Screening and identification of potential MERS-CoV papain-like protease (PLpro) inhibitors; Steady-state kinetic and Molecular dynamic studies

Abstract

MERS-CoV belongs to the coronavirus group. Recent years have seen a rash of coronavirus epidemics. In June 2012, MERS-CoV was discovered in the Kingdom of Saudi Arabia, with 2,591 MERSA cases confirmed by lab tests by the end of August 2022 and 894 deaths at a case-fatality ratio (CFR) of 34.5% documented worldwide. Saudi Arabia reported the majority of these cases, with 2,184 cases and 813 deaths (CFR: 37.2%), necessitating a thorough understanding of the molecular machinery of MERS-CoV. To develop antiviral medicines, illustrative investigation of the protein in coronavirus subunits are required to increase our understanding of the subject. In this study, recombinant expression and purification of MERS-CoV (PLpro), a primary goal for the development of 22 new inhibitors, were completed using a high throughput screening methodology that employed fragment-based libraries in conjunction with structure-based virtual screening. Compounds 2, 7, and 20, showed significant biological activity. Moreover, a docking analysis revealed that the three compounds had favorable binding mood and binding free energy. Molecular dynamic simulation demonstrated the stability of compound 2 (2-((Benzimidazol-2-yl) thio)-1-arylethan-1-ones) the strongest inhibitory activity against the PLpro enzyme. In addition, disubstitutions at the meta and para locations are the only substitutions that may boost the inhibitory action against PLpro. Compound 2 was chosen as a MERS-CoV PLpro inhibitor after passing absorption, distribution, metabolism, and excretion studies; however, further investigations are required.

1. Introduction

In June 2012, Coronavirus associated with Middle East Respiratory Syndrome (MERS-CoV) was encountered in a Saudi Arabian patient. The patient had been hospitalized with a severe case of pneumonia. A sputum sample was examined to isolate the virus. In September 2012, different cases were reported, and more than 2,040 laboratories confirmed MERS-CoV infection cases with 712 fatalities in 27 countries from September 2012 to March 2019. Between April 2012 and August 2022, an amount of 2,591 cases of the Middle East respiratory syndrome have been confirmed by laboratories and 894 deaths at a case-fatality ratio (CFR) of 34.5 % were reported worldwide. The bulk of recorded cases, 2184 cases, and 813 deaths originated in Saudi Arabia (CFR: 37.2 %)(WHO 2022). Coronavirus clinical survivors and discharged patients have functional impairment of the lungs, heart, liver, and other health problems which need further medical assistance (Chan et al., 2003, Han et al., 2003). Coronaviruses are etiological agents of many deadly diseases that spread among animals, leading to catastrophic harm in farming (Vlasova et al. 2014). Moreover, Coronaviruses have high recombination and mutation rates, making it possible for them to infect new hosts of a different species. (Denison et al. 2011). MERS-CoV is still triggering the alarm, particularly in the KSA, and some other middle east countries and represents an epidemic threat without effective remedies. Therefore, the disease requires accurate screening for the development of effective remedies.

