | LPS |
| 5 | UDP-2-acetamido-2-deoxy-d-glucuronic acid 3-dehydrogenase WbpB | 100 % | VFG014100 (wbpB) UDP-N-acetyl-2-amino-2-deoxy-D-glucuronate oxidase | LPS |
| 6 | Chemotaxis-specific methylesterase | 29 % | VFG043015 (PA1459) chemotaxis-specific methylesterase | Flagella |
| 26 % | VFG001232 (chpB) probable methylesterase | Type IV pili | ||
| 30 % | VFG000119 (algR) alginate biosynthesis regulatory protein AlgR | Alginate | ||
| 34 % | VFG001226 (pilH) twitching motility protein PilH | Type IV pili | ||
| 7 | Phospho-N-acetylmuramoyl-pentapeptide- transferase | 25 % | VFG014113 (wbpL) glycosyltransferase WbpL | LPS |
| 8 | 3-deoxy-D-manno-octulosonic-acid transferase | 100 % | VFG000141 (waaA) lipopolysaccharide core biosynthesis protein WaaP | LPS |
| 9 | Lipopolysaccharide kinase WaaP | 95 % | VFG000140 (waaP) UDP-glucose:(heptosyl) LPS alpha 1,3-glucosyltransferase WaaG |
LPS |
| 10 | UDP-glucose:(heptosyl) LPS alpha 1,3-glucosyltransferase WaaG | 95 % | VFG000139 (waaG) B-band O-antigen polymerase | LPS |
3.5.2. Analysis of resistance genes
The 45 distinct targets were subsequently investigated using the CARD database to determine their involvement in antibiotic resistance in Pseudomonas species. This examination revealed five proteins responsible for conferring resistance to multiple drug classes, such as cephalosporins, sulphonamides, penams, as well as disinfectants and antiseptics, within the P. aeruginosa PAO1 strain (Table 7).
Table 7.
Identification of proteins related to resistance genes in the Comprehensive Antibiotic Resistance Database (CARD).
| S. No | Protein Name | % identity | ARO tag | Name of the gene | Species | Resistant to drug class | Resistance mechanism |
|---|---|---|---|---|---|---|---|
| 1 | Tetraacyldisaccharide 4′-kinase | 35 | 3004077 | PmpM | Pseudomonas aeruginosa PAO1 | Disinfecting Agents And Antiseptics, Aminoglycoside Antibiotic, Fluoroquinolone Antibiotic | Antibiotic Efflux |
| 2 | Tetrahydrodipicolinate succinylase | 35 | 3003699 | mexQ | Pseudomonas aeruginosa | Carbapenem, Macrolide Antibiotic, Disinfecting Agents And Antiseptics, Tetracycline Antibiotic, Phenicol Antibiotic, Diaminopyrimidine Antibiotic | Antibiotic Efflux |
| 3 | Chemotaxis-specific methylesterase | 27 | 3003688 | PvrR | Pseudomonas aeruginosa | Aminoglycoside Antibiotic, Penam, Tetracycline Antibiotic | Resistance By Absence |
| 25 | 3004054 | CpxR | Pseudomonas aeruginosa | Peptide Antibiotic, Aminoglycoside Antibiotic, Diaminopyrimidine Antibiotic, Sulfonamide Antibiotic, Aminocoumarin Antibiotic, Penam, Fluoroquinolone Antibiotic, Cephalosporin, Carbapenem, Macrolide Antibiotic, Monobactam, Tetracycline Antibiotic, Phenicol Antibiotic, Cephamycin, Penem | Antibiotic Efflux | ||
| 30 | 3005068 | ParR | Pseudomonas aeruginosa PAO1 | Aminoglycoside Antibiotic, Carbapenem, Penem, Cephamycin, Monobactam, Phenicol Antibiotic, Macrolide Antibiotic, Fluoroquinolone Antibiotic, Disinfecting Agents And Antiseptics, Tetracycline Antibiotic, Penam, Cephalosporin | Reduced Permeability To Antibiotic, Antibiotic Efflux | ||
| 23 | 3003895 | Pseudomonas mutant PhoP conferring resistance to colistin | Pseudomonas aeruginosa PAO1 | Peptide Antibiotic, Macrolide Antibiotic | Antibiotic Target Alteration, Antibiotic Efflux, Resistance By Absence | ||
| 36 | 3001014 | TEM-147 | Pseudomonas aeruginosa | Penam, Penem, Cephalosporin, Monobactam | Antibiotic Inactivation | ||
| 24 | 3005063 | cprR | Pseudomonas aeruginosa PAO1 | Peptide Antibiotic | Antibiotic Target Alteration, Antibiotic Efflux | ||
| 36 | 3001382 | TEM-205 | Pseudomonas aeruginosa | Penam, Penem, Monobactam, Cephalosporin | Antibiotic Inactivation | ||
| 36 | 3007451 | TEM-247 | Pseudomonas alloputida | Penam, Cephalosporin, Monobactam, Penem | Antibiotic Inactivation | ||
| 36 | 3005265 | TEM-234 | Pseudomonas aeruginosa | Penam, Cephalosporin, Monobactam, Penem | Antibiotic Inactivation | ||
| 36 | 3005271 | TEM-241 | Pseudomonas aeruginosa | Penam, Penem, Cephalosporin, Monobactam | Antibiotic Inactivation | ||
| 36 | 3,001,390 | TEM-213 | Pseudomonas aeruginosa | Penam, Monobactam, Penem, Cephalosporin | Antibiotic Inactivation | ||
| 36 | 3000874 | TEM-2 | Pseudomonas aeruginosa | Penam, Penem, Cephalosporin, Monobactam | Antibiotic Inactivation | ||
| 4 | NalC protein | 100 | 3000818 | nalC | Pseudomonas aeruginosa PAO1 | Peptide Antibiotic, Diaminopyrimidine Antibiotic, Sulfonamide Antibiotic, Macrolide Antibiotic, Monobactam, Tetracycline Antibiotic, Fluoroquinolone Antibiotic, Cephalosporin, Carbapenem, Phenicol Antibiotic, Penam, Aminocoumarin Antibiotic, Cephamycin, Penem | Antibiotic Efflux |
| 30 | 3003710 | MexL | Pseudomonas aeruginosa PAO1 | Macrolide Antibiotic, Disinfecting Agents And Antiseptics, Tetracycline Antibiotic | Antibiotic Efflux | ||
| 34 | 3000819 | nalD | Pseudomonas aeruginosa PAO1 | Peptide Antibiotic, Sulfonamide Antibiotic, Diaminopyrimidine Antibiotic, Cephalosporin, Macrolide Antibiotic, Aminocoumarin Antibiotic, Fluoroquinolone Antibiotic, Tetracycline Antibiotic, Carbapenem, Penam, Phenicol Antibiotic, Monobactam, Penem, Cephamycin | Antibiotic Efflux | ||
| 31 | 3003709 | MexZ | Pseudomonas aeruginosa PAO1 | Aminoglycoside Antibiotic, Fluoroquinolone Antibiotic, Macrolide Antibiotic, Tetracycline Antibiotic, Carbapenem, Disinfecting Agents And Antiseptics, Phenicol Antibiotic, Cephamycin, Cephalosporin, Penam | Antibiotic Efflux | ||
| 5 | Preprotein translocase subunit SecD | 22 | 3004075 | MuxC | Pseudomonas aeruginosa PAO1 | Macrolide Antibiotic, Monobactam, Aminocoumarin Antibiotic, Tetracycline Antibiotic | Antibiotic Efflux |
| 25 | 3003699 | mexQ | Pseudomonas aeruginosa | Phenicol Antibiotic, Macrolide Antibiotic, Diaminopyrimidine Antibiotic, Tetracycline Antibiotic, Carbapenem, Disinfecting Agents And Antiseptics | Antibiotic Efflux | ||
| 26 | 3003693 | MexK | Pseudomonas aeruginosa PAO1 | Disinfecting Agents And Antiseptics, Tetracycline Antibiotic, Macrolide Antibiotic | Antibiotic Efflux | ||
| 26 | 3003033 | mexY | Pseudomonas aeruginosa PAO1 | Aminoglycoside Antibiotic, Phenicol Antibiotic, Fluoroquinolone Antibiotic, Macrolide Antibiotic, Tetracycline Antibiotic, Disinfecting Agents And Antiseptics, Carbapenem, Cephamycin, Cephalosporin, Penam | Antibiotic Efflux | ||
| 19 | 3000804 | MexF | Pseudomonas aeruginosa PAO1 | Phenicol Antibiotic, Diaminopyrimidine Antibiotic, Fluoroquinolone Antibiotic | Antibiotic Efflux | ||
| 20 | 3000801 | MexD | Pseudomonas aeruginosa | Phenicol Antibiotic, Diaminopyrimidine Antibiotic, Aminocoumarin Antibiotic, Tetracycline Antibiotic, Aminoglycoside Antibiotic, Macrolide Antibiotic, Cephalosporin, Penam, Fluoroquinolone Antibiotic | Antibiotic Efflux | ||
| 24 | 3000378 | MexB | Pseudomonas aeruginosa | Peptide Antibiotic, Diaminopyrimidine Antibiotic, Sulfonamide Antibiotic, Phenicol Antibiotic, Penam, Macrolide Antibiotic, Carbapenem, Cephalosporin, Tetracycline Antibiotic, Monobactam, Fluoroquinolone Antibiotic, Aminocoumarin Antibiotic, Penem, Cephamycin | Antibiotic Efflux | ||
| 19 | 3000378 | MexB | Pseudomonas aeruginosa | Peptide Antibiotic, Diaminopyrimidine Antibiotic, Sulfonamide Antibiotic, Phenicol Antibiotic, Penam, Macrolide Antibiotic, Carbapenem, Cephalosporin, Tetracycline Antibiotic, Monobactam, Fluoroquinolone Antibiotic, Aminocoumarin Antibiotic, Penem, Cephamycin | Antibiotic Efflux |
3.6. Broad-spectrum target identification
By conducting a comparative sequence analysis of the identified targets with medically important species and various bacterial pathogens, we can assess their viability as potential broad-spectrum therapeutic targets. A BLASTp comparison was conducted against the full proteomes of 240 bacterial species (Table 8) identified several promising candidates. All the targets were found to have close homologs in more than 400 proteins, and their presence ranged from 167 to 235 pathogens, respectively. Notably, the list of proteins included those that share homology with proteins in various virulent strains of Pseudomonas, such as P. aeruginosa PAO1, PA7, LESB58, UCBPP-PA14, Pf0-1, P. mendocina ymp, F1, GB-1, KT2440, and W619 (Table 9). All the 14 protein targets with homology to over 167 pathogens were identified as broad-spectrum candidates. Targeting these proteins with drug molecules has the potential to address a wide range of pathogens, offering a strategic approach to developing broad-spectrum antimicrobial therapies. Our investigation revealed ten proteins linked to virulence and five associated with resistance, including one protein common to both analyses. Moreover, 14 proteins were conserved across all Pseudomonas virulent strains, highlighting their potential as broad-spectrum drug targets.
Table 8.
List of 240 infectious bacteria used in broad spectrum analysis.
| S.No | Name of Infectious bacteria |
|---|---|
| 1 | Acinetobacter_baumannii_ACICU |
| 2 | Acinetobacter_baumannii_ATCC_17978 |
| 3 | Acinetobacter_baumannii_AYE |
| 4 | Acinetobacter_baumannii_SDF |
| 5 | Acinetobacter_sp_ADP1 |
| 6 | Aeromonas_hydrophila_subsp_hydrophila_ATCC_7966 |
| 7 | Anaplasma_phagocytophilum_HZ |
| 8 | Bacillus_anthracis_str_Ames |
| 9 | Bacillus_anthracis_str_Sterne |
| 10 | Bacillus_cereus_ATCC_10987 |
| 11 | Bacillus_cereus_ATCC_14579 |
| 12 | Bacillus_thuringiensis_serovar_konkukian |
| 13 | Bacteroides_fragilis_NCTC_9343 |
| 14 | Bacteroides_fragilis_YCH46 |
| 15 | Bartonella_bacilliformis_KC583 |
| 16 | Bartonella_henselae_str_Houston-1 |
| 17 | Bartonella_quintana_str_Toulouse |
| 18 | Bordetella_bronchiseptica |
| 19 | Bordetella_pertussis |
| 20 | Borrelia_burgdorferi_group |
| 21 | Borrelia_garinii_PBi |
| 22 | Burkholderia_ambifaria_MC40-6 |
| 23 | Burkholderia_cenocepacia |
| 24 | Burkholderia_cenocepacia_AU_1054 |
| 25 | Burkholderia_cenocepacia_HI2424 |
| 26 | Burkholderia_cenocepacia_MC0-3 |
| 27 | Burkholderia_mallei_ATCC_23344 |
| 28 | Burkholderia_mallei_NCTC_10229 |
| 29 | Burkholderia_mallei_NCTC_10247 |
| 30 | Burkholderia_mallei_SAVP1 |
| 31 | Burkholderia_multivorans_ATCC_17616 |
| 32 | Burkholderia_pseudomallei_1106a |
| 33 | Burkholderia_pseudomallei_1710b |
| 34 | Burkholderia_pseudomallei_668 |
| 35 | Burkholderia_pseudomallei_K96243 |
| 36 | Burkholderia_sp_383 |
| 37 | Burkholderia_xenovorans_LB400 |
| 38 | Campylobacter_concisus_13826 |
| 39 | Campylobacter_curvus_525.92 |
| 40 | Campylobacter_fetus_subsp_fetus_82-40 |
| 41 | Campylobacter_jejuni |
| 42 | Campylobacter_jejuni_RM1221 |
| 43 | Campylobacter_jejuni_subsp_jejuni_81116 |
| 44 | Campylobacter_jejuni_subsp_jejuni_81-176 |
| 45 | Chlamydia_muridarum |
| 46 | Chlamydia_trachomatis |
| 47 | Chlamydia_trachomatis_434/Bu |
| 48 | Chlamydia_trachomatis_A/HAR-13 |
| 49 | Chlamydia_trachomatis_L2b/UCH-1/proctitis |
| 50 | Chlamydophila_abortus_S26/3 |
| 51 | Chlamydophila_caviae |
| 52 | Chlamydophila_felis_Fe/C-56 |
| 53 | Chlamydophila_pneumoniae_AR39 |
| 54 | Chlamydophila_pneumoniae_CWL029 |
| 55 | Chlamydophila_pneumoniae_J138 |
| 56 | Chlamydophila_pneumoniae_TW-183 |
| 57 | Citrobacter_koseri_ATCC_BAA-895 |
| 58 | Clostridium_acetobutylicum |
| 59 | Clostridium_beijerinckii_NCIMB_8052 |
| 60 | Clostridium_botulinum_A |
| 61 | Clostridium_botulinum_A_str_ATCC_19397 |
| 62 | Clostridium_botulinum_A_str_Hall |
| 63 | Clostridium_botulinum_A3_str_Loch_Maree |
| 64 | Clostridium_botulinum_B_str_Eklund_17B |
| 65 | Clostridium_botulinum_B1_str_Okra |
| 66 | Clostridium_botulinum_F_str_Langeland |
| 67 | Clostridium_difficile_630 |
| 68 | Clostridium_perfringens |
| 69 | Clostridium_perfringens_ATCC_13124 |
| 70 | Clostridium_perfringens_SM101 |
| 71 | Clostridium_tetani_E88 |
| 72 | Clostridium_thermocellum_ATCC_27405 |
| 73 | Corynebacterium_diphtheriae |
| 74 | Corynebacterium_efficiens_YS-314 |
| 75 | Corynebacterium_jeikeium_K411 |
| 76 | Corynebacterium_urealyticum_DSM_7109 |
| 77 | Coxiella_burnetii |
| 78 | Coxiella_burnetii_RSA_331 |
| 79 | Ehrlichia_chaffeensis_str_Arkansas |
| 80 | Enterococcus_faecalis_V583 |
| 81 | Escherichia_coli_O157:H7 |
| 82 | Escherichia_coli_O157:H7_str_EDL933 |
| 83 | Francisella_tularensis_subsp_holarctica |
| 84 | Francisella_tularensis_subsp_holarctica_OSU18 |
| 85 | Francisella_tularensis_subsp_mediasiatica_FSC147 |
| 86 | Francisella_tularensis_subsp_tularensis |
| 87 | Francisella_tularensis_subsp_tularensis_FSC198 |
| 88 | Francisella_tularensis_subsp_tularensis_WY96-3418 |
| 89 | Fusobacterium_nucleatum |
| 90 | Haemophilus_ducreyi_35000HP |
| 91 | Haemophilus_influenzae |
| 92 | Haemophilus_influenzae_86-028NP |
| 93 | Haemophilus_influenzae_PittEE |
| 94 | Haemophilus_influenzae_PittGG |
| 95 | Haemophilus_somnus_129 PT |
| 96 | Haemophilus_somnus_2336 |
| 97 | Helicobacter_hepaticus |
| 98 | Helicobacter_pylori_26695 |
| 99 | Helicobacter_pylori_HPAG1 |
| 100 | Helicobacter_pylori_J99 |
| 101 | Klebsiella_pneumoniae_subsp_pneumoniae_MGH_78578 |
| 102 | Legionella_pneumophila_str_Corby |
| 103 | Legionella_pneumophila_str_Lens |
| 104 | Legionella_pneumophila_str_Paris |
| 105 | Legionella_pneumophila_subsp_pneumophila_Philadelphia_1 |
| 106 | Leptospira_borgpetersenii_serovar Hardjo-bovis JB197 |
| 107 | Leptospira_borgpetersenii_serovar_Hardjo-bovis_L550 |
| 108 | Leptospira_interrogans_serovar_Copenhageni |
| 109 | Leptospira_interrogans_serovar_Lai |