It is likely that the molecular dynamics and pathophysiology of MERS-CoV, which has RNA with a positive-sense, single-stranded, have been studied extensively because of its similarity to other types of dangerous human coronaviruses that are known to cause a number of respiratory complications syndrome, including HCoV-229E, HCoV-NL63, HCoV-OC43(van Boheemen et al., 2012, Kraaij-Dirkzwager et al., 2014, Sipulwa et al., 2016). The structure of the genome is a single-stranded RNA (ssRNA) which encode11 different proteins; 2 replicase polyproteins (open reading frames [ORFs] 1a and 1b), 4 structural proteins (S, E, N, and M), and 5 nonstructural proteins (ORFs 3, 4a, 4b, and 5) (Eckerle et al., 2013, Wang et al., 2013). The two ORFs (ORF1a, ORF1b) encode a replicase complex, considering that the remaining accessory ORFs each code for one of five different accessory proteins which are essential to infection and pathogenesis (Menachery et al. 2017). The spike protein resides on the surface plays an important part in the transmission of zoonotic pathogens through the mediation of virus-receptor recognition and the subsequent beginning stages of an infection caused by a virus. The MERS-S CoV protein is a transmembrane protein with pair subunits, subunits 1(S1) and subunits 2(S2). The first subunit is equipped with a receptor-binding domain (RBD) that interacts with the host’s dipeptidyl peptidase 4 (DPP4) receptor (Zumla et al., 2015, Mustafa et al., 2018). MERS-CoV utilizes the host cell’s DPP4 receptor for cell entrance by binding its S protein. Double heptad repeats, denoted by the designations H1 and H2, from the S2 subunit make up the primary membrane fusion unit. All three stages of the MERS-CoV life cycle, budding, intracellular transport, and assembly are dependent on the envelope (E) protein. of MERS-CoV, while the morphogenesis process cannot occur without the membrane (M) protein. but not necessary for viral replication (Liu et al., 2010, Raj et al., 2013, Xia et al., 2014, Surya et al., 2015).

MERS-CoV has 11 open reading frames (ORF) that get translated within the infected cells to different proteins to start the infection cycle (Zhang et al. 2016). ORF1a and ORF1b encode polyproteins, polyprotein1a and polyprotein 1b, which undergo processing by a papain-like protease (PLpro) and a 3-C-like protease (3CLpro) (Lei and Hilgenfeld 2016). The former usually exists in two isoforms, PLP1 and PLP2, in many coronaviruses. MERS-CoV and SARS-CoV, however, express sole PLpro enzyme, which is a component of a big nonstructural protein 3 (nsp3) that includes four other domains, including a ubiquitin-like fold (UB1), an ADP-ribose-1d-phosphatase (ADRP) domain, a SARS-unique domain (SUD), and a transmembrane (TM) domain. (Yang et al., 2014, Lee et al., 2015). PLpro is the enzyme that cleaves the polyprotein at the first three positions, and 3CLpro is the enzyme that cleaves the polyprotein at the remaining 11 locations, causing the release of a combined total of 16 different non-structural proteins (NSPs) in MERS-CoV and SARS-CoV (Lei and Hilgenfeld 2016).

Once MERS-CoV enters a host cell, it begins its life cycle through endocytosis or the fusing of the spike protein of the virus with the endosomal membrane of the host cell. Inside the host cell, a double membrane vesicle is formed, liberating the RNA enclosed in the nucleocapsid, after which transcription of the viral genome takes place. Thereafter, viral RNA is liberated into the cytoplasm, and the infected cells produce and release the progeny virus through exocytosis (Wang et al., 2016b, Skariyachan et al., 2019).

The various stages that occur throughout the life cycle of viruses present a potential opportunity for the discovery of antiviral inhibitors. (Liang et al. 2018). (Liang et al. 2018) summarized new strategies to increase the efficiency of screening small-molecule inhibitors, which are expected to lessen the likelihood of future MERS-CoV infections.

The luciferase-based biosensor assay strategy selects PLpro and 3CLpro inhibitors as potential targets via transfection of two plasmids into HEK293T cells utilizing a cell-based screening test for MERS-CoV; the expression contains a MERS-CoV PLpro sequence, nsp4, and nsp5 nonstructural proteins, and the N-terminal 6 region. In addition, circularly permuted Photuris pennsylvanica luciferase and the amino sequence of the PLpro or 3CLpro cleavage site are both contained in biosensor expression plasmids (Kilianski et al. 2013).

(Gierer et al. 2013) developed pseudovirus-based assays for screening antiviral compounds targeting part of the virus life cycle using lentiviral particles as non-infectious viruses. This makes it possible to infect multiple types of cells in a single cycle. that express dipeptidyl peptidase-4 (DPP4), and the results were consistent with those obtained from an inhibition assay based on live MERS-CoV.

In structure-guided design, a piperidine moiety is incorporated into the design to provide the best possible pharmacological activity and protein kinase properties through a binding interaction by 3CLpro's S3 and S4 subsites (Galasiti Galasiti Kankanamalage et al. 2018).

The ubiquitin-like domain 2 (Ubl2) strategy also occurs in coronavirus polyproteins. Ubl2 is close to the N-terminus of the PLpro domain. However, evidence suggests that the MERS PLpro can either act as an interferon antagonist or perform its primary function, which is to process the polyprotein, which includes removing ubiquitin and removing ISGyl groups from cellular proteins. (Clasman et al. 2017).

The antiviral inhibitors strategy (Josset et al., 2013) analyzes the way by which the MERS-CoV infection influence and interferes with the host cells by infecting the respiratory system epithelial cell with MERS-CoV and analyzing the transcriptome to find blocking substances in the host.

MERS-CoV poses a considerable challenge due to the lack of therapeutics and effective countermeasures. Current treatments depend on supportive care and complication control. Only a few compounds are reported to have PLpro inhibitory activity. These compounds include N-ethylmaleimide (NEM), mycophenolic acid, 8-(trifluoromethyl)-9H-purin-6-amine, 6-thioguanine (6TG) and 6-mercaptopurine (6MP) (Cheng et al. 2015). Therefore, developing therapies is highly important to saving lives and slowing down or even stopping the spread of MERS-CoV. One of the most important drug targets in the field of virology is protease. (Bannwarth and Reboud-Ravaux, 2007, Skoreński and Sieńczyk, 2013, Wang et al., 2016a). Coronavirus proteases, especially 3CLpro and PLpro, are considered attractive antiviral targets since they are in charge of breaking down nonstructural polyproteins (pp1a and pp1b) that play an indispensable role in viral maturation (Hilgenfeld, 2014, Lin et al., 2014).

Three amino acids, Cys111, His278, and Asp293, work together as a catalytic triad in the MERS-CoV PLpro. (Lin et al. 2014). Cys111, which is found in the PLpro of the MERS-CoV, is positioned at the N-terminus of α4 and points into the substrate-binding cleft which is located between the thumb and palm domains. During the process of enzyme crystallization, a disulfide bond was formed between the catalytic cysteine and β-mercaptoethanol (BME), as indicated by the obvious presence of electron density indicating this modification. His278 is situated at the N-terminus of β16; the distance separating its Nδ 1 atom and the sulfur of Cys111 is 4.4 Å. This long distance is probably because of the mercaptoethanol -modification of Cys111; in the majority of structures of (PLpro), the distances are between 3.7 and 4.Å. Despite the fact that the His278 side chain is moved from its normal position in the catalytic triad, Asp293 and Cys111 get along extremely well with their equivalents in SARS-CoV PLpro and USP14.

Through the donation of two hydrogen bonds, papain-like cysteine proteases are able to maintain the intermediate form that the proteolytic reaction takes. that they are responsible for catalyzing inside the oxyanion hole, the first one from the amide of the catalytic cysteine residue, while the second one from the amide of the side-chain of a Gln or residues of Asn at positions 5 and 6 N-terminal to the catalytic Cys residue. For instance, the last amino acid in USP14 and HAUSP is Asn. (Hu et al. 2005), whereas in (UCH)-L3 and UCH-L, Gln in the ubiquitin C-terminal hydrolases is the last residue (Johnston et al. 1997).