| 110 | Listeria_monocytogenes |
| 111 | Listeria_monocytogenes_serotype_4b_str_F2365 |
| 112 | Mycobacterium_asiaticum |
| 113 | Mycobacterium_avium |
| 114 | Mycobacterium_avium_104 |
| 115 | Mycobacterium_avium complex_(MAC) |
| 116 | Mycobacterium_avium_paratuberculosis |
| 117 | Mycobacterium_celatum |
| 118 | Mycobacterium_chelonae |
| 119 | Mycobacterium_conspicuum |
| 120 | Mycobacterium_fortuitum |
| 121 | Mycobacterium_gastri |
| 122 | Mycobacterium_genavense |
| 123 | Mycobacterium_gordonae |
| 124 | Mycobacterium_haemophilum |
| 125 | Mycobacterium_immunogenum |
| 126 | Mycobacterium_intracellulare |
| 127 | Mycobacterium_kansasii |
| 128 | Mycobacterium_leprae |
| 129 | Mycobacterium_malmoense |
| 130 | Mycobacterium_marinum |
| 131 | Mycobacterium_mucogenicum |
| 132 | Mycobacterium_nonchromogenicum |
| 133 | Mycobacterium_scrofulaceum |
| 134 | Mycobacterium_shimoidei |
| 135 | Mycobacterium_simiae |
| 136 | Mycobacterium_smegmatis |
| 137 | Mycobacterium_szulgai |
| 138 | Mycobacterium_terrae |
| 139 | Mycobacterium_terrae_complex |
| 140 | Mycobacterium_tuberculosis_CDC1551 |
| 141 | Mycobacterium_tuberculosis_F11 |
| 142 | Mycobacterium_tuberculosis_H37Ra |
| 143 | Mycobacterium_tuberculosis_H37Rv |
| 144 | Mycobacterium_ulcerans_Agy99 |
| 145 | Mycobacterium_xenopi |
| 146 | Mycoplasma_capricolum_subsp_capricolum_ATCC_27343 |
| 147 | Mycoplasma_gallisepticum |
| 148 | Mycoplasma_genitalium |
| 149 | Mycoplasma_penetrans |
| 150 | Mycoplasma_pneumoniae |
| 151 | Neisseria_gonorrhoeae_FA_1090 |
| 152 | Neisseria_meningitidis_053442 |
| 153 | Neisseria_meningitidis_FAM18 |
| 154 | Neisseria_meningitidis_MC58 |
| 155 | Neisseria_meningitidis_Z2491 |
| 156 | Neorickettsia_sennetsu_str_Miyayama |
| 157 | Nitrosospira_multiformis_ATCC_25196 |
| 158 | Nocardia_farcinica_IFM_10152 |
| 159 | Orientia_tsutsugamushi_Boryong |
| 160 | Orientia_tsutsugamushi_Ikeda |
| 161 | Pasteurella_multocida |
| 162 | Propionibacterium_acnes_KPA171202 |
| 163 | Pseudomonas_aeruginosa |
| 164 | Pseudomonas_aeruginosa_PA7 |
| 165 | Pseudomonas_aeruginosa_UCBPP-PA14 |
| 166 | Pseudomonas_entomophila_L48 |
| 167 | Pseudomonas_mendocina_ymp |
| 168 | Rickettsia_conorii |
| 169 | Rickettsia_felis_URRWXCal2 |
| 170 | Rickettsia_rickettsii_Iowa |
| 171 | Rickettsia_rickettsii_Sheila_Smith |
| 172 | Rickettsia_typhi_wilmington |
| 173 | Salmonella_enterica_subsp_arizonae_serovar_62:z4,z23: - |
| 174 | Salmonella_enterica_subsp_enterica_serovar_Choleraesuis |
| 175 | Salmonella_enterica_subsp_enterica_serovar_Paratyphi_A_str_ATCC_9150 |
| 176 | Salmonella_enterica_subsp_enterica_serovar_Paratyphi_B_str_SPB7 |
| 177 | Salmonella_typhi_ (Schroeter 1886)_Warren_and_Scott_1930 |
| 178 | Shigella_boydii_CDC_3083-94 |
| 179 | Shigella_boydii_Sb227 |
| 180 | Shigella_dysenteriae |
| 181 | Shigella_flexneri_2a |
| 182 | Shigella_flexneri_2a_str_2457T |
| 183 | Shigella_flexneri_5_str_8401 |
| 184 | Staphylococcus_aureus_RF122 |
| 185 | Staphylococcus_aureus_subsp_aureus_COL |
| 186 | Staphylococcus_aureus_subsp_aureus_JH1 |
| 187 | Staphylococcus_aureus_subsp_aureus_JH9 |
| 188 | Staphylococcus_aureus_subsp_aureus_MRSA252 |
| 189 | Staphylococcus_aureus_subsp_aureus_MSSA476 |
| 190 | Staphylococcus_aureus_subsp_aureus_Mu3 |
| 191 | Staphylococcus_aureus_subsp_aureus_Mu50 |
| 192 | Staphylococcus_aureus_subsp_aureus_MW2 |
| 193 | Staphylococcus_aureus_subsp_aureus_N315 |
| 194 | Staphylococcus_aureus_subsp_aureus_NCTC_8325 |
| 195 | Staphylococcus_aureus_subsp_aureus_USA300 |
| 196 | Staphylococcus_aureus_subsp_aureus_USA300_TCH1516 |
| 197 | Staphylococcus_epidermidis_ATCC_12228 |
| 198 | Staphylococcus_epidermidis_RP62A |
| 199 | Staphylococcus_haemolyticus |
| 200 | Staphylococcus_saprophyticus |
| 201 | Streptococcus_gordonii_str_Challis_substr_CH1 |
| 202 | Streptococcus_pneumoniae_CGSP14 |
| 203 | Streptococcus_pneumoniae_D39 |
| 204 | Streptococcus_pneumoniae_Hungary19A-6 |
| 205 | Streptococcus_pneumoniae_R6 |
| 206 | Streptococcus_pneumoniae_TIGR4 |
| 207 | Streptococcus_pyogenes_M1_GAS |
| 208 | Streptococcus_pyogenes_Manfredo |
| 209 | Streptococcus_pyogenes_MGAS10270 |
| 210 | Streptococcus_pyogenes_MGAS10394 |
| 211 | Streptococcus_pyogenes_MGAS10750 |
| 212 | Streptococcus_pyogenes_MGAS2096 |
| 213 | Streptococcus_pyogenes_MGAS315 |
| 214 | Streptococcus_pyogenes_MGAS5005 |
| 215 | Streptococcus_pyogenes_MGAS6180 |
| 216 | Streptococcus_pyogenes_MGAS8232 |
| 217 | Streptococcus_pyogenes_MGAS9429 |
| 218 | Streptococcus_pyogenes_SSI-1 |
| 219 | Streptococcus_sanguinis_SK36 |
| 220 | Streptococcus_suis_05ZYH33 |
| 221 | Streptococcus_suis_98HAH33 |
| 222 | Treponema_pallidum |
| 223 | Treponema_pallidum_subsp_pallidum_SS14 |
| 224 | Tropheryma_whipplei_TW08/27 |
| 225 | Tropheryma_whipplei_Twist |
| 226 | Ureaplasma_parvum_serovar_3_str_ATCC_27815 |
| 227 | Vibrio_cholerae |
| 228 | Vibrio_cholerae_O395 |
| 229 | Vibrio_parahaemolyticus |
| 230 | Vibrio_vulnificus_CMCP6 |
| 231 | Vibrio_vulnificus_YJ016 |
| 232 | Yersinia_enterocolitica_subsp_enterocolitica_8081 |
| 233 | Yersinia_pestis_Angola |
| 234 | Yersinia_pestis_Antiqua |
| 235 | Yersinia_pestis_CO92 |
| 236 | Yersinia_pestis_Nepal516 |
| 237 | Yersinia_pestis_Pestoides_F |
| 238 | Yersinia_pseudotuberculosis_IP_31758 |
| 239 | Yersinia_pseudotuberculosis_IP_32953 |
| 240 | Yersinia_pseudotuberculosis_YPIII |
Table 9.
Broad-spectrum analysis of proteins in virulent strains of Pseudomonas Species.
| S.NO | Protein Name | Virulent Strains from VFDB | Broad spectrum analysis |
|---|---|---|---|
| 1 | HxcU pseudopilin | P. aeruginosa PAO1, | 183 |
| P. aeruginosa PA7, | |||
| P. aeruginosa LESB58, | |||
| P. aeruginosa UCBPP-PA14, | |||
| P. entomophila L48, | |||
| P. fluorescens Pf-5, | |||
| P. fluorescens Pf0-1, | |||
| P. fluorescens SBW25, | |||
| P. mendocina ymp, | |||
| P. putida F1, | |||
| P. putida GB-1, | |||
| P. putida KT2440, | |||
| P. putida W619, | |||
| P. syringae pv. tomato str. DC3000 | |||
| 2 | Nucleotide sugar epimerasedehydratase WbpM | P. aeruginosa PAO1, | 207 |
| P. aeruginosa PA7, | |||
| P. aeruginosa LESB58, | |||
| P. aeruginosa UCBPP-PA14, | |||
| P. entomophila L48, | |||
| P. fluorescens Pf-5, | |||
| P. fluorescens Pf0-1, | |||
| P. fluorescens SBW25, | |||
| P. mendocina ymp, | |||
| P. putida F1, | |||
| P. putida GB-1, | |||
| P. putida KT2440, | |||
| P. putida W619, | |||
| P. stutzeri A1501, | |||
| P. syringae pv. phaseolicola 1448A, | |||
| P. syringae pv. tomato str. DC3000 | |||
| 3 | LPS biosynthesis protein WbpG | P. aeruginosa PAO1, | 180 |
| P. aeruginosa PA7, | |||
| P. aeruginosa LESB58, | |||
| P. aeruginosa UCBPP-PA14, | |||
| P. entomophila L48, | |||
| P. fluorescens Pf-5, | |||
| P. fluorescens Pf0-1, | |||
| P. fluorescens SBW25, | |||
| P. mendocina ymp, | |||
| P. putida F1, | |||
| P. putida GB-1, | |||
| P. putida KT2440, | |||
| P. putida W619, | |||
| P. syringae pv. tomato str. DC3000 | |||
| 4 | Imidazole glycerol phosphate synthase subunit HisF2 | P. aeruginosa PAO1, | 215 |
| P. aeruginosa PA7, | |||
| P. aeruginosa LESB58, | |||
| P. aeruginosa UCBPP-PA14, | |||
| P. entomophila L48, | |||
| P. fluorescens Pf-5, | |||
| P. fluorescens Pf0-1, | |||
| P. fluorescens SBW25, | |||
| P. mendocina ymp, | |||
| P. putida F1, | |||
| P. putida GB-1, | |||
| P. putida KT2440, | |||
| P. putida W619, | |||
| P. syringae pv. tomato str. DC3000 | |||
| 5 | UDP-2-acetamido-2-deoxy-d-glucuronic acid 3-dehydrogenase WbpB | P. aeruginosa PAO1, | 199 |
| P. aeruginosa PA7, | |||
| P. aeruginosa LESB58, | |||
| P. aeruginosa UCBPP-PA14, | |||
| P. entomophila L48, | |||
| P. fluorescens Pf-5, | |||
| P. fluorescens Pf0-1, | |||
| P. fluorescens SBW25, | |||
| P. mendocina ymp, | |||
| P. putida F1, | |||
| P. putida GB-1, | |||
| P. putida KT2440, | |||
| P. putida W619, | |||
| P. syringae pv. tomato str. DC3000 | |||
| 6 | Chemotaxis-specific methylesterase | P. aeruginosa PAO1, | 230 |
| P. aeruginosa PA7, | |||
| P. aeruginosa LESB58, | |||
| P. aeruginosa UCBPP-PA14, | |||
| P. entomophila L48, | |||
| P. fluorescens Pf-5, | |||
| P. fluorescens Pf0-1, | |||
| P. fluorescens SBW25, | |||
| P. mendocina ymp, | |||
| P. putida F1, | |||
| P. putida GB-1, | |||
| P. putida KT2440, | |||
| P. putida W619, | |||
| P. stutzeri A1501, | |||
| P. syringae pv. tomato str. DC3000 | |||
| 7 | Phospho-N-acetylmuramoyl-pentapeptide- transferase | P. aeruginosa PAO1, | 235 |
| P. aeruginosa PA7, | |||
| P. aeruginosa LESB58, | |||
| P. aeruginosa UCBPP-PA14, | |||
| P. entomophila L48, | |||
| P. fluorescens Pf-5, | |||
| P. fluorescens Pf0-1, | |||
| P. fluorescens SBW25, | |||
| P. mendocina ymp, | |||
| P. putida F1, | |||
| P. putida GB-1, | |||
| P. putida KT2440, | |||
| P. putida W619, | |||
| P. stutzeri A1501, | |||
| P. syringae pv. tomato str. DC3000 | |||
| 8 | 3-deoxy-D-manno-octulosonic-acid transferase | P. aeruginosa PAO1, | 206 |
| P. aeruginosa PA7, | |||
| P. aeruginosa LESB58, | |||
| P. aeruginosa UCBPP-PA14, | |||
| P. entomophila L48, | |||
| P. fluorescens Pf-5, | |||
| P. fluorescens Pf0-1, | |||
| P. fluorescens SBW25, | |||
| P. mendocina ymp, | |||
| P. putida F1, | |||
| P. putida GB-1, | |||
| P. putida KT2440, | |||
| P. putida W619, | |||
| P. stutzeri A1501, | |||
| P. syringae pv. tomato str. DC3000 | |||
| 9 | Lipopolysaccharide kinase WaaP | P. aeruginosa PAO1, | 167 |
| P. aeruginosa PA7, | |||
| P. aeruginosa LESB58, | |||
| P. aeruginosa UCBPP-PA14, | |||
| P. entomophila L48, | |||
| P. fluorescens Pf-5, | |||
| P. fluorescens Pf0-1, | |||
| P. fluorescens SBW25, | |||
| P. mendocina ymp, | |||
| P. putida F1, | |||
| P. putida GB-1, | |||
| P. putida KT2440, | |||
| P. putida W619, | |||
| P. stutzeri A1501, | |||
| P. syringae pv. tomato str. DC3000 | |||
| 10 | UDP-glucose:(heptosyl) LPS alpha 1,3-glucosyltransferase WaaG | P. aeruginosa PAO1, | 200 |
| P. aeruginosa PA7, | |||
| P. aeruginosa LESB58, | |||
| P. aeruginosa UCBPP-PA14, | |||
| P. entomophila L48, | |||
| P. fluorescens Pf-5, | |||
| P. fluorescens Pf0-1, | |||
| P. fluorescens SBW25, | |||
| P. mendocina ymp, | |||
| P. putida F1, | |||
| P. putida GB-1, | |||
| P. putida KT2440, | |||
| P. putida W619, | |||
| P. stutzeri A1501, | |||
| P. syringae pv. tomato str. DC3000 | |||
| 11 | Tetraacyldisaccharide 4′-kinase | P. aeruginosa PAO1, | 205 |
| P. aeruginosa PA7, | |||
| P. aeruginosa LESB58, | |||
| P. aeruginosa UCBPP-PA14, | |||
| P. entomophila L48, | |||
| P. fluorescens Pf-5, | |||
| P. fluorescens Pf0-1, | |||
| P. fluorescens SBW25, | |||
| P. mendocina ymp, | |||
| P. putida F1, | |||
| P. putida GB-1, | |||
| P. putida KT2440, | |||
| P. putida W619, | |||
| P. stutzeri A1501, | |||
| P. syringae pv. tomato str. DC3000 | |||
| 12 | Tetrahydrodipicolinate succinylase | P. aeruginosa PAO1, | 183 |
| P. aeruginosa PA7, | |||
| P. aeruginosa LESB58, | |||
| P. aeruginosa UCBPP-PA14, | |||
| P. entomophila L48, | |||
| P. fluorescens Pf-5, | |||
| P. fluorescens Pf0-1, | |||
| P. fluorescens SBW25, | |||
| P. mendocina ymp, | |||
| P. putida F1, | |||
| P. putida GB-1, | |||
| P. putida KT2440, | |||
| P. putida W619, | |||
| P. stutzeri A1501, | |||
| P. syringae pv. tomato str. DC3000 | |||
| 13 | NalC protein | P. aeruginosa PAO1, | 200 |
| P. aeruginosa PA7, | |||
| P. aeruginosa LESB58, | |||
| P. aeruginosa UCBPP-PA14, | |||
| P. entomophila L48, | |||
| P. fluorescens Pf-5, | |||
| P. fluorescens Pf0-1, | |||
| P. fluorescens SBW25, | |||
| P. mendocina ymp, | |||
| P. putida F1, | |||
| P. putida GB-1, | |||
| P. putida KT2440, | |||
| P. putida W619, | |||
| P. stutzeri A1501, | |||
| P. syringae pv. tomato str. DC3000 | |||
| 14 | Preprotein translocase subunit SecD | P. aeruginosa PAO1, | 231 |
| P. aeruginosa PA7, | |||
| P. aeruginosa LESB58, | |||
| P. aeruginosa UCBPP-PA14, | |||
| P. entomophila L48, | |||
| P. fluorescens Pf-5, | |||
| P. fluorescens Pf0-1, | |||
| P. fluorescens SBW25, | |||
| P. mendocina ymp, | |||
| P. putida F1, | |||
| P. putida GB-1, | |||
| P. putida KT2440, | |||
| P. putida W619, | |||
| P. stutzeri A1501, | |||
| P. syringae pv. tomato str. DC3000 |
3.7. Protein physicochemical property analysis
To enhance the prioritization process, we assessed various factors that aid in identifying pharmacological targets from a pool of 14 proteins. These factors included lower molecular weight, a reduced isoelectric point, and increased hydrophobicity, which suggests lower polarity. The proteins exhibited isoelectric points (pI) ranging from 5.56 to less than 11.42, which are dependent on the pH of their amino acids. Most of the proteins had instability indices exceeding 40, indicating their likely instability, as a value below 40 generally signifies stability. The Grand Average Hydropathicity (GRAVY) scores were mostly negative, suggesting that the proteins are hydrophilic in their natural state. Furthermore, the proteins displayed an Aliphatic Index (Ai) greater than 78, pointing to their stability across a broad temperature range. The parameters were set with specific cut-offs: molecular weight below 100 kDa, pI around 7.2, instability index under 40, Ai between 78 and 108, and hydrophobicity greater than −0.117. Out of the 14 proteins, only 6 met the physicochemical criteria, while the remaining 8 were disqualified due to instability (Table 10).