Ratia et al. (Ratia et al. 2006) showed that the SARS-PLpro tryptophan W107 located underneath the catalytic cysteine has a crucial function in creating a hydrogen bond (H-bond) with transitional as H-bond donor in the active site by demonstrating that the SARS-PLpro Trp107Ala mutant completely devoid of all catalytic activity. But, Leu106 occupies the corresponding position in the active site of MERS-PLpro, which cannot function as H-bond donor (Lee et al. 2015). MERS-CoV PL(pro) and SARS-CoV PLpro are exactly equivalent, on the other hand, there are specificity sites differences in oxyanion hole, S3, and S5. These variations may explain why MERS-CoV PLpro has lower in vitro peptide hydrolysis and deubiquitinating activity than SARS PLpro. The Leu106Trp mutation increases the in vitro catalytic activity of MERS-CoV PLpro(Lei et al. 2014). Surprisingly, coronaviruses in the bat which are part of the same lineage group C as MERS-CoV have a high degree of conservation of the leucine residue at this position. However, similar to SARS-PLpro, the PLpro enzymes of human coronaviruses (229E and NL63) feature a residue (Thr or Gln) that functions as H-bond donor. As a result, this transitional-stabilizing role in MERS-PLpro must be played by a different residue. Ratia et al. presumed that SARS-asparagine PLpro's (N110), which is highly preserved amongst many coronavirus PLpro enzymes, would play a role in oxyanion hole stabilization with W107. (Ratia et al. 2006). Lee et al. observed, according to the structural orientation of the active site, that the N109 of MERS-PLpro overlaps the N110 of SARS-PLpro. This proposed that the residue N109, positioned atop catalytic cysteine, may perform this crucial character in MERS-PLpro; two possible mechanisms were proposed. (1) The amine group that is located on the side chain of N109 has the potential to form an H-bond with the oxyanion that is located on the intermediate donor, or (2) N109 carbonyl group has the potential to form a hydrogen bond with H2O, which would then turn another H-bond into the water, with an intermediate that is negatively charged. After investigating the two above hypotheses, the results show that N109 is a key amino acid for stabilizing the intermediate, by making H-bond with the amine group on the side chain of N109 (Lee et al. 2015). Residues 101–108 which are part of the short loop close to the active site in MERS-PLpro are vital for its catalytic activity by regulating active site access and holding a key residue that stabilizes the oxyanion. Hydrogen bonding between D109 and W94 constrains the loop conformation, stopping it from shifting to obstruct accession to the active site(Ratia et al. 2006).

In this study, we optimized the production methodology of MERS-CoV PLpro as a drug target. Moreover, we report the discovery of 22 new small molecules with promising PLpro inhibitory activities. In addition, molecular docking studies were conducted to explore the structural requirements for inhibitory activity against PLpro, which is expected to help design new molecules with improved activities. Furthermore, the ADMET parameters of the molecules were determined and analyzed to evaluate their pharmacokinetic and toxicity effects.

2. Materials and methods

Open-reading frame 1 of the MERS-CoV PLpro, which encoded on the polyprotein1a, was cloned into a pET28a plasmid. The nucleotide sequence is optimized to enhance transcriptional and translational levels under the control of a strong T7 promoter (Lin et al. 2014). The codon was cloned between NcoI and XhoI in the C-terminal His tag of the vector using Genescript. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was obtained from GenScript, and E. coli DE3-pLysS was purchased from EMD4 Biosciences (San Diego, CA, USA). Glycerol and NaCl were purchased from Scharlau Chemie, S.A. (Sentmenat, Spain); chicken egg lysozyme was obtained from Honeywell Research Chemicals (Morris Plains, NJ, USA); ANS, kanamycin, and benzonase was obtained from Sigma Aldrich (St. Louis, MO, UNA). The AKTA purification system and Superdex 75 columns were obtained from Amersham Biosciences (St. Giles, UK). PAGE 4–12 % bis-tris-gel was obtained from Life Technologies (Pleasanton, CA, USA); prepacked Ni-NTA columns were purchased from GE Healthcare. Agilent Technologies (USA) supplied the Cary 60 UV–vis spectrometer. The New Brunswick Innova 44R shaking incubator and Thermomixer were obtained from Eppendorf (Hamburg, Germany).

2.1. Chemicals

2-((Benzimidazol-2-yl)thio)-1-arylethan-1-ones have been prepared as reported previously(Abdel-Aziz et al. 2015).

2.2. Expression of MERS-CoV PLpro

Escherichia coli BL21(DE3) is a frequently used host for recombinant protein high-level expression because of its deficiency in both lon and ompT proteases and its compatibility with the T7 lacO promoter system. Moreover, E. coli BL21(DE3) shelters a prophage DE3 obtained from a bacteriophage λ which contains a genomic copy of the T7 RNA polymerase gene controlled by lacUV5 promoters and is induced with isopropyl-β-d-1-thiogalactopyranoside (IPTG). T7 RNA polymerase is not produced under repressive conditions, and the transcription of the target gene is very low. After induction with IPTG, the T7 RNA polymerase is synthesized, and most of the cellular protein synthesis machinery gets activated to produce the target protein (Gräslund et al., 2008, Kortmann et al., 2015). The codon-optimized pET28a-MPLpro clone (GeneScript) was transformed into chemically competent E. coli BL 21 (DE3) pLysS cells and selected on LB_kan (200 µg/mL) plates. The protein (MERS-PLpro) was expressed and extracted following the method outlined by (Lin et al. 2014). The recombinant protein (MERS-CoV PLpro) was produced on a large scale. E.coli BL 21 (DE3) pLysS cells harboring pET28a-MPLpro were inoculated into LB_kan medium and grown overnight at 37 °C. An isolated colony was inoculated into 60 mL LB media containing Kanamicyn (200 µg mL⁻¹) and then kept in the incubator shaker overnight at 37 °C and 150 rpm. Next, 1 % overnight culture was added to 5-L flasks containing 1 L of LB_kan. The flasks were then incubated at 37 °C and 150-rpm shaking. Following an initial incubation period of 2.5 h at 20 °C, the cultures were induced with 0.4 mM of IPTG and kept in the incubator overnight. Thereafter, the cultures were harvested at 5,000 rpm for 15 min at 4 °C, the supernatant was thrown away, and the wet biomass was stored at −80 °C until further use.

2.3. Cell lysis and Extraction of MERS-CoV PLpro

Initially, (∼5 g) of the thawed E. coli wet cells were added to 20 mL of lysis buffer (20 mM Tris pH 8.0, 2 mg mL⁻¹ lysozyme, and 1 mM EDTA). The mixture was shaken and incubated at 4 °C for 30 min, then 0.2 % Triton X 100, 5 % glycerol, 300 mM NaCl, and 1 mM DTT were added. Subsequently, 6 mM MgCl² and 1 μL benzonase were added and incubated at 4 °C until viscosity disappeared, and centrifugation at 13,000 rpm was used to pellet the cellular debris for 30 min at 4 °C. Finally, the cleared lysate was composed for a further purification step.

2.4. Purification of MERS-CoV PLpro by Immobilized metal affinity chromatography

Purification of MERS-CoV PLpro was conducted with a slight deviation from the procedure described in (Malik and Alsenaidy 2017). Nickel-chelating affinity resin (Ni²⁺–Sepharose prepacked, 1 mL; GE Healthcare) was used to purify recombinant MERS-CoV PLpro. Initially, the column was equilibrated with 10 times column volumes of buffer (20 mM Tris pH 8.0, 500 mM NaCl, and 20 % glycerol), different imidazole concentrations (20, 30, and 40 mM) to determine the optimal imidazole concentration required to suppress non-specific binding on the Ni-NTA column at a flow rate of 1 mL min⁻¹. After filtering the cell extract using a 0.45-μm filter, the filtrate was passed through the column and further washed out using an equilibrium buffer to remove unbound proteins. Afterward, elution buffer (Ni-NTA equilibration buffer and 500 mM imidazole) was added to release the bound protein.

2.5. Gel filtration chromatography

Proteins of known molecular weight were used to calibrate the Superdex 75 column equilibrated with 20 mM Tris, pH 8.5, 100 mM NaCl, and 1 mM DTT. Afterward, Ni-NTA-purified MERS-CoV PLpro was additionally purified by the Superdex 75 column. The protein-containing part was pooled and evaluated by SDS-PAGE. Pure fractions were collected and stored at −80 °C. Quantification of the protein was conducted following thawing and centrifuging for 15 min at 13,000 rpm and 4 °C. the concentration of the protein was measured using a spectrophotometer at 280 nm.