Table 10.
Physico-chemical properties of Broad-spectrum Proteins.
| S.NO | Protein Name | Molecular weight in Da | Theoretical pI | Instability index | Aliphatic index | GRAVY | Number of amino acids |
|---|---|---|---|---|---|---|---|
| 1 | HxcU pseudopilin | 16437.06 | 11.42 | 37.42 stable | 102.21 | −0.117 | 149 |
| 2 | Nucleotide sugar epimerasedehydratase WbpM | 74415.82 | 9.33 | 42.22 unstable | 108.60 | 0.096 | 665 |
| 3 | LPS biosynthesis protein WbpG | 43503.78 | 8.28 | 44.00 unstable | 78.67 | −0.368 | 377 |
| 4 | Imidazole glycerol phosphate synthase subunit HisF | 27448.65 | 5.62 | 29.72 stable | 104.06 | 0.082 | 251 |
| 5 | UDP-2-acetamido-2-deoxy-d-glucuronic acid 3-dehydrogenase WbpB | 35717.51 | 6.11 | 40.03 unstable | 85.82 | −0.266 | 316 |
| 6 | Chemotaxis-specific methylesterase | 35697.39 | 6.13 | 30.17 stable | 105.46 | 0.223 | 335 |
| 7 | Phospho-N-acetylmuramoyl-pentapeptide- transferase | 39653.46 | 9.51 | 27.49 stable | 128.31 | 0.823 | 360 |
| 8 | 3-deoxy-D-manno-octulosonic-acid transferase | 46486.98 | 9.01 | 42.40 unstable | 104.82 | 0.102 | 425 |
| 9 | Lipopolysaccharide kinase WaaP | 31311.07 | 9.87 | 42.91 unstable | 87.39 | −0.553 | 268 |
| 10 | UDP-glucose:(heptosyl) LPS alpha 1,3-glucosyltransferase WaaG | 42156.35 | 7.10 | 53.16 unstable | 95.55 | −0.199 | 373 |
| 11 | Tetraacyldisaccharide 4′-kinase | 36746.29 | 6.77 | 46.09 unstable | 95.96 | −0.119 | 332 |
| 12 | Tetrahydrodipicolinate succinylase | 35973.29 | 5.74 | 33.99 stable | 106.13 | 0.208 | 344 |
| 13 | NalC protein | 23532.93 | 5.56 | 48.15 unstable | 89.01 | −0.065 | 213 |
| 14 | Preprotein translocase subunit SecD | 67674.72 | 8.88 | 34.17 stable | 107.76 | 0.126 | 620 |
3.8. Functional annotation and evolutionary analysis
3.8.1. Domain analysis
Functional and evolutionary patterns of the shortlisted proteins were identified using several databases, including CDD, InterPro, Pfam, SCOP-Superfamily, and Motif. Each analysis was conducted using the default settings, and Table 11 provides a detailed breakdown of the results. These resources offered significant insights and were crucial in the subsequent downstream analysis. The proteins listed in Table 11 were analyzed for functional domains across multiple databases, revealing key insights into their biological roles and evolutionary history. HxcU pseudopilin and Chemotaxis-specific methylesterase are involved in the Type II secretion system, associated with pili formation and chemotaxis, sharing common structural motifs related to methylation and secretion. Imidazole glycerol phosphate synthase subunit HisF plays a critical role in histidine biosynthesis, exhibiting multiple conserved domains, including binding motifs for phosphate and ribulose-phosphate. Phospho-N-acetylmuramoyl-pentapeptide transferase and Tetrahydrodipicolinate succinylase are involved in glycosyl transferase and enzymatic functions essential for bacterial cell wall synthesis, characterized by distinctive motifs in their sequences. Finally, Preprotein translocase subunit SecD is a key component in protein export and membrane protein translocation, displaying significant conserved regions related to protein translocase functionality.
Table 11.
Functional annotation of proteins.
| S.NO | Protein Name | Conserved domain database | InterPro | Pfam | SCOP-Superfamily | MOTIF |
|---|---|---|---|---|---|---|
| 1 | HxcU pseudopilin | Type II secretion system protein H | Type II secretion system protein GspH | Type II transport protein GspH | Pili subunits | Prokaryotic N-terminal methylation motif (Position: 3–28) |
| Type II transport protein GspH (Position: 44–138) | ||||||
| 2 | Imidazole glycerol phosphate synthase subunit HisF | AglZ/HisF2 family acetamidino modification protein | Histidine biosynthesis protein | Phosphate binding site TIM barrel family | Ribulose-phoshate binding barrel | Histidine biosynthesis protein (Position: 5–232) |
| Dihydrouridine synthase (Dus) (Position:146–226) | ||||||
| Nitronate monooxygenase (Position: 186–215) | ||||||
| Dihydroorotate dehydrogenase (Position:187–237) | ||||||
| Ketopantoate hydroxymethyltransferase (Position:147–207) | ||||||
| SLOG in TRPM (Position:187–217) | ||||||
| 3 | Chemotaxis-specific methylesterase | Chemotaxis-specific protein-glutamate methyltransferase CheB | Protein-glutamate methylesterase/protein-glutamine glutaminase, CheB type | Response regulator | Methylesterase CheB, C-terminal domain/CheY-like | CheB methylesterase (Position:154–330) |
| Response regulator receiver domain (Position: 4–103) | ||||||
| Prolyl oligopeptidase family (Position: 150–188) | ||||||
| 4 | Phospho-N-acetylmuramoyl-pentapeptide- transferase | UDP-D-N-acetylhexosamine:polyprenol phosphate D-N-acetylhexosamine-1-phosphate transferases | Glycosyl transferase family 4 | Glycosyl transferase family 4 | Glycosyl transferase family 4 (Position:99–284) | |
| Phospho-N-acetylmuramoyl-pentapeptide-transferase signature 1 (Position: 68–80) | ||||||
| 5 | Tetrahydrodipicolinate succinylase | Tetrahydrodipicolinate N-succinyltransferase | Trimeric LpxA-like enzymes | Tetrahydrodipicolinate N-succinyltransferase middle (Position:132–172) | ||
| Hexapeptide repeat of succinyl-transferase (Position:254–286) | ||||||
| Tetrahydrodipicolinate N-succinyltransferase N-terminal (Position:78–129) | ||||||
| Bacterial transferase hexapeptide (six repeats) (Position:256–282) | ||||||
| Prophage tail length tape measure protein (Position:48–108) | ||||||
| 6 | Preprotein translocase subunit SecD | Preprotein translocase subunit SecD, SecD export protein N-terminal TM region | SecD export protein N-terminal TM region (Position: 2–104) | |||
| Protein translocase subunit SecDF, P1 domain, N-terminal (Position:230–287) | ||||||
| Protein export membrane protein (Position: 443–606) | ||||||
| MMPL family (Position:481–607) | ||||||
| SecD/SecF GG Motif (Position:117–141) | ||||||
| AcrB/AcrD/AcrF family (Position:472–610) |
3.8.2. Phylogenetic analysis
The evolutionary relationships of the selected proteins, including HxcU pseudopilin, Imidazole glycerol phosphate synthase subunit HisF, Chemotaxis-specific methylesterase, Phospho-N-acetylmuramoyl-pentapeptide transferase, Tetrahydrodipicolinate succinylase, and Preprotein translocase subunit SecD, were grouped into three distinct categories: RED (Group 1), PINK (Group 2), and BLUE (Group 3). The phylogenetic analysis revealed two primary clades: Clade 1 (RED Group) includes HxcU pseudopilin and Chemotaxis-specific methylesterase, which are closely related with moderate bootstrap support of 0.66, suggesting a relatively strong evolutionary connection. This group is further linked to Preprotein translocase subunit SecD, expanding the clade. Clade 2 (PINK Group) consists of Imidazole glycerol phosphate synthase subunit HisF and Tetrahydrodipicolinate succinylase, which show a weaker evolutionary connection with a low bootstrap support of 0.31, indicating less confidence in their relationship. This clade is connected to Phospho-N-acetylmuramoyl-pentapeptide transferase (BLUE Group), forming another distinct group. The RED group demonstrates a relatively stronger evolutionary relationship, while the PINK group shows a more distant evolutionary connection. The BLUE group (Phospho-N-acetylmuramoyl-pentapeptide transferase) appears to be evolutionarily distinct, suggesting a separate trajectory from the other proteins (Fig. 3).
Fig. 3.
Visualization of the phylogenetic tree of six proteins. The RED (HxcU pseudopilin - Q9I5P7, and Chemotaxis-specific methylesterase - Q9HXT8), PINK (Imidazole glycerol phosphate synthase subunit HisF- P72139, and Tetrahydrodipicolinate succinylase - G3XD76), and BLUE (Phospho-N-acetylmuramoyl-pentapeptide transferase – Q9HVZ8).
3.9. Structural prediction and validation
3.9.1. Secondary structure prediction
The secondary structure of the proteins was predicted using PSIPRED and SOPMA. According to the SOPMA analysis, the random coil was the most common structure found in HxcU pseudopilin (40.94 %), Imidazole glycerol phosphate synthase subunit HisF2 (33.07 %), Chemotaxis-specific methylesterase (36.42 %), and tetrahydrodipicolinate succinylase (34.30 %). On the other hand, the alpha helix was the predominant structure in Phospho-N-acetylmuramoyl-pentapeptide transferase (46.67 %) and Preprotein translocase subunit SecD (47.26 %). The secondary structure predictions made by PSIPRED are shown in Fig. 4.1 and 4.2.
Fig. 4.1.
Secondary structure of essential non-homologous proteins
Fig. 4.2 secondary structure of essential non-homologous proteins.
3.9.2. Tertiary structure prediction and druggability assessment
Among the six proteins shortlisted, only one had an experimentally determined 3D structure, while the remaining five had their 3D structure models predicted using the Alphafold protein structure database. To evaluate the quality of these models, we used the Ramachandran plot, which showed that over 90 % of the residues were in the most favorable regions, confirming the reliability and accuracy of the 3D structures (Fig. 5). Subsequently, binding sites were identified using the SiteMap tool in Schrodinger-suite 2023 software. The Site and D scores were analyzed to determine the druggability of the proteins. Proteins with Site and D scores below 0.8 were considered undruggable due to the absence of suitable ligand binding sites, while those with scores above 0.8 were deemed druggable. Four of the proteins were found to be druggable (Table 12), including Phospho-N-acetylmuramoyl-pentapeptide transferase (MraY) from PAO1 and PA14 strains of P. aeruginosa, which was identified as a potential target in our literature review [56,57]. Furthermore, we suggest that three proteins—Preprotein translocase subunit SecD, Imidazole glycerol phosphate synthase subunit HisF2, and Chemotaxis-specific methyl esterase—are present in all virulent Pseudomonas species, making them suitable as pan-drug targets. These findings indicate that these proteins could serve as promising drug targets for combating multidrug resistance and virulence in various virulent Pseudomonas species.
Fig. 5.
Analysis of 3D structures of novel proteins obtained from AlphaFold Protein Structure database and Protein Data Bank, along with Ramachandran plots proving the validity of the structures.
Table 12.
Structural evaluation of identified targets in P. aeruginosa.
| S.NO | Name of the Target protein | Uniprot ID | Druggable Site Identification |
|||
|---|---|---|---|---|---|---|
| Site Score | D-score | Druggable target | Binding site residues | |||
| 1 | HxcU pseudopilin | Q9I5P7 | 0.840 | 0.667 | No | – |
| 2 | Imidazole glycerol phosphate synthase subunit HisF2 | P72139 | 1.185 | 0.887 | Yes | ARG176, ASP177, GLY178, VAL179, GLN180, PHE183, CYS202, GLY203, GLY204, ALA205, ALA224, ALA225, GLY226, SER227, LEU228 |
| 3 | Chemotaxis-specific methylesterase | Q9HXT8 | 0.993 | 0.922 | Yes | ILE83, ASP100, ALA101, VAL102, ASN103, PRO118, ARG121, LYS122, ASN125, HIS186, VAL187, ASP188, VAL190, PHE191, ASN227, ARG245, PHE247, VAL248, TYR249, ARG250, PRO251 |
| 4 | Phospho-N-acetylmuramoyl-pentapeptide- transferase | Q9HVZ8 | 1.124 | 1.237 | Yes | LEU179, PHE182, VAL183, VAL185, GLY186, SER187, ASN189, ALA190, VAL191, LEU193, GLY274, LEU |
| 5 | Tetrahydrodipicolinate succinylase | G3XD76 | 0.715 | 0.702 | No | – |
| 6 | Preprotein translocase subunit SecD | Q9HXI1 | 1.024 | 1.056 | Yes | PHE21, SER24, ALA25, ASN27, LEU28, PRO30, ASP31, PRO33, GLN36, SER38, GLY39, ALA40, SER41, THR42, LEU44, GLN45, VAL46, LEU70, SER71, LYS72, LYS73, GLY74, LEU76, GLN82, GLN85, LYS89, ARG93, ASP98, ASP99, TYR100, VAL101, VAL102, ALA103, LEU104, ASN105, LEU106, ALA107, GLN108, THR110, ARG115, GLY118, GLY119, SER120, PRO121, MET122, LEU123, LEU124, GLY125, LEU126, ASP127, LEU128, SER129, VAL132, HIS133, LEU135, GLU243, LEU244, GLY245, VAL246, SER247, GLU248, PRO249, LEU250, GLN254, VAL260, GLU262, PRO264, GLY265, VAL266, GLN267, ASP268, GLU271, ALA272, ARG274, ILE275, SER327, ALA328, SER329, PHE330, PRO420, GLY421, SER423, SER424, GLU425, ALA427, LEU428, ARG431, GLU443, ARG445, THR446, ILE447, PRO449, GLY452, ALA453, ILE456, GLY459, ILE460, SER463, MET493, VAL497, SER501, ILE502, GLY504, ALA505, THR506, LEU507, THR508, ILE512, THR515, TYR573, THR577, GLY578, PRO579, LYS581, GLY582, VAL585 |
3.9.2.1. Significance of identified drug targets
-
i.
Imidazole glycerol phosphate synthase subunit HisF2: HisF2 is essential in P. aeruginosa's histidine biosynthesis pathway, catalyzing the conversion of histidine phosphate to histidine, a critical step for histidine production [58]. This capability enables the bacterium to grow in low-histidine environments, which supports its survival and virulence [59]. Histidine is vital for protein synthesis and numerous metabolic functions, so disrupting its biosynthesis could significantly impair bacterial growth and pathogenicity [60]. Targeting HisF2 may offer a therapeutic approach to inhibit histidine production, thereby weakening P. aeruginosa's ability to survive in nutrient-limited conditions and potentially reducing its antibiotic resistance [61]. Histidine synthesis is regulated by his genes and various mechanisms, including transcription factors, RNA-based systems, and feedback loops, highlighting its roles in protein production, cellular metabolism, and nitrogen and purine synthesis [62]. Our research identified the Imidazole glycerol phosphate synthase subunit HisF2 as an LPS-linked virulence factor involved in immune modulation and inflammatory signaling, as confirmed in the VFDB database. LPS-related virulence elements, including HisF2, are vital in conferring resistance to immune responses such as serum killing and phagocytosis. Furthermore, HisF2 may bind to the normal cystic fibrosis transmembrane conductance regulator (CFTR), aiding bacterial invasion of host cells, which could contribute to ocular infections in humans. This process entails CFTR-mediated internalization by airway epithelial cells, followed by the shedding infected cells as a defense mechanism. These findings underscore HisF2's role in facilitating Pseudomonas infection (https://www.mgc.ac.cn/VFs/main.htm).
-
ii.
Chemotaxis-specific methylesterase: Chemotaxis, a type of cellular motility predominantly observed in prokaryotes, is directed by chemical gradients composed of attractants or repellents. This mechanism is crucial during the early stages of infection, where identifying the main chemo effectors and corresponding chemoreceptors could pave the way for interventions to block bacterial migration. Genomic studies indicate that numerous bacteria, including P. aeruginosa, harbor an extensive array of chemoreceptors [63]. These organisms are inherently equipped to detect and respond to complex chemical gradients in their environment [64,65], mediated by a two-component system (TCS) that includes a sensor histidine kinase (HK) and a response regulator (RR), a common signaling module in bacteria and archaea [66,67]. Chemotaxis is closely linked to virulence traits such as flagella, type IV pili, and alginate production, as noted in the VFDB database. Flagella, classified under motility and assembly, facilitate swimming motility, biofilm formation, and other pathogenic adaptations. Type IV pili, categorized as adherence factors, enable bacterial attachment to host cells (but not to mucin) and induce twitching motility, allowing movement along cell surfaces and biofilm formation. Alginate, involved in biofilm formation, enhances bacterial persistence in the cystic fibrosis lung by acting as an adhesin, preventing bacterial expulsion, and creating a protective slime layer that inhibits phagocytosis. Chemotaxis-related methylesterases are also associated with resistance genes like PvrR, CpxR, ParR, PhoP, and multiple TEM alleles (TEM-147, TEM-205, TEM-247, TEM-234, TEM-241, TEM-213, TEM-2), conferring multidrug resistance to various antimicrobial classes, including aminoglycosides, penams, tetracyclines, peptides, diaminopyrimidines, sulfonamides, aminocoumarins, fluoroquinolones, cephalosporins, carbapenems, macrolides, monobactams, phenicols, cephamycins, penems, as well as disinfectants and antiseptics (https://card.mcmaster.ca/).
-
iii.
Preprotein translocase subunit SecD: The secretion systems of bacteria function as sophisticated nanomachines that enable the export of proteins from the bacterial cytosol to specific external environments, playing a critical role in immune evasion through the secretion of effectors [68]. Targeting these virulence mechanisms may enhance pathogen clearance by the host's immune system. Within this context, SecD is integral to the protein secretion system of P. aeruginosa, significantly influencing both antimicrobial resistance and virulence [69,70]. As a key component of the Sec machinery, SecD facilitates the translocation of various virulence factors, including toxins and enzymes, across the inner membrane, essential for evading host immune responses and establishing infections [71]. This efficient secretion system enhances the bacterium's ability to form biofilmsbut also protects bacterial cells from antimicrobial agents, contributing to antimicrobial resistance and persistent infections [72]. Moreover, SecD is involved in mechanisms conferring antibiotic resistance by enabling the export of proteins that can degrade or modify their targets, thereby reinforcing P. aeruginosa's notorious multi-drug resistance profile [73]. Overall, SecD's pivotal role in both virulence factor secretion and antibiotic resistance mechanisms highlights its significance in the pathogenicity of P. aeruginosa, suggesting that targeting the secretion system could be a promising strategy to combat bacterial infections without directly selecting for resistant mutations. Our investigation revealed that HisF2 shares similarities with several resistance-associated genes in P aeruginosa, including MuxC, MexQ, MexK, MexY, MexF, MexD, and MexB. These genes contribute to resistance against a diverse range of antimicrobial agents such as macrolides, monobactams, aminocoumarins, tetracyclines, phenicols, diaminopyrimidines, carbapenems, disinfectants, antiseptics, aminoglycosides, fluoroquinolones, cephamycins, cephalosporins, penams, peptide antibiotics, sulfonamides, and penems (https://card.mcmaster.ca/). This similarity strongly suggests that HisF2 may play a significant role in drug resistance.
The identified three targets were conserved in all the virulent species of Pseudomonas, i.e. P. aeruginosa PAO1, P. aeruginosa PA7, P. aeruginosa LESB58, P. aeruginosa UCBPP-PA14, P. fluorescens Pf0-1, P. mendocina ymp, P. putida F1, P. putida GB-1, P. putida KT2440, and P. putida W619 which facilitates their use as pan drug targets for Pseudomonads infection.
3.10. Virtual screening
Receptor-based virtual screening was conducted on three target proteins—Imidazole glycerol phosphate synthase subunit HisF2, Chemotaxis-specific methylesterase, and Preprotein translocase subunit SecD—using a ligand dataset containing 4,648,867 molecules. For each protein, the top five hits were identified through molecular docking analysis. The top hits for Imidazole glycerol phosphate synthase subunit HisF2 exhibited binding affinities ranging from −11.460 to −3.854 kcal/mol and glide e-model scores between −67.136 and −36.635 kcal/mol. For Chemotaxis-specific methylesterase, the top hits had binding affinities from −8.843 to −7.894 kcal/mol and glide e-model scores ranging from −70.918 to −78.428 kcal/mol. The Preprotein translocase subunit SecD docking analysis revealed top hits with binding scores between −8.706 and −8.100 kcal/mol and glide model scores from −65.651 to −80.481 kcal/mol. The top docked complexes of Imidazole glycerol phosphate synthase subunit HisF2 showed hydrogen bonds and salt bridge interactions (Fig. 6). The docked complexes of Chemotaxis-specific methylesterase interacted with ARG121, ASP188, and ARG250, with additional salt bridge interactions with THR104 and ASN145 (Fig. 7). For SecD, the docked complexes formed halogen bonds, hydrogen bonds, and salt bridge interactions with SER247, LEU104, and LYS89 (Fig. 8). Ciprofloxacin was used as the control drug for all target proteins (Table 13)
Fig. 6.
Protein-Ligand interaction diagrams of Top 5 hits and a control drug obtained from virtual screening of compound library with Imidazole glycerol phosphate synthase subunit HisF2.
Fig. 7.
Protein-Ligand interaction diagrams of Top 5 hits and a control drug obtained from virtual screening of compound library with Chemotaxis-specific methylesterase.
Fig. 8.
Protein-Ligand interaction diagrams of Top 5 hits and a control drug obtained from virtual screening of compound library with of Preprotein translocase subunit SecD.
Table 13.
Docking-based Inverse virtual screening of VITAS-M small molecule library against the identified targets.
| S.NO | Compound ID | Compound | Docking Score (kcal/mol) | Glide Emodel (kcal/mol) | Protein-ligand Interactions |
|---|---|---|---|---|---|
| Imidazole glycerol phosphate synthase subunit HisF2 | |||||
| 1 | HIT 1 | STL146296 2,2,3,3-tetrahydroxy-2,3-dihydronaphthalene-1,4-dione |
−11.46 | −67.136 | 2XH-bond with THR104 |
| 2XH-bond with ASN145 | |||||
| HIT 2 | STL346340 6-(1H-indol-3-yl)-1,3,5-triazinane-2,4-dione |
−10.178 | −60.993 | 2X H-bond with GLY81 | |
| H-bond with ILE83 | |||||
| H-bond with THR104 | |||||
| H-bond with ALA105 | |||||
| HIT 3 | STL490336 1-[(3-bromopropanoyl)oxy]pyrrolidine-2,5-dione |
−7.69 | −40.03 | H-bond with GLY81 | |
| H-bond with ASN103 | |||||
| H-bond with ALA105 | |||||
STK199996 3-methyl-4-(2-(4-(2-nitro-4-(trifluoromethyl)phenyl)piperazin-1-yl)ethoxy)-1,2,5-oxadiazole |
−7.657 | −67.972 | H-bond with ASN145 | ||
| HIT 4 | 2X Salt bridge with ASP130 | ||||
| HIT 5 | STK417467 N-(pyrimidin-2-yl)tetrahydrofuran-2-carboxamide |
−3.854 | −36.635 | H-bond with ASN145 | |
| 2X Salt bridge with ASP130 | |||||
| Control |
 Ciprofloxacin |
−0.413 | −27.141 | H-bond with SER101 | |
| Chemotaxis-specific methylesterase | |||||
| 2 | HIT 1 | STL321942 1,4-dihydroxyoctahydroquinoxaline-2,3-dione |
−8.843 | −70.918 | Salt bridge with ASP100 |
| Salt bridge with ARG121 | |||||
| Salt bridge with LYS122 | |||||
| H-bond with ASN125 | |||||
| 2X Salt bridge with ASP188 | |||||
| H-bond with ARG250 | |||||
| HIT 2 | STL190843 2-[2-(1H-pyrrol-1-yl)-1,3-thiazol-5-yl]ethanol |
−8.695 | −42.239 | Salt bridge with ARG121 | |
| Salt bridge with ASP188 | |||||
| Salt bridge with ARG250 | |||||
| HIT 3 | STL069540 2,3-bis(hydroxymethyl)-1-oxoquinoxalin-1-ium-4(1H)-olate |
−8.477 | −72.743 | Salt bridge with ASP100 | |
| Salt bridge with LYS122 | |||||
| H-bond with ASN125 | |||||
| 2XSalt bridge with ASP188 | |||||
| H-bond with ARG250 | |||||
| HIT 4 | STL513090 2-(1-oxidopyridin-2-yl)ethanol |
−7.978 | −43.743 | Salt bridge with ASP100 | |
| Salt bridge with ARG121 | |||||
| Salt bridge with LYS122 | |||||
| Salt bridge with ASN125 | |||||
| Salt bridge with ASP188 | |||||
| HIT 5 | STL321396 5,5a,6a,7,8,9,10,10a-octahydro-4H- [1,2,5]oxadiazolo [3,4-c]carbazole-6,10b-diol3-oxide |
−7.894 | −78.428 | Salt bridge with ASP100 | |
| 2XSalt bridge with ARG121 | |||||
| Salt bridge with LYS122 | |||||
| H-bond with ASN125 | |||||
| 2XSalt bridge with ASP188 | |||||
| 2X H-bond with ARG250 | |||||
| Control |
 Ciprofloxacin |
−3.757 | −34.021 | H-bond with ALA106 | |
| H-bond with ASP188 | |||||
| Salt bridge with ASP188 | |||||
| Preprotein translocase subunit SecD | |||||
| 3 | HIT 1 | STK394282 4-/(E)-(4-amino-1,2,5-oxadiazol-3-yl)-NNO-azoxy/-1,2,5-oxadiazol-3-amine |
−8.706 | −65.651 | – |
| HIT 2 | STK232493 1-methylbicyclo[2.2.0]hex-2-yl {[6-amino-4-oxo-1-(prop-2-en-1-yl)-1,4-dihydropyrimidin-2-yl]sulfanyl}acetate |
−8.265 | −79.677 | H-bond with GLU271 | |
| H-bond with SER247 | |||||
| H-bond with ASP268 | |||||
| H-bond with GLN267 | |||||
| HIT 3 | STL243336 4-(1,2-benzothiazol-3-yl)-N-[2-oxo-2-(thiomorpholin-4-yl)ethyl]piperazine-1-carboxamide |
−8.204 | −70.908 | Salt bridge with LYS89 | |
| H-bond with LEU104 | |||||
| H-bond with SER247 | |||||
| HIT 4 | STK205602 2-oxo-2-(piperidin-1-yl)ethyl pyrrolidine-1-carbodithioate |
−8.193 | −72.579 | H-bond with LEU104 | |
| HIT 5 | STK368220 4-[(2-chloroethyl)amino]-N-methyl-1,2,5-oxadiazole-3-carboxamide |
−8.100 | −80.481 | Halogen bond with LYS89 | |
| H-bond with ASN105 | |||||
| H-bond with SER247 | |||||
| Control |
 Ciprofloxacin |
−5.611 | −45.968 | H-bond with LEU104 | |
| H-bond with PRO121 | |||||
| H-bond with LEU124 | |||||
3.11. Druggability analysis of hit compounds
The pharmacokinetic properties of the hit molecules were evaluated using the QikProp module of Schrodinger 2023. Most of the molecules were found to fall within the acceptable ranges, with a few exceptions. The detailed results are shown in Table 14.
Table 14.