2.6. MERS PLpro concentration calculation for reaction mixture for the plate reader

2.6.1. PL-pro stock

In the optimization reaction, 500 µg/mL PLpro stock was 2-fold serially diluted in 50 mM phosphate buffer pH 6.5. In each reaction, 100 µL of different dilutions of PLpro was used. Thus, the final concentrations per reaction were 50, 25, 12.5, 6.25, 3.125, 1.5, and 0.78 µg. In the enzyme inhibition assays, MERS PLpro was diluted to 100 µg/mL in 50 mM phosphate buffer, pH 6.5, after which 100 µL was used in the assay; thus, the final concentration of PLpro in each reaction was 10 µg/ reaction.

2.6.2. Inhibitor stock

100 mM inhibitor stock solution was made in DMSO; the inhibitors were diluted 50-fold in 50 mM phosphate buffer (pH 6.5). Subsequently, inhibitors concentration were tenfold serially diluted in 50 mM phosphate buffer (pH 6.5). The final concentrations of inhibitors were 2000, 200, 20, and 2 µM. Subsequently, 100 µL diluted inhibitors were added to the reaction mixture.

2.6.3. Peptide substrate stock

400 µM Peptide stock solution was prepared in DMSO. Next, 10 µL substrate was added to 200 µL reaction mixture 50 mM phosphate buffer pH 6.5. The final concentration of substrate was 20 µM.

Positive control: PLpro and substrate.

Negative control: inhibitor and substrate.

Blank: buffer and substrate.

Test: PLpro, substrate, and inhibitor.

To run the reaction, the full set was duplicated.

2.7. MERS-CoV PLpro Concentration and samples treatment

Aliquots of purified MERS-CoV PLpro samples were centrifuged for 15 min at 13,000 rpm at 4 °C with buffer exchange (10 mM Tris pH 7.0, 50 mM NaCl, and 0.1 mM DTT). The absorbance of MERS-CoV PLpro was estimated at 280 nm, (Chou et al. 2008). A theoretical extinction coefficient of 42,400 M⁻¹ cm was used to determine the concentration of the protein.

2.8. MERS-CoV PLpro activity

In this research, we measured steady-state kinetics according to the procedure described by (Malik and Alsenaidy 2017). A 20-µM substrate was mixed with 10 µg MERS-CoV PLpro in various concentrations of 10-fold serially diluted inhibitors at 25 °C using 50 mM phosphate as a buffer at pH 6.5. Measurement was conducted using a 340-nm excitation and 535-nm emission filter for 30 min. MERS-CoV PLpro steady-state kinetic was evaluated using the fluorogenic peptidyl substrate,

Dabcyl–FRLKGGAPIKGV–Edans, to study the activity of the enzyme.

2.9. Docking study

The MOE program was employed to perform docking studies for all the screened compounds. The model of MERS-CoV PLpro (Protein Data Bank [PDB] ID: 4RNA, chains A) was utilized for a docking process (Lee et al., 2015). Protonation was performed on the crystal structure of the protein of interest utilizing the Protonate 3D technique (Labute 2009), and energy minimization was achieved with the help of the MOE-applied AMBER12 force field. The active site was identified using the site finder module of Molecular Operating Environment (MOE) 2019 (Chemical Computing Group, Montreal, Canada). As a method of placement during the docking procedure, the triangle matcher algorithm was also employed. The generalized Born volume integral/weighted surface area dG rescoring function and the London dG scoring function were also utilized in this study. Each compound was docked to its respective interaction binding site. Following this, the interaction of vital residues between compounds and proteins was fingerprinted using a protein–ligand interaction fingerprint (PLIF). (Da and Kireev 2014). Further binding positions were examined visually by MOE 2019.