Druggability analysis of hit compounds.
| S.No | Compound ID | mol MW | donorHB | accptHB | QPlogPo/w | QPlogS | QPlogHERG | QPPCaco | Percent Human Oral Absorption |
|---|---|---|---|---|---|---|---|---|---|
| 1 | STL146296 | 224.170 | 4 | 7 | −0.971 | −1.297 | −3.551 | 56.312 | 52.590 |
| 2 | STL346340 | 222.162 | 0.5 | 3.5 | 0.157 | −1.22 | −3.356 | 378.509 | 74.009 |
| 3 | STL490336 | 241.985 | 0 | 6 | −1.117 | 1.451 | −2.196 | 1156.739 | 75.23 |
| 4 | STK199996 | 289.209 | 2 | 4 | −0.471 | −0.664 | −3.866 | 22.466 | 48.375 |
| 5 | STK417467 | 235.158 | 0 | 6.5 | −1.337 | 0.465 | −2.782 | 253.357 | 62.141 |
| 6 | STL321942 | 188.099 | 0 | 3.5 | −0.547 | 0.235 | −2.535 | 452.933 | 71.28 |
| 7 | STL190843 | 194.251 | 1 | 2.2 | 0.479 | 0.629 | 3.000 | 2661.414 | 91.053 |
| 8 | STL069540 | 212.121 | 0 | 6 | −1.786 | 1.047 | −3.075 | 158.449 | 55.86 |
| 9 | STL513090 | 131.09 | 1 | 1.75 | 1.368 | −0.779 | −3.821 | 643.144 | 85.217 |
| 10 | STL321396 | 251.158 | 1 | 4.75 | 0.408 | −2.14 | −4.197 | 117.872 | 66.406 |
| 11 | STK394282 | 391.57 | 0 | 2 | 2.832 | −3.906 | −3.487 | 1978.84 | 100 |
| 12 | STK232493 | 487.288 | 0 | 4 | 3.226 | −3.923 | −4.6 | 2442.388 | 100 |
| 13 | STL243336 | 383.358 | 1 | 2.25 | 2.95 | −4.255 | −4.1 | 985.084 | 100 |
| 14 | STK205602 | 348.276 | 0 | 2.75 | 3.233 | −4.072 | −5.47 | 1851.434 | 100 |
| 15 | STK368220 | 350.249 | 2 | 6.5 | 0.157 | −2.431 | −4.184 | 93.229 | 63.116 |
| 16 | Control (Ciprofloxacin) | 331.346 | 5 | 3.75 | 1.063 | −3.253 | −4.556 | 752.718 | 71.697 |
3.12. Molecular dynamics simulation
A 100 ns molecular dynamics simulation was performed on selected hits to further examine the binding stability of the docked complexes.
-
i.
RMSD Analysis
RMSDs were analyzed to evaluate the equilibrium of the MD trajectories and assess the stability of the protein-ligand complex systems.
-
a)
RMSD analysis of Imidazole glycerol phosphate synthase subunit HisF2 docked complexes
The Imidazole glycerol phosphate synthase subunit HisF2 protein's unbound RMSD was determined to be 1.8 Å. When complexed with hit1, the ligand's RMSD varied from 1.2 to 7 Å, showing fluctuations over time due to instability. In the case of the protein-hit2 complex, a stable interaction was observed, with the ligand's RMSD ranging from 1.5 to 2.4 Å, demonstrating good convergence during the simulation. The simulation results indicate that the HisF2 protein-hit3 complex is unstable, as reflected by a wide range in the ligand's RMSD from 0.5 to 7.0 Å. For the hit4 complex, the ligand RMSD ranged between 0.6 and 5.6 Å, with the ligand protruding from the binding pocket after 40 ns, suggesting low stability. The ligand RMSD in the hit5 complex was observed to range from 1.25 to 3.0 Å, with convergence and stability detected in the final 20 ns of the simulation. When the control drug was complexed with the protein, it initially deviated from the binding pocket for 40 ns before stabilizing, with the ligand maintaining an RMSD between 1.5 and 4.0 Å (Fig. 9).
-
b)
RMSD analysis of Chemotaxis-specific methylesterase docked complexes
Fig. 9.
RMSD plots obtained from Molecular dynamics simulation of top hits. The Superimposed RMSD graph spectrum of the unbound Imidazole glycerol phosphate synthase subunit HisF2 protein, the Control (Ciprofloxacin) and the 5 promising compounds Hit 1 (STL146296), Hit 2 (STL346340), HIT 3 (STL490336), Hit 4 (STK199996), Hit 5 (STK417467) in complex with Imidazole glycerol phosphate synthase subunit HisF2 protein.
The Chemotaxis-specific methylesterase protein's unbound RMSD was determined to be 1.7 Å. In the presence of hit1, the ligand's RMSD ranged from 1.7 to 3.0 Å, and the simulation over 100 ns showed convergence between the ligand and protein, indicating binding stability. When hit2 was bound, the simulation showed an unstable complex, with the hit2 RMSD fluctuating between 0.75 and 7.5 Å. The hit3-Chemotaxis-specific methylesterase protein complex demonstrated stability over the 100 ns simulation, with the ligand's RMSD remaining below 3.0 Å. In contrast, with hit4, the ligand RMSD ranged widely from 1.25 to 56 Å, suggesting that the ligand likely diffused out of the binding pocket, as its RMSD continued to vary up to 50 ns. For the hit5 complex, stability was observed due to the convergence of the protein and ligand RMSD plots, with the ligand RMSD between 1.5 and 4.0 Å. The control drug exhibited instability at the binding site, with the ligand RMSD fluctuating from 0.25 to 8 Å until around 50 ns (Fig. 10).
-
c)
RMSD analysis of Preprotein translocase subunit SecD docked complexes
Fig. 10.
RMSD plots obtained from Molecular dynamics simulation of top hits. The Superimposed RMSD graph spectrum of the unbound Chemotaxis-specific methylesterase protein, the Control (Ciprofloxacin) and the 5 promising compounds Hit 1 (STL321942), Hit 2 (STL190843), HIT 3 (STL069540), Hit 4 (STL513090), Hit 5 (STL321396) in complex with Chemotaxis-specific methylesterase protein.
The unbound RMSD of the Preprotein translocase subunit SecD protein was determined to be 2.4 Å. In complex with hit1, the ligand's RMSD ranged from 2.4 to 5 Å, with the simulation showing that the ligand was well-fitted in the binding pocket. For the hit2-protein complex, the ligand RMSD was between 1.6 and 3.2 Å, indicating stability. When hit3 was present, its RMSD ranged from 1.6 to 4.0 Å, with minimal deviations observed, suggesting a stable interaction. The hit4 complex, however, proved unstable, as the ligand withdrew from the binding pocket after 10 ns, indicating a lack of stability. In the presence of hit5, the ligand RMSD varied between 1.8 and 4.8 Å, with convergence observed in the final stages of the simulation, suggesting complex stability. When bound to the control, the ligand RMSD fluctuated significantly, ranging from 2.4 to 14 Å, indicating instability in the dynamic setting (Fig. 11). Stable complexes were chosen based on their RMSDs to further examine the simulation characteristics, such as protein and ligand RMSF and protein-ligand interactions during the simulation.
-
ii.
Protein and Ligand RMSF Analysis
Fig. 11.
RMSD plots were obtained from the molecular dynamics simulation of top hits. The Superimposed RMSD graph spectrum of the unbound Preprotein translocase subunit SecD protein, the Control (Ciprofloxacin) and the 5 promising compounds Hit 1 (STK394282), Hit 2 (STK232493), HIT 3 (STL243336), Hit 4 (STK205602), Hit 5 (STK368220) in complex with Preprotein translocase subunit SecD protein.
The RMSF values for proteins and ligands provide insight into the flexibility and stability of the docked complexes during MD simulations. A higher RMSF value denotes increased flexibility of residues or ligand atoms, while lower RMSF values reflect a stable interaction, contributing to overall system stability. Here, we examine the Protein RMSF and Ligand RMSF values for three stable docked protein-ligand complexes.
-
a)
RMSF Analysis of Imidazole Glycerol Phosphate Synthase Subunit HisF2 protein
In the 100 ns MD simulations, the protein backbone RMSF values indicated strong stability across the docked complexes. For the complex with hit 2, the protein RMSF values ranged between 0.5 and 1.0 Å, reflecting minimal residue fluctuations and robust stability within the binding site. Similarly, the hit 5 complex exhibited protein RMSF values between 0.5 and 1.0 Å, underscoring a stable interaction with minimal flexibility. For the control drug, the protein residues consistently showed low flexibility, with RMSF values in the same range of 0.5–1.0 Å, further supporting the structural integrity of the complex (Fig. 12).
-
b)
RMSF Analysis of Chemotaxis-Specific Methylesterase protein
Fig. 12.
RMSF graph of Imidazole glycerol phosphate synthase subunit HisF2, showing residue flexibility for the protein bound with the control drug (ciprofloxacin) and the promising hit compounds (Hits 2 and 5).
In the MD simulations, the protein backbone RMSF values across the complexes with hit 1, hit 3, and hit 5 indicated strong stability. For the hit 1 complex, the RMSF values for the protein residues ranged from 0.5 to 1.5 Å, showing minimal fluctuation and suggesting stable binding. The hit 3 complex demonstrated even lower RMSF values, ranging from 0.5 to 1.5 Å, highlighting an enhanced level of stability compared to hit 1. Similarly, the protein in the Hit 5 complex maintained RMSF values between 0.5 and 1.5 Å, further confirming the overall stability of the protein-ligand interaction (Fig. 13).
-
c)
RMSF Analysis of Preprotein Translocase Subunit SecD protein
Fig. 13.
RMSF graph of Chemotaxis-specific methylesterase in complex with the control drug (ciprofloxacin) and the promising hit compounds (Hits 1, 3, and 5), highlighting residue flexibility across ligand interactions.
In the MD simulations, the protein RMSF values across the complexes with hit 1, hit 2 and hits 3 and 5 demonstrated varying levels of stability. For the hit 1 complex, the protein residues exhibited minimal flexibility, with RMSF values ranging from 0.8 to 1.6 Å, suggesting a stable interaction. The hit 2 complex displayed similar RMSF values ranging from 0.8 to 1.6 Å, indicating a stable binding environment. In contrast, the protein RMSF values for the hits 3 and 5 complexes were slightly higher, ranging from 1.2 to 2.4 Å for hit 3 and 1.6 to 1.8 Å for hit 5, which, while reflecting some flexibility, remained within acceptable limits, indicating stable complexes overall (Fig. 14).
-
d)
RMSF Analysis of hit compounds in presence of Imidazole Glycerol Phosphate Synthase Subunit HisF2
Fig. 14.
RMSF graph of Preprotein translocase subunit SecD, illustrating residue flexibility for the protein in complex with the control drug (ciprofloxacin) and the promising hit compounds (Hits 1, 2, 3, and 5) across different ligand-binding interactions.
In the MD simulations, the ligand RMSF values for the complexes with hit 2, hit 5, and the control drug highlighted varying degrees of stability. For the hit 2 complex, the ligand RMSF values ranged from 1.0 to 1.2 Å, indicating minimal fluctuation and a stable binding interaction. The hit 5 complex showed slightly higher variability, with ligand RMSF values ranging from 0.75 to 1.75 Å, but still remaining within acceptable limits for stability. In contrast, the control drug exhibited a broader RMSF range of 1.0–2.0 Å, suggesting moderate structural variation, though the protein-ligand interaction remained stable overall (Fig. 15).
-
e)
RMSF Analysis of hit compounds in presence of Chemotaxis-Specific Methylesterase
Fig. 15.
RMSF graph of the control drug (ciprofloxacin) and hit compounds (Hits 2 and 5) in the presence of Imidazole glycerol phosphate synthase subunit HisF2, depicting atom flexibility within the ligand-binding site.
In the MD simulations, the ligand RMSF values for the complexes with hit 1, hit 3, and hit 5 demonstrated varying degrees of stability. For the hit 1 complex, the ligand RMSF values were minimal, ranging from 0.7 to 1.0 Å, indicating a stable fit within the binding pocket. In the hit 3 complex, the ligand RMSF ranged from 1.25 to 1.75 Å, showing slight fluctuations but reinforcing a stable interaction. The hit 5 complex exhibited slightly higher RMSF values, ranging from 1.25 to 2.0 Å, yet these values remained within acceptable limits, suggesting stability in the overall protein-ligand interaction (Fig. 16).
-
f)
RMSF Analysis of hit compounds in presence of Preprotein Translocase Subunit SecD
Fig. 16.
RMSF graph of the control drug (ciprofloxacin) and hit compounds (Hits 1, 3, and 5) in complex with Chemotaxis-specific methylesterase, illustrating atom flexibility within the ligand-binding site.
In the MD simulations, the ligand RMSF values for the complexes with hit 1, hit 2 and hits 3 and 5 indicated varying levels of stability. For the hit 1 complex, the ligand RMSF values ranged from 1.25 to 1.75 Å, showing minor fluctuations but maintaining stable binding. In the hit 2 complex, the ligand RMSF values ranged from 1.2 to 2.0 Å, which remained within acceptable limits, suggesting no significant impact on the structural integrity of the complex. For the hit 3 and hit 5 complexes, the ligand RMSF values ranged from 1.25 to 2.0 Å for hit 3, while hit 5 exhibited slightly higher fluctuations between 2.25 and 3.0 Å. Despite these variations, both complexes showed stable interactions overall (Fig. 17).
-
iii)
Radius of Gyration Analysis
Fig. 17.
RMSF graph of the control drug (ciprofloxacin) and hit compounds (Hits 1, 2, 3, and 5) in complex with Preprotein translocase subunit SecD, highlighting atom flexibility within the ligand-binding site.
The Rg reflects the structural stability and spatial compactness of a ligand, offering insights into its degree of "extendedness" or distribution around its center of mass. In the context of ligand stability at the binding site, a lower Rg may indicate a more stable, compact conformation, whereas a higher Rg suggests a more extended and potentially less stable configuration, which could impact its interaction dynamics and binding efficiency.
-
a)
Rg analysis of hit compounds at the binding site of imidazole glycerol phosphate synthase subunit HisF2
The analysis of the Rg over 100 ns of molecular dynamics simulations demonstrated notable differences in the stability of the binding site across the control and ligand-bound systems. The control compound consistently displayed higher Rg values (∼4 Å), indicating reduced compactness and minimal structural stabilization. In contrast, both hit 2 and hit 5 showed significantly lower and more stable Rg values, reflecting their ability to enhance binding site stability. Hit 2 exhibited a Rg of approximately 3 Å, indicating moderate stabilization, while hit 5 demonstrated the highest level of compactness and stability, with a Rg slightly below 3 Å. These results highlight the superior stabilizing potential of hit 5, making it a strong candidate for further exploration (Fig. 18).
-
b)
Rg Analysis of hit compounds at the binding site of Chemotaxis-Specific Methylesterase
Fig. 18.
Rg graph of the control drug (ciprofloxacin) and hit compounds (Hits 2 and 5) at the Imidazole glycerol phosphate synthase subunit HisF2 binding site during a 100 ns molecular dynamics simulation.
The Rg analysis over 100 ns of MD simulations revealed significant differences in stability between the control and ligand-bound systems. The control drug displayed consistently higher Rg values (∼4 Å), indicating a lack of compactness and structural stabilization at the binding site. In contrast, the ligand-bound systems demonstrated enhanced stability, with lower and more stable Rg values throughout the simulation. Among the tested ligands, hit 1 exhibited the most compact and stable behavior, maintaining an Rg of ∼2.5 Å, while hit 3 and hit 5 showed slightly higher but comparable stability, with Rg values around ∼3 Å. These results highlight the ability of the ligands, particularly hit 1, to effectively stabilize the binding site, suggesting their potential as promising candidates for further investigation (Fig. 19).
-
c)
Rg Analysis of hit compounds at the binding site of Preprotein Translocase Subunit SecD
Fig. 19.
Rg plot showing the structural stability of the control drug (ciprofloxacin) and hit compounds (Hits 1, 3, and 5) at the binding site of Chemotaxis-specific methylesterase during a 100 ns molecular dynamics simulation.
The Rg values for the control remain consistent around 4 Å, suggesting structural stability. hit 1 shows a lower and stable Rg (∼2 Å), indicating compactness with the SecD protein. hit 2 and hit 3 exhibit similar profiles, maintaining Rg values around 4 Å, comparable to the control, which reflects stable conformational behavior. In contrast, hit 5 displays slightly higher Rg values, suggesting relatively less compactness. These results demonstrate the dynamic stability and conformational characteristics of the studied systems (Fig. 20).
-
iv)
Molecular Surface Area Analysis
Fig. 20.
Rg graph depicting the structural dynamics of the control drug (ciprofloxacin) and hit compounds (Hits 1, 2, 3, and 5) at the binding site of the Preprotein translocase subunit SecD over a 100 ns molecular dynamics simulation.
The molecular surface calculation, performed using a 1.4 Å probe radius, represents the van der Waals surface area. In the context of ligand stability at the binding site, this measurement reflects the ligand's accessible surface area available for interactions, providing insights into how well the ligand fits and stabilizes within the binding pocket through van der Waals and hydrophobic interactions.
-
a)
MolSA Analysis of Hit Compounds at the Binding Site of Imidazole Glycerol Phosphate Synthase Subunit HisF2
The MolSA profiles of the control and selected compounds (hit 2 and hit 5) were analyzed over a 100-ns simulation to assess their structural dynamics in a solvent environment. The control exhibited consistently higher MolSA values (∼300 Å2), indicative of a more open conformation and extensive solvent interaction. In contrast, hits 2 and 5 displayed significantly lower MolSA values (∼200 Å2), reflecting a more compact structural organization with limited solvent exposure. The stability of these MolSA trends throughout the simulation underscores the conformational steadiness of the systems, with the reduced solvent-accessible surface area of the hits potentially aligning with enhanced structural stability and minimized solvent interaction (Fig. 21).
-
b)
MolSA Evaluation of Hit Compounds at the Chemotaxis-Specific Methylesterase Binding Site
Fig. 21.
MolSA plot depicting the stability and structural changes of the control drug (ciprofloxacin) and hit compounds (Hits 2 and 5) at the binding site of Imidazole glycerol phosphate synthase subunit HisF2 over a 100 ns molecular dynamics simulation.
The MolSA profiles of the control and selected hit compounds (hit 1, hit 3, and hit 5) were monitored over a 100-ns simulation period. The control consistently exhibited the highest MolSA (∼300 Å2), indicative of its pronounced solvent exposure and extended conformation. In contrast, hit 1 demonstrated the lowest MolSA (∼150 Å2), reflecting its compact structure and minimal solvent-accessible surface. hits 3 and 5 displayed intermediate MolSA values (∼200 Å2), suggesting moderate solvent exposure and structural compactness compared to the control. These findings underscore the distinct solvent-accessibility characteristics of the compounds, which may influence their functional stability and potential binding interactions (Fig. 22).
-
c)
MolSA Assessment of Hit Compounds at the Binding Site of Preprotein Translocase Subunit SecD
Fig. 22.
MolSA plot showing the structural dynamics of the control drug (ciprofloxacin) and hit compounds (Hits 1, 3, and 5) at the Chemotaxis-specific methylesterase binding site during a 100 ns molecular dynamics simulation.
The MolSA analysis highlights the differences in conformational stability between the control and five potential hits during a 100 ns molecular dynamics simulation. The control displayed consistently higher MolSA values, averaging around 300 Å2 throughout the simulation, indicative of its robust structural stability. In comparison, the hits exhibited relatively lower MolSA values with minor fluctuations, reflecting distinct binding conformations. hit 1 maintained a MolSA value averaging approximately 200 Å2, demonstrating stable structural behavior. hit 2 exhibited a slightly higher average MolSA of around 220 Å2, indicating a moderate binding interface. Similarly, hit 3 displayed values near 230 Å2, while hit 5 maintained the lowest MolSA among the hits, averaging close to 180 Å2. Despite these variations, the stability of MolSA values for all hits underscores minimal structural deviations and reliable complex formation under dynamic conditions (Fig. 23).
-
v)
Solvent Accessible Surface Area Analysis
Fig. 23.
MolSA plot illustrating the stability and structural behavior of the control drug (ciprofloxacin) and hit compounds (Hits 1, 2, 3, and 5) at the binding site of the Preprotein translocase subunit SecD during a 100 ns molecular dynamics simulation.
The surface area of a molecule accessible to a water molecule represents the regions of the ligand exposed to solvent. From the perspective of ligand stability at the binding site, this parameter highlights the balance between buried and exposed regions, influencing the ligand's ability to form stable interactions with the binding pocket while maintaining solvation dynamics.
-
a)
SASA Analysis of Hit Compounds at the Binding Site of Imidazole Glycerol Phosphate Synthase Subunit HisF2
In the 100 ns MD simulations, the SASA was analyzed for the control and two different hits. The control exhibited a gradual increase in SASA values, reaching approximately 25 Å2 by the end of the simulation, indicating progressive exposure to the solvent. In contrast, hit 2 showed a stable profile in the initial 70 ns, with minimal SASA values, followed by a steep rise around 80 ns, peaking at nearly 45 Å2, and suggesting significant solvent exposure in the latter stages of the simulation. However, hit 5 maintained consistently low SASA values throughout the simulation, with only minor fluctuations and an endpoint near 10 Å2, reflecting stable structural compactness and limited solvent interaction (Fig. 24).
-
b)
SASA Evaluation of Hit Compounds at the Chemotaxis-Specific Methylesterase Binding Site
Fig. 24.
SASA plot showing the solvent exposure and structural dynamics of the control drug (ciprofloxacin) and hit compounds (Hits 2 and 5) at the binding site of Imidazole glycerol phosphate synthase subunit HisF2 during a 100 ns molecular dynamics simulation.
The SASA analysis provides a detailed examination of the conformational dynamics and solvent exposure for the control and three selected hits during a 100 ns molecular dynamics simulation. The control exhibited significantly higher SASA values throughout the simulation, with fluctuations ranging from approximately 250 Å2 to 350 Å2, indicating a greater degree of solvent exposure. Among the hits, hit 1 maintained a relatively consistent SASA value averaging around 50 Å2, signifying minimal solvent exposure and compact binding. hit 3 displayed slightly higher SASA values, averaging close to 60 Å2, reflecting moderate accessibility. hit 5 exhibited the lowest SASA values, maintaining a stable average near 40 Å2, indicative of strong encapsulation within the binding pocket. These observations suggest that the hits exhibit distinct interaction profiles and structural behaviors compared to the control, with reduced solvent exposure correlating with potentially tighter binding and stable complex formation under dynamic conditions (Fig. 25).
-
c)
SASA Assessment of Hit Compounds at the Binding Site of Preprotein Translocase Subunit SecD
Fig. 25.
SASA plot illustrating the solvent accessibility and conformational changes of the control drug (ciprofloxacin) and hit compounds (Hits 1, 3, and 5) at the Chemotaxis-specific methylesterase binding site over a 100 ns molecular dynamics simulation.
The SASA values exhibited distinct behaviors across the control and the various hits. The control displayed a significant increase in SASA, peaking around 550 Å2 near the midpoint of the simulation, followed by a gradual decline towards the end. This behavior highlights substantial exposure to the solvent, indicative of structural rearrangements. In contrast, hit 1 maintained consistently low SASA values, with minor fluctuations around 50 Å2 throughout the simulation, reflecting stable compactness. Similarly, hit 2 showed a slightly elevated yet stable profile, with SASA values remaining around 70 Å2. hit 3 exhibited behavior akin to hit 2, maintaining a steady SASA near 80 Å2, suggesting limited solvent exposure. Finally, hit 5 demonstrated the lowest SASA values, remaining under 50 Å2 for the entirety of the simulation, indicating minimal solvent interaction and robust structural stability (Fig. 26).
-
vi)
Polar Surface Area Analysis
Fig. 26.
SASA plot depicting the solvent exposure and structural stability of the control drug (ciprofloxacin) and hit compounds (Hits 1, 2, 3, and 5) at the binding site of Preprotein translocase subunit SecD during a 100 ns molecular dynamics simulation.
The solvent-accessible surface area of a molecule, contributed solely by oxygen and nitrogen atoms, represents the regions capable of forming hydrogen bonds or polar interactions. From a ligand stability perspective, this parameter provides critical insights into the ligand's potential for establishing stable polar and hydrogen bonding interactions within the binding site.
-
a)
PSA Analysis of Hit Compounds at the Binding Site of Imidazole Glycerol Phosphate Synthase Subunit HisF2
In the 100 ns molecular dynamics simulations, the PSA values showed distinct behaviors among the control and the hits. The control exhibited relatively stable PSA values, fluctuating slightly around 150 Å2 throughout the simulation, indicating consistent exposure of polar regions. Hit 2 displayed slightly higher PSA values compared to the control, maintaining a steady profile around 160 Å2, suggesting an increased but stable level of polar surface exposure. In contrast, hit 5 showed considerably lower PSA values, consistently averaging around 100 Å2, indicating reduced polar surface exposure and potentially greater structural compactness. These variations in PSA profiles reflect the differing dynamic properties and solvent interaction behaviors of the hits relative to the control (Fig. 27).
-
b)
PSA Assessment of Hit Compounds at the Chemotaxis-Specific Methylesterase Binding Site
Fig. 27.
PSA plot showing the exposure of polar surface areas and structural dynamics of the control drug (ciprofloxacin) and hit compounds (Hits 2 and 5) at the binding site of Imidazole glycerol phosphate synthase subunit HisF2 over a 100 ns molecular dynamics simulation.
The PSA values demonstrated consistent trends across the control and the various hits. The control maintained a stable PSA around 200 Å2 throughout the simulation, indicating minimal fluctuations and a consistent level of polar surface exposure. hit 1 exhibited a similar profile, with PSA values closely matching those of the control, suggesting comparable structural stability and solvent interactions. hit 3 followed a comparable trend, with PSA values remaining steady at approximately 200 Å2, highlighting its uniform behavior over time. Likewise, hit 5 showed a nearly identical pattern, maintaining PSA values around the same range, reflecting stable polar surface exposure. These observations suggest that the hits and the control displayed minimal deviation in PSA dynamics during the simulation, indicative of conserved polar characteristics across all tested molecules (Fig. 28).
-
c)
PSA Assessment of Hit Compounds at the Preprotein Translocase Subunit SecD Binding Site
Fig. 28.
PSA plot illustrating the accessibility of polar regions and conformational stability of the control drug (ciprofloxacin) and hit compounds (Hits 1, 3, and 5) at the Chemotaxis-specific methylesterase binding site during a 100 ns molecular dynamics simulation.
Significant differences were observed in the PSA values between the control and the investigated hits. The control exhibited a consistent PSA of 150 Å2 throughout the simulation, reflecting stable exposure to polar regions. Similarly, hit 1 displayed PSA values comparable to the control, indicating analogous structural dynamics. In contrast, hit 2 consistently showed markedly higher PSA values, averaging around 350 Å2, likely due to increased solvent accessibility and greater exposure to polar regions. On the other hand, hits 3 and 5 demonstrated consistently lower PSA values, averaging approximately 100 Å2, suggesting reduced polar surface exposure and a more compact structural configuration. These findings underscore the variability in solvent interaction behaviors and stability among the compounds during the simulation (Fig. 29).
Fig. 29.
PSA plot depicting the exposure of polar solvent-accessible regions and structural behavior of the control drug (ciprofloxacin) and hit compounds (Hits 1, 2, 3, and 5) at the binding site of Preprotein translocase subunit SecD during a 100 ns molecular dynamics simulation.
In conclusion, hits 2 and 5 demonstrated strong stability with Imidazole glycerol phosphate synthase subunit HisF2, characterized by low RMSD, minimal RMSF fluctuations, compact structural dynamics as indicated by Rg and MolSA analyses, and limited solvent exposure observed through SASA and PSA profiles. Similarly, hits 1, 2, 3, and 5 exhibited exceptional stability with Preprotein translocase subunit SecD, showing minimal structural deviation, robust compactness, and reliable interaction profiles under dynamic conditions. Additionally, hits 1, 3, and 5 displayed robust stability with Chemotaxis-specific methylesterase, as reflected by consistent RMSD and RMSF values, reduced solvent exposure, and favorable structural compactness. Collectively, these findings position these hits as the most promising compounds for their respective protein targets, offering significant potential in the development of novel antibiotics against multidrug-resistant P. aeruginosa.
3.13. MMPBSA analysis
To evaluate the stability of final protein-drug complexes obtained from MD simulations, MMPBSA calculations were conducted using the farPPI web server. For stable complexes with Imidazole glycerol phosphate synthase subunit HisF2, hits 2 and 5 demonstrated binding energies of −5.11 kcal/mol and −10.01 kcal/mol, respectively. In the case of Chemotaxis-specific methylesterase, hits 1, 3, and 5 showed binding energies of −8.63 kcal/mol, −2.14 kcal/mol, and −10.99 kcal/mol, respectively. Meanwhile, Preprotein translocase subunit SecD, complexed with hits 1, 2, 3, and 5, had binding energies of −10.01 kcal/mol, −8.25 kcal/mol, −28.26 kcal/mol, and −16.97 kcal/mol, respectively. Notably, the strongest binding energies were seen with Chemotaxis-specific methylesterase and hit 5 (STL321396) at −10.99 kcal/mol, and with Imidazole glycerol phosphate synthase subunit HisF2, hit 5 (STK417467) at −10.01 kcal/mol. Additionally, Preprotein translocase subunit SecD exhibited firm binding with hit 3 (STL243336) at −28.26 kcal/mol. These findings highlight these compounds as highly stable and promising candidates with robust interactions across their target proteins, suggesting potential for further development as antibiotics against multidrug-resistant P. aeruginosa.
3.14. Density Functional Theory calculations
DFT calculations were conducted to explore the electronic properties of three predicted inhibitors: STK417467 (hit 5 against Imidazole Glycerol Phosphate Synthase subunit HisF2), STL243336 (hit 5 targeting chemotaxis-specific methylesterase), and STL321396 (hit 3 for Preprotein Translocase subunit SecD). These compounds were selected for their potential inhibitory effects on their respective bacterial targets. The analyses encompassed key parameters, including the HOMO-LUMO energy gap (ΔE), Electrophilicity Index (ω), electrostatic potential, and electron density, providing a comprehensive evaluation of their reactivity, stability, and binding characteristics (Table 15). Among the three inhibitors, STK417467 exhibited the largest HOMO-LUMO gap (ΔE = 0.1956 eV, Fig. 30A and B), indicating its superior stability and minimal reactivity, making it a promising candidate for HisF2 inhibition. STL243336 demonstrated a moderate ΔE of 0.1672 eV (Fig. 32A and B), reflecting a balanced profile of stability and reactivity, which suits its interaction potential with SecD. On the other hand, STL321396 had the smallest ΔE (0.15598 eV, Fig. 31A and B), marking it as the most reactive compound, ideal for targeting chemotaxis-specific methylesterase. The Electrophilicity Index values supported these observations. STL321396 showed the highest electrophilicity (ω = 0.09773 eV), reflecting its enhanced reactivity, while STL243336 had the lowest electrophilicity (ω = 0.06192 eV), indicating reduced reactivity. STK417467 exhibited intermediate values, consistent with its stability and suitability for HisF2 inhibition. Electrostatic potential maps further revealed distinct interaction profiles. STK417467 displayed a uniform electrostatic potential distribution (Fig. 30D), highlighting its stability in interactions. In contrast, STL321396 showed a pronounced dipole moment (Fig. 31D), underscoring its high reactivity toward electrophilic centers. STL243336, with a neutral electrostatic potential distribution (Fig. 32D), indicated balanced interactions with both nucleophiles and electrophiles. Electron density analyses aligned with these findings. STK417467 (Fig. 30C) and STL243336 (Fig. 32C) exhibited concentrated electron densities, signifying their stability, whereas STL321396 (Fig. 31C) displayed a dispersed electron density, consistent with its high reactivity. These DFT analyses provide a detailed understanding of the electronic properties of the selected inhibitors, reinforcing their potential as therapeutic candidates for their respective bacterial targets.
Table 15.
DFT analysis of Predicted Inhibitors.
| Compound | HOMO (eV) | LUMO (eV) | ΔE (eV) | Electrophilicity Index (ω) (eV) |
|---|---|---|---|---|
| STL243336 | −0.227488 | −0.060288 | 0.1672 | 0.06192 |
| STL321396 | −0.252589 | −0.096609 | 0.15598 | 0.09773 |
| STK417467 | −0.260798 | −0.065222 | 0.195576 | 0.06799 |
Fig. 30.
DFT analysis of hit 5 (STK417467) for Imidazole glycerol phosphate synthase subunit HisF2: A) HOMO, B) LUMO, C) Electron density distribution, and D) Electrostatic potential map.