2.10. PLIF analysis

PLIF investigation was conducted by MOE 2019.10′s PLIF module. Through the process of changing 3D structural binding data to 1D binary strings, this analysis provided important data about protein–ligand interactions (El-Hasab et al. 2018). Protease preparation steps included hydrogen atom addition, disulfide fixation, bond order, and assignment of protonation state, after which energy minimization, applying the AMBER10: extended Hückel theory force field, was conducted, then the protein structures were superimposed. Finally, the binding site residues of the proteases were labeled to create the interaction fingerprints. These fingerprints offer insightful data that can be applied to clustering, virtual screening, and creating pharmacophore models.

2.11. Molecular Dynamic (MD) simulations

MD simulations were executed for the docked pose of lead 2 reported MERS-CoV PLpro inhibitor, using NAMD_Git-2021–09-06_Linux-x86_64-multicore(Phillips et al. 2005), and the crystal structure of the MERS-CoV PLpro enzyme at a temperature of 303.15 K. The CHARMM-GUI (https://www.charmm-gui.org/) was a website used to generate the configuration files that were used for MD simulation. The ligand was parameterized with the help of the CHARMM General Force Field (CGenFF) web application (https://cgenff.umaryland.edu/)(Phillips et al., 2005, Jo et al., 2008, Brooks, Brooks et al., 2009, Lee et al., 2016). The transferable intermolecular potential water molecules (TIP3P) model has utilized in order to solvate all system. The system was then neutralized by the addition of 0.15 M counter ions (Na + and Clâ'), and energy minimization was achieved by taking advantage of the steepest decline and conjugate gradients (25000 steps) for all the neutralized systems. followed by conjugate gradients. The systems' energy was minimized for 10,000 steps during equilibration on a 5 ns timeframe using the NVT ensemble, followed by production on a 25 ns timescale using the NPT ensemble. The LINC method was followed to constrain the bonds and angles. The complexes were subjected to molecular dynamics simulations (25 ns). All post-MD analyses, including RMSD, RMSF, and Rg, were carried out with NAMD tools. Trajectory visualizations and images were created using VMD-1.9.1 (Humphrey et al. 1996).

2.12. ADMET Forecasting

ADMET forecasting was conducted utilizing the ADMET technique in Discovery Studio 2.5 (Accelrys, San Diego, CA, USA) and a prediction method developed by Hakami et al. (Hakami et al. 2022). Ligands were prepared as stated in the preceding section. ADMET prediction was accomplished by computing both polar surface area and AlogP 98, and then plotting it on a 2 dimensional plane via pattern recognition utilizing information on the bioavailability of pharmaceuticals (Ghazwani et al. 2021). Molecules with greater than 90 % absorbability were distinguished from those that were absorbed or partially absorbed.

3. Result and discussion

3.1. Expression and purification of recombinant MERS-CoV PL pro in E. Coli

MERS-CoV PLpro was expressed in E. coli BL21 (DE3) pLysS and purified via double-steps chromatography as defined in (Malik and Alsenaidy 2017). To determine the optimum growth time for producing MERS-CoV PLpro, two shake flasks were examined, and at various intervals, 0.4 mM IPTG was used to induce cultures. As explained in the Method section, the E. coli cell pellet was subjected to a gentle chemical lysis procedure to extract soluble proteins. Soluble MERS-CoV PLpro was found in the supernatant after centrifugation. Next, nickel column affinity chromatography was used to purify recombinant MERS-CoV PLpro, which contained 6xHis-Tag in its C-terminal. After passing the soluble cell extract through the Ni-NTA column, it was washed with Ni-NTA equilibration buffer to remove unbound and loosely bound proteins. Subsequently, His-tagged MERS-CoV PLpro was eluted with a buffer containing 500 mM imidazole (Fig. 1). When the Ni-NTA eluate passed through the Superdex 75 gel filtration column, an insignificant impurity peak and a significant symmetrical peak was found. The symmetrical sharp peak was obtained (Fig. 2A). On SDS-PAGE, the eluted fractions were examined to determine their level of purity. MERS-CoV PLpro was expressed at a right size of 36.5 (Fig. 2.B). A 1-L shake flask yielded approximately 50 mg of pure MERS-CoV PLpro.

Fig. 1.

His-tagged MERS-CoV PLpro purification. Eluted with a buffer containing 500 mM imidazole.