Fig. 32.
DFT analysis of hit 3 (STKL4336) for Preprotein translocase subunit SecD: A) HOMO, B) LUMO, C) Electron density distribution, and D) Electrostatic potential map.
Fig. 31.
DFT analysis of hit 5 (STK321396) for Chemotaxis-specific methylesterase: A) HOMO, B) LUMO, C) Electron density distribution, and D) Electrostatic potential map.
4. Discussion
Antimicrobial resistance (AMR) remains a profound global health challenge [75,76], exacerbated by the rapid emergence of multidrug-resistant (MDR) pathogens such as P. aeruginosa [77]. This pathogen's capacity to evolve resistance mechanisms, including the production of beta-lactamases, activation of efflux pumps, and biofilm formation, significantly complicates treatment regimens and contributes to high mortality rates, particularly in immunocompromised individuals or those undergoing invasive medical procedures [78,79]. Addressing this issue necessitates innovative approaches to identify new therapeutic targets and develop strategies to combat resistant strains.
This study presents a comprehensive in-silico approach that integrates multiple computational techniques to identify and evaluate promising drug targets within P. aeruginosa. Our workflow incorporates protein data retrieval, comparative analyses, metabolic pathway assessments, druggability evaluations, and virulence factor identification, among other methodologies, which collectively allow for the refinement of potential drug targets. A key strength of our approach lies in its hierarchical framework, enabling the identification of non-homologous proteins, which minimizes the risk of off-target effects and enhances the specificity of drug development efforts. The initial analysis of 5563 proteins from P. aeruginosa PAO1 revealed 5403 proteins as non-homologous to human proteins, providing a promising starting point for the selection of drug targets with reduced risk of cross-reactivity with human proteins.
Further narrowing of our target selection involved focusing on essential proteins with critical roles in bacterial viability. Through an examination of metabolic pathways and subcellular localization, we identified 149 essential proteins, including preprotein translocase subunit SecD, imidazole glycerol phosphate synthase subunit HisF2, and chemotaxis-specific methylesterase, as prime candidates for drug development. These proteins are highly conserved across various P. aeruginosa strains, underscoring their potential as broad-spectrum therapeutic targets.
Integrating druggability assessments and virulence factor analyses provided deeper insights into the biological roles of these proteins, revealing their involvement in key bacterial processes such as protein export, chemotaxis, and metabolism. By examining the structural and functional characteristics of these proteins, we were able to identify promising small molecules that could effectively inhibit their activity. Specifically, STK417467 emerged as a potential inhibitor for HisF2, STL321396 for chemotaxis-specific methylesterase, and STL243336 for SecD. These compounds exhibited favorable docking scores and demonstrated stability in molecular dynamics simulations, further validating their potential as therapeutic agents.
Our findings align with the growing body of literature on novel drug target identification for P. aeruginosa. Recent studies have leveraged in-silico techniques, including homology modeling and molecular docking, to predict druggable sites in proteins implicated in resistance mechanisms [80]. Our approach builds upon this body of work, distinguishing itself by combining multiple computational methods—ranging from druggability assessments to molecular dynamics simulations—to enhance the robustness of our target selection process. Moreover, while pangenome analyses have been used to identify conserved genetic markers for diagnostics, our study goes beyond diagnostic applications by focusing on identifying protein targets that are both essential for bacterial survival and critical to the pathogen's virulence [81].
Additionally, alternative therapeutic strategies, such as phage therapy, are gaining attention as potential solutions to combat MDR P. aeruginosa infections. High-throughput platforms for personalized phage therapy, which integrate genomics, proteomics, and computational biology, have been developed to identify bacterial vulnerabilities specific to individual patient strains [82]. While promising, these strategies are still in the experimental phase and face challenges related to scalability and regulatory approval. Our work complements these efforts by focusing on druggable proteins that could be targeted by small molecules, thereby providing an alternative therapeutic strategy that could be implemented more rapidly in clinical settings.
A critical aspect of our study lies in the identification of P. aeruginosa proteins that are involved in essential bacterial processes, such as protein export (SecD), metabolism (HisF2), and chemotaxis (methylesterase), as therapeutic targets. These proteins are integral to bacterial survival and pathogenicity, which makes them ideal candidates for novel antimicrobial therapies. By targeting these processes, it may be possible to hinder the bacterium's ability to establish infections and reduce its capacity to develop resistance. Furthermore, the identification of virulence-associated proteins enhances the therapeutic potential of the selected targets, as drugs designed to inhibit these proteins could both limit bacterial survival and attenuate pathogenicity, offering dual therapeutic benefits.
Despite the promise of computational approaches, several limitations must be acknowledged. The accuracy of in-silico predictions is contingent upon the availability of accurate structural data for target proteins, as well as a comprehensive understanding of resistance mechanisms. While our study employed a robust computational framework, future work should prioritize experimental validation of the identified targets to rigorously assess their efficacy, biocompatibility, and safety in vitro and in vivo. The identification of promising inhibitors, such as STK417467, STL321396, and STL243336, warrants further investigation, including pharmacokinetic profiling and toxicity evaluations, to determine their suitability for clinical development. This study presents a novel and comprehensive in-silico approach to identify and evaluate potential drug targets and inhibitors for P. aeruginosa. Our findings highlight key proteins involved in essential bacterial processes, which represent promising candidates for broad-spectrum antimicrobial therapies. The integration of computational predictions with experimental validation has the potential to expedite the development of targeted therapies capable of overcoming the growing challenge of antimicrobial resistance. Furthermore, the methodologies employed in this study could be extended to other MDR pathogens, contributing to the broader global effort to combat AMR.
5. Conclusion
The rise in multi-drug resistant strains of Pseudomonas species presents a growing global health threat, underscoring the urgent need for new therapeutic strategies. While the traditional approach of developing new antibiotics remains important, this study shifts focus to identifying the underlying molecular targets responsible for the virulence and resistance mechanisms of Pseudomonas species, which are the true drivers of infection persistence and resistance. By employing subtractive proteomics, we have successfully identified three potential druggable targets—preprotein translocase subunit SecD, imidazole glycerol phosphate synthase subunit HisF2, and chemotaxis-specific methylesterase—in Pseudomonas. These targets play critical roles in bacterial survival, pathogenicity, and resistance, making them prime candidates for therapeutic intervention. Furthermore, through computational screening, we have identified novel small-molecule inhibitors (STK417467, STL321396, and STL243336) capable of targeting these proteins, offering a promising avenue for the development of new drugs. This study marks a significant advancement in the search for novel treatments against Pseudomonas infections, particularly in the context of multidrug resistance. These findings provide valuable insights into the mechanisms of Pseudomonas pathogenesis and antibiotic resistance, paving the way for the development of targeted therapeutic strategies that have the potential to enhance clinical outcomes in individuals at high risk of severe infections. However, the identified targets and inhibitors require further experimental validation, including in-vitro and in-vivo studies, to confirm their efficacy, safety, and potential for clinical application. With continued research, these findings could provide a foundation for the development of effective therapeutic strategies against Pseudomonas infections, addressing the critical challenges posed by antimicrobial resistance.
CRediT authorship contribution statement
Divya Vemula: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Methodology, Formal analysis, Data curation, Conceptualization. Vasundhra Bhandari: Supervision, Resources, Project administration.
Data availability statement
The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.
Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors thank the National Institute of Pharmaceutical Education and Research (NIPER) Hyderabad for the infrastructure, overall support, and software availability.
Footnotes
Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2025.e42584.
Appendix A. Supplementary data
The following is the Supplementary data to this article:
References
- 1.Magill S.S., Edwards J.R., Bamberg W., Beldavs Z.G., Dumyati G., Kainer M.A., Lynfield R., Maloney M., McAllister-Hollod L., Nadle J., Ray S.M. Multistate point-prevalence survey of health care–associated infections. N. Engl. J. Med. 2014 Mar 27;370(13):1198–1208. doi: 10.1056/NEJMoa1306801. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Jesudason T. WHO publishes updated list of bacterial priority pathogens. The Lancet Microbe. 2024 Sep 1;5(9) doi: 10.1016/j.lanmic.2024.07.003. [DOI] [PubMed] [Google Scholar]
- 3.Azam M.W., Khan A.U. Updates on the pathogenicity status of Pseudomonas aeruginosa. Drug Discov. Today. 2019 Jan 1;24(1):350–359. doi: 10.1016/j.drudis.2018.07.003. [DOI] [PubMed] [Google Scholar]
- 4.Das T., Manoharan A., Whiteley G., Glasbey T., Manos J. InNew and Future Developments in Microbial Biotechnology and Bioengineering: Microbial Biofilms. Elsevier; 2020 Jan 1. Pseudomonas aeruginosa biofilms and infections: roles of extracellular molecules; pp. 29–46. [DOI] [Google Scholar]
- 5.Shao X., Xie Y., Zhang Y., Liu J., Ding Y., Wu M., Wang X., Deng X. Novel therapeutic strategies for treating Pseudomonas aeruginosa infection. Expet Opin. Drug Discov. 2020 Dec 1;15(12):1403–1423. doi: 10.1080/17460441.2020.1803274. [DOI] [PubMed] [Google Scholar]
- 6.Weiner L.M., Webb A.K., Limbago B., Dudeck M.A., Patel J., Kallen A.J., Edwards J.R., Sievert D.M. Antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of data reported to the national healthcare safety network at the centers for disease control and prevention, 2011–2014. Infect. Control Hosp. Epidemiol. 2016;37(11):1288–1301. doi: 10.1017/ice.2016.174. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Williams B.J., Dehnbostel J., Blackwell T.S. Pseudomonas aeruginosa: host defence in lung diseases. Respirology. 2010;15(7):1037–1056. doi: 10.1111/j.1440-1843.2010.01819.x. [DOI] [PubMed] [Google Scholar]
- 8.Parker C.M., Kutsogiannis J., Muscedere J., Cook D., Dodek P., Day A.G., Heyland D.K., Canadian Critical Care Trials Group Ventilator-associated pneumonia caused by multidrug-resistant organisms or Pseudomonas aeruginosa: prevalence, incidence, risk factors, and outcomes. J. Crit. Care. 2008;23(1):18–26. doi: 10.1016/j.jcrc.2008.02.001. [DOI] [PubMed] [Google Scholar]
- 9.Vincent J.L., Sakr Y., Singer M., Martin-Loeches I., Machado F.R., Marshall J.C., Finfer S., Pelosi P., Brazzi L., Aditianingsih D., Timsit J.F. Prevalence and outcomes of infection among patients in intensive care units in 2017. JAMA. 2020;323(15):1478–1487. doi: 10.1001/jama.2020.2717. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Thaden J.T., Park L.P., Maskarinec S.A., Ruffin F., Fowler Jr V.G., Van Duin D. Results from a 13-year prospective cohort study show increased mortality associated with bloodstream infections caused by Pseudomonas aeruginosa compared to other bacteria. Antimicrob. Agents Chemother. 2017;61(6) doi: 10.1128/AAC.02671-16. 16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Williams F.N., Herndon D.N., Hawkins H.K., Lee J.O., Cox R.A., Kulp G.A., Finnerty C.C., Chinkes D.L., Jeschke M.G. The leading causes of death after burn injury in a single pediatric burn center. Crit. Care. 2009;13:1–7. doi: 10.1186/cc8170. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Ci K. Bloodstream infections caused by antibiotic-resistant gram-negative bacilli: risk factors for mortality and impact of inappropriate initial antimicrobial therapy on outcome. Antimicrob. Agents Chemother. 2005;49:760–766. doi: 10.1128/AAC.49.2.760-766.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Pachori P., Gothalwal R., Gandhi P. Emergence of antibiotic resistance Pseudomonas aeruginosa in intensive care unit; a critical review. Genes & diseases. 2019 Jun 1;6(2):109–119. doi: 10.1016/j.gendis.2019.04.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Mulcahy L.R., Isabella V.M., Lewis K. Pseudomonas aeruginosa biofilms in disease. Microb. Ecol. 2014;68(1):1–12. doi: 10.1007/s00248-013-0297-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Witzany C., Bonhoeffer S., Rolff J. Is antimicrobial resistance evolution accelerating? PLoS Pathog. 2020;16(10) doi: 10.1371/journal.ppat.1008905. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Magiorakos A.P., Srinivasan A., Carey R.B., Carmeli Y., Falagas M.E., Giske C.G., Harbarth S., Hindler J.F., Kahlmeter G., Olsson-Liljequist B., Paterson D.L. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infection. 2012 Mar 1;18(3):268–281. doi: 10.1111/j.1469-0691.2011.03570.x. [DOI] [PubMed] [Google Scholar]
- 17.Bassetti M., Castaldo N., Cattelan A., Mussini C., Righi E., Tascini C., Menichetti F., Mastroianni C.M., Tumbarello M., Grossi P., Artioli S. Ceftolozane/tazobactam for the treatment of serious Pseudomonas aeruginosa infections: a multicentre nationwide clinical experience. Int. J. Antimicrob. Agents. 2019 Apr 1;53(4):408–415. doi: 10.1016/j.ijantimicag.2018.11.001. [DOI] [PubMed] [Google Scholar]
- 18.Bassetti M., Garau J. Current and future perspectives in the treatment of multidrug-resistant Gram-negative infections. J. Antimicrob. Chemother. 2021 Nov 1;76(Supplement_4):iv23–37. doi: 10.1093/jac/dkab352. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Elfadadny A., Ragab R.F., AlHarbi M., Badshah F., Ibáñez-Arancibia E., Farag A., Hendawy A.O., De los Ríos-Escalante P.R., Aboubakr M., Zakai S.A., Nageeb W.M. Antimicrobial resistance of Pseudomonas aeruginosa: navigating clinical impacts, current resistance trends, and innovations in breaking therapies. Front. Microbiol. 2024 Apr 5;15 doi: 10.3389/fmicb.2024.1374466. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Kaur H., Modgil V., Chaudhary N., Mohan B., Taneja N. Computational guided drug targets identification against extended-spectrum beta-lactamase-producing multi-drug resistant uropathogenic Escherichia coli. Biomedicines. 2023 Jul 19;11(7):2028. doi: 10.3390/biomedicines11072028. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Serral F., Castello F.A., Sosa E.J., Pardo A.M., Palumbo M.C., Modenutti C., Palomino M.M., Lazarowski A., Auzmendi J., Ramos P.I.P., Nicolás M.F. From genome to drugs: new approaches in antimicrobial discovery. Front. Pharmacol. 2021;12 doi: 10.3389/fphar.2021.647060. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Ramos P.I.P., Fernández Do Porto D., Lanzarotti E., Sosa E.J., Burguener G., Pardo A.M., Klein C.C., Sagot M.F., De Vasconcelos A.T.R., Gales A.C., Marti M. An integrative, multi-omics approach towards the prioritization of Klebsiella pneumoniae drug targets. Sci. Rep. 2018;8(1) doi: 10.1038/s41598-018-28916-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Wadood A., Jamal A., Riaz M., Khan A., Uddin R., Jelani M., Azam S.S. Subtractive genome analysis for in silico identification and characterization of novel drug targets in Streptococcus pneumonia strain JJA. Microb. Pathog. 2018 Feb 1;115:194–198. doi: 10.1016/j.micpath.2017.12.063. [DOI] [PubMed] [Google Scholar]
- 24.Uddin R., Jamil F. Prioritization of potential drug targets against P. aeruginosa by core proteomic analysis using computational subtractive genomics and Protein-Protein interaction network. Comput. Biol. Chem. 2018 Jun 1;74:115–122. doi: 10.1016/j.compbiolchem.2018.02.017. [DOI] [PubMed] [Google Scholar]
- 25.Solanki V., Tiwari V. Subtractive proteomics to identify novel drug targets and reverse vaccinology for the development of chimeric vaccine against Acinetobacter baumannii. Sci. Rep. 2018 Jun 13;8(1):9044. doi: 10.1038/s41598-018-26689-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Hossain T., Kamruzzaman M., Choudhury T.Z., Mahmood H.N., Nabi A.N., Hosen M.I. Application of the subtractive genomics and molecular docking analysis for the identification of novel putative drug targets against Salmonella enterica subsp. enterica serovar Poona. BioMed Res. Int. 2017;2017(1) doi: 10.1155/2017/3783714. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Ahmad S., Navid A., Akhtar A.S., Azam S.S., Wadood A., Pérez-Sánchez H. Subtractive genomics, molecular docking and molecular dynamics simulation revealed LpxC as a potential drug target against multi-drug resistant Klebsiella pneumoniae. Interdiscipl. Sci. Comput. Life Sci. 2019 Sep 1;11:508–526. doi: 10.1007/s12539-018-0299-y. [DOI] [PubMed] [Google Scholar]
- 28.Harvey A.L. Natural products in drug discovery. Drug Discov. Today. 2008 Oct 1;13(19–20):894–901. doi: 10.1016/j.drudis.2008.07.004. [DOI] [PubMed] [Google Scholar]
- 29.Chowdhury U.F., Al Saba A., Sufi A.S., Khan A.M., Sharmin I., Sultana A., Islam M.O. Subtractive proteomics approach to Unravel the druggable proteins of the emerging pathogen Waddlia chondrophila and drug repositioning on its MurB protein. Heliyon. 2021 Jun 1;7(6) doi: 10.1016/j.heliyon.2021.e07320. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Urra G., Valdés-Muñoz E., Suardiaz R., Hernández-Rodríguez E.W., Palma J.M., Ríos-Rozas S.E., Flores-Morales C.A., Alegría-Arcos M., Yáñez O., Morales-Quintana L., D'Afonseca V. From proteome to potential drugs: integration of subtractive proteomics and ensemble docking for drug repurposing against Pseudomonas aeruginosa RND superfamily proteins. Int. J. Mol. Sci. 2024 Jul 23;25(15):8027. doi: 10.3390/ijms25158027. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Barh D., Tiwari S., Jain N., Ali A., Santos A.R., Misra A.N., Azevedo V., Kumar A. In silico subtractive genomics for target identification in human bacterial pathogens. Drug Dev. Res. 2011 Mar;72(2):162–177. doi: 10.1002/ddr.20413. [DOI] [Google Scholar]
- 32.UniProt Consortium T. UniProt: the universal protein knowledgebase. Nucleic acids research. 2018 Mar 16;46(5):2699. doi: 10.1093/nar/gkw1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Collins J.F., Coulson A.F.W., Lyall A. The significance of protein sequence similarities. Bioinformatics. 1988;4(1):67–71. doi: 10.1093/bioinformatics/4.1.67. [DOI] [PubMed] [Google Scholar]
- 34.Luo, et al. DEG 15, an update of the Database of Essential Genes that includes built-in analysis tools. Nucleic Acids Res. 2021;49(D1):D677–D686. doi: 10.1093/nar/gkaa917. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Pearson W.R. vol. 266. Academic Press; 1996. [15] Effective protein sequence comparison; pp. 227–258. (Methods in Enzymology). [DOI] [PubMed] [Google Scholar]
- 36.Johnson, et al. NCBI BLAST: a better web interface. Nucleic Acids Res. 2008;36(suppl_2):W5–W9. doi: 10.1093/nar/gkn201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Pearson W.R. Comparison of methods for searching protein sequence databases. Protein Sci. 1995;4(6):1145–1160. doi: 10.1002/pro.5560040613. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Moriya Y., Itoh M., Okuda S., Yoshizawa A.C., Kanehisa M. KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res. 2007;35(suppl_2):W182–W185. doi: 10.1093/nar/gkm321. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Kanehisa M., Furumichi M., Tanabe M., Sato Y., Morishima K. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 2017;45(D1):D353–D361. doi: 10.1093/nar/gkw1092. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Gardy J.L., Spencer C., Wang K., Ester M., Tusnady G.E., Simon I., Hua S., DeFays K., Lambert C., Nakai K., Brinkman F.S. PSORT-B: improving protein subcellular localization prediction for Gram-negative bacteria. Nucleic Acids Res. 2003;31(13):3613–3617. doi: 10.1093/nar/gkg602. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Yu C.S., Lin C.J., Hwang J.K. Predicting subcellular localization of proteins for Gram-negative bacteria by support vector machines based on n-peptide compositions. Protein Sci. 2004;13(5):1402–1406. doi: 10.1110/ps.03479604. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Wishart D.S., Feunang Y.D., Guo A.C., Lo E.J., Marcu A., Grant J.R., Sajed T., Johnson D., Li C., Sayeeda Z., Assempour N. DrugBank 5.0: a major update to the DrugBank database for 2018. Nucleic Acids Res. 2018;46(D1):D1074–D1082. doi: 10.1093/nar/gkx1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Chen L., Zheng D., Liu B., Yang J., Jin Q. Vfdb 2016: hierarchical and refined dataset for big data analysis—10 years on. Nucleic Acids Res. 2016;44(D1):D694–D697. doi: 10.1093/nar/gkv1239. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Alcock B.P., Huynh W., Chalil R., Smith K.W., Raphenya A.R., Wlodarski M.A., Edalatmand A., Petkau A., Syed S.A., Tsang K.K., Baker S.J. Card 2023: expanded curation, support for machine learning, and resistome prediction at the Comprehensive Antibiotic Resistance Database. Nucleic acids research. 2023 Jan 6;51(D1):D690–D699. doi: 10.1093/nar/gkac920. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Xie R., Shao N., Zheng J. Integrated co-functional network analysis on the resistance and virulence features in Acinetobacter baumannii. Front. Microbiol. 2020;11 doi: 10.3389/fmicb.2020.598380. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.Raman K., Yeturu K., Chandra N. targetTB: a target identification pipeline for Mycobacterium tuberculosis through an interactome, reactome, and genome-scale structural analysis. BMC Syst. Biol. 2008;2:109. doi: 10.1186/1752-0509-2-109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Shanmugham B., Pan A. Identification and characterization of potential therapeutic candidates in emerging human pathogen Mycobacterium abscessus: a novel hierarchical in silico approach. PLoS One. 2013;8(3) doi: 10.1371/journal.pone.0059126. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Duvaud S., Gabella C., Lisacek F., Stockinger H., Ioannidis V., Durinx C. Expasy, the Swiss bioinformatics resource portal, as designed by its users. Nucleic acids research. 2021 Jul 2;49(W1):W216–W227. doi: 10.1093/nar/gkab225. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Burley S.K., Berman H.M., Kleywegt G.J., Markley J.L., Nakamura H., Velankar S. Protein Data Bank (PDB): the single global macromolecular structure archive. Protein crystallography: methods and protocols. 2017:627–641. doi: 10.1007/978-1-4939-7000-1_26. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Pieper U., Webb B.M., Dong G.Q., Schneidman-Duhovny D., Fan H., Kim S.J., Khuri N., Spill Y.G., Weinkam P., Hammel M., Tainer J.A. ModBase, a database of annotated comparative protein structure models and associated resources. Nucleic acids research. 2014 Jan 1;42(D1):D336–D346. doi: 10.1093/nar/gkt1144. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51.Lu S., Wang J., Chitsaz F., Derbyshire M.K., Geer R.C., Gonzales N.R., Gwadz M., Hurwitz D.I., Marchler G.H., Song J.S., Thanki N. CDD/SPARCLE: the conserved domain database in 2020. Nucleic acids research. 2020 Jan 8;48(D1):D265–D268. doi: 10.1093/nar/gkz991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52.Paysan-Lafosse T., Blum M., Chuguransky S., Grego T., Pinto B.L., Salazar G.A., Bileschi M.L., Bork P., Bridge A., Colwell L., Gough J. InterPro in 2022. Nucleic acids research. 2023 Jan 6;51(D1):D418–D427. doi: 10.1093/nar/gkac993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 53.Wolf Y.I. IMPALA/RPS‐BLAST/PSI‐BLAST in protein sequence analysis. Encyclopedia of Genetics, Genomics. Proteomics and Bioinformatics. 2004 Oct 15 doi: 10.1002/047001153X.g403411. [DOI] [Google Scholar]
- 54.Paysan-Lafosse T., Andreeva A., Blum M., Chuguransky S.R., Grego T., Pinto B.L., Salazar G.A., Bileschi M.L., Llinares-López F., Meng-Papaxanthos L., Colwell L.J. The Pfam protein families database: embracing AI/ML. Nucleic Acids Res. 2025 Jan 6;53(D1):D523–D534. doi: 10.1093/nar/gkae997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55.Andreeva A., Kulesha E., Gough J., Murzin A.G. The SCOP database in 2020: expanded classification of representative family and superfamily domains of known protein structures. Nucleic acids research. 2020 Jan 8;48(D1):D376–D382. doi: 10.1093/nar/gkz1064. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 56.Tamura K., Stecher G., Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 2021 Jul 1;38(7):3022–3027. doi: 10.1093/molbev/msab120. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 57.Letunic I., Bork P. Interactive Tree of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021;49(W1):W293–W296. doi: 10.1093/nar/gkab301. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 58.Buchan D.W., Moffat L., Lau A., Kandathil S.M., Jones D.T. Deep learning for the PSIPRED protein analysis workbench. Nucleic Acids Res. 2024 May;15 doi: 10.1093/nar/gkae328. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 59.Angamuthu K., Piramanayagam S. Evaluation of in silico protein secondary structure prediction methods by employing statistical techniques. Biomedical and Biotechnology Research Journal (BBRJ) 2017 Jul 1;1(1):29–36. doi: 10.4103/bbrj.bbrj_28_17. [DOI] [Google Scholar]
- 60.Burley S.K., Berman H.M., Kleywegt G.J., Markley J.L., Nakamura H., Velankar S. Protein Data Bank (PDB): the single global macromolecular structure archive. Protein crystallography: methods and protocols. 2017:627–641. doi: 10.1007/978-1-4939-7000-1_26. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 61.Varadi M., Anyango S., Deshpande M., Nair S., Natassia C., Yordanova G., Yuan D., Stroe O., Wood G., Laydon A., Žídek A. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 2022;50(D1):D439–D444. doi: 10.1093/nar/gkab1061. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 62.Halgren T. New method for fast and accurate binding‐site identification and analysis. Chem. Biol. Drug Des. 2007;69(2):146–148. doi: 10.1111/j.1747-0285.2007.00483.x. [DOI] [PubMed] [Google Scholar]
- 63.Vemula D., Maddi D.R., Bhandari V. Homology modeling, virtual screening, molecular docking, and dynamics studies for discovering Staphylococcus epidermidis FtsZ inhibitors. Front. Mol. Biosci. 2023;10 doi: 10.3389/fmolb.2023.1087676. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 64.Yang Y., Yao K., Repasky M.P., Leswing K., Abel R., Shoichet B.K., Jerome S.V. Efficient exploration of chemical space with docking and deep learning. J. Chem. Theor. Comput. 2021;17(11):7106–7119. doi: 10.1021/acs.jctc.1c00810. [DOI] [PubMed] [Google Scholar]
- 65.Vemula D., Mohanty S., Bhandari V. Repurposing of Food and Drug Administration (FDA) approved library to identify a potential inhibitor of trypanothione synthetase for developing an antileishmanial agent. Heliyon. 2024;10(6) doi: 10.1016/j.heliyon.2024.e27602. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 66.Schrödinger Release 2024-1: QikProp. Schrödinger, LLC; New York, NY: 2024. [Google Scholar]
- 67.Nosé S. A unified formulation of the constant temperature molecular dynamics methods. J. Chem. Phys. 1984;81(1):511–519. doi: 10.1063/1.447334. [DOI] [Google Scholar]
- 68.Shivakumar D., Williams J., Wu Y., Damm W., Shelley J., Sherman W. Prediction of absolute solvation free energies using molecular dynamics free energy perturbation and the OPLS force field. J. Chem. Theor. Comput. 2010;6(5):1509–1519. doi: 10.1021/ct900587b. [DOI] [PubMed] [Google Scholar]
- 69.Wang Z., Wang X., Li Y., Lei T., Wang E., Li D., Kang Y., Zhu F., Hou T. farPPI: a webserver for accurate prediction of protein-ligand binding structures for small-molecule PPI inhibitors by MM/PB (GB) SA methods. Bioinformatics. 2019;35(10):1777–1779. doi: 10.1093/bioinformatics/bty879. [DOI] [PubMed] [Google Scholar]
- 70.Vivekanandan S., Vetrivel U., Hanna L.E. Design of human immunodeficiency virus-1 neutralizing peptides targeting CD4-binding site: an integrative computational biologics approach. Front. Med. 2022;9 doi: 10.3389/fmed.2022.1036874. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 71.Singh N., Chaput L., Villoutreix B.O. Virtual screening web servers: designing chemical probes and drug candidates in the cyberspace. Briefings Bioinf. 2021;22(2):1790–1818. doi: 10.1093/bib/bbaa034. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 72.James N., Shanthi V., Ramanathan K. Density functional theory and molecular simulation studies for prioritizing anaplastic lymphoma kinase inhibitors. Applied biochemistry and biotechnology. 2020 Apr;190:1127–1146. doi: 10.1007/s12010-019-03156-1. [DOI] [PubMed] [Google Scholar]
- 73.Zhao B., Wang C., Zhao S., Qin M., Zhou Z., Sun Y. Density functional theory study on the structure and vibrational frequencies of glycylglycine. Spectrochim. Acta Mol. Biomol. Spectrosc. 2008 Jul 1;70(2):301–306. doi: 10.1016/j.saa.2007.08.009. [DOI] [PubMed] [Google Scholar]
- 74.Bochevarov A.D., Harder E., Hughes T.F., Greenwood J.R., Braden D.A., Philipp D.M., Rinaldo D., Halls M.D., Zhang J., Friesner R.A. Jaguar: a high‐performance quantum chemistry software program with strengths in life and materials sciences. Int. J. Quant. Chem. 2013 Sep 15;113(18):2110–2142. doi: 10.1002/qua.24481. [DOI] [Google Scholar]
- 75.Ferri M., Ranucci E., Romagnoli P., Giaccone V. Antimicrobial resistance: a global emerging threat to public health systems. Crit. Rev. Food Sci. Nutr. 2017;57(13):2857–2876. doi: 10.1080/10408398.2015.1077192. [DOI] [PubMed] [Google Scholar]
- 76.Aslam B., Wang W., Arshad M.I., Khurshid M., Muzammil S., Rasool M.H., Nisar M.A., Alvi R.F., Aslam M.A., Qamar M.U., Salamat M.K.F. Antibiotic resistance: a rundown of a global crisis. Infect. Drug Resist. 2018:1645–1658. doi: 10.2147/IDR.S173867. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 77.Kunz Coyne AJ., El Ghali A., Holger D., Rebold N., Rybak M.J. Therapeutic strategies for emerging multidrug-resistant Pseudomonas aeruginosa. Infectious diseases and therapy. 2022 Apr;11(2):661–682. doi: 10.1007/s40121-022-00591-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 78.Breidenstein E.B., de la Fuente-Núñez C., Hancock R.E. Pseudomonas aeruginosa: all roads lead to resistance. Trends Microbiol. 2011 Aug 1;19(8):419–426. doi: 10.1016/j.tim.2011.04.005. [DOI] [PubMed] [Google Scholar]
- 79.Meletis G., Bagkeri M. Pseudomonas aeruginosa: multi-drug-resistance development and treatment options. Infect. Control. 2013 May 29;2:34–45. doi: 10.5772/55616. [DOI] [Google Scholar]
- 80.Babic N., Kovacic F. Predicting drug targets by homology modelling of Pseudomonas aeruginosa proteins of unknown function. PLoS One. 2021 Oct 14;16(10) doi: 10.1371/journal.pone.0258385. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 81.Rouli L., Merhej V., Fournier P.E., Raoult D. The bacterial pangenome as a new tool for analysing pathogenic bacteria. New microbes and new infections. 2015 Sep 1;7:72–85. doi: 10.1016/j.nmni.2015.06.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 82.Bayat F., Hilal A., Thirugnanasampanthar M., Tremblay D., Filipe C.D., Moineau S., Didar T.F., Hosseinidoust Z. High throughput platform technology for rapid target identification in personalized phage therapy. Nat. Commun. 2024 Jul 11;15(1):5626. doi: 10.1038/s41467-024-49710-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Data Availability Statement
The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.