Fig. 2.

A. Ni-NTA PLpro elute was purified via gel Superdex 75. small impurity peak and one major symmetrical sharp peak was obtained. B. SDS-PAGE. Lane 1& 2 marker& pooled fractions of MERS-CoV PLpro.

3.2. Inhibition of MERS-CoV PLpro activity

Dabcyl–FRLKGGAPIKGV–Edans is a fluorogenic substrate for the MERS-CoV papain-like protease (PLpro) that recognizes LKGG consensus cleavage sites. When PLpro cuts between glycine and alanine, the resulting APIKGV-Edans fluorescence can be seen at excitation wavelengths of 355 nm and emission wavelengths of 538 nm. When the MERS-CoV PLpro mixed with substrate and the stock solution of 22 inhibitors defined concentrations, we noticed that compounds 2,7 and 20 showed significant loss of PLpro activity at 1,000 µM (Fig. 3).

Fig. 3.

Inhibitors 2,7 and 20 shows clear loss of activity at 1000 µM,

3.3. Identification of ligand binding sites

The potential drug-binding sites were identified on the X-ray crystal 3 structure of PLpro using the site finder module of MOE. Regarding PLpro, we discovered a representative drug-binding site near the critical residuals (Cys111, His278, and Asp293). (Fig. 4).

Fig. 4.

Comprehensive examination of fingerprint of PLpro protein–ligand interaction. (A) The x-axis displays the residue’s numbers corresponding to every group of fingerprints and is marked with random colors. (B) The frequency of occurrence of fingerprint bits at the top of the largest bar is shown by the bit count.

3.4. PLIF analysis

As illustrated in Fig. 4, using PLIF analysis allows for the construction of an automatic picture of the significant interactions that occur with the residues. The fingerprint of the compounds showed multiple interactions, comprising backbone acceptor and surface, ionic, and side chain interactions. Furthermore, the fingerprint brought to light some important residues, such as Asn109, Cys111, Pro163, Asp164, Thr274, Ala275, Val276, and His278, which contributed to the interaction between proteins and ligands.

Within highly potent compounds, His278 and Cys111, besides Asn109, revealed side chain and surface interactions. Moreover, in the majority of complexes, Cys111 was found to exhibit side chain acceptor, bond donor, and backbone acceptor interactions. Finally, the aromatic ring of His278 caused surface interactions with ligands. The ligand display graphic in Table 1 shows the ligands of the specific entries, in diagrammatic form, with a simultaneous alignment representative of bound conformation, and also shows the interactions in which it participates.

Table 1.

Binding energies and hydrogen bond interactions of specific ligands with active site residues of MERS-CoV PLpro.

Inhibitor	Ligand	Receptor	Interaction	Distance	E (kcal/mol)	Docking score1	Chemical structure
1	N 11	SD CYS 111 (A)	H-donor	3.62	−0.2	−7.071
	S 13	O ALA 275 (A)	H-donor	3.74	−2.3
	N 15	O VAL 276 (A)	H-donor	2.97	−0.6
	5-ring	CB PRO 163 (A)	pi-H	3.33	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.31	−0.5
	6-ring	CE1 PHE 269 (A)	pi-H	4.86	−0.2
2	C 2	O VAL 276 (A)	H-donor	3.14	−0.2	−9.651
	N 11	SD Cys 111 (A)	H-donor	3.59	−0.2
	N 17	O VAL 276 (A)	H-donor	2.99	−0.6
	5-ring	CB PRO 163 (A)	pi-H	3.34	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.3	−0.5
	6-ring	CB HIS 278 (A)	pi-H	3.82	−0.2
3	C 2	O VAL 276 (A)	H-donor	3.14	−0.2	−9.031
	N 11	SD CSS 111 (A)	H-donor	3.59	−0.2
	N 17	O VAL 276 (A)	H-donor	3	−0.6
	C 27	O THR 274 (A)	H-donor	3.46	−0.2
	5-ring	CB PRO 163 (A)	pi-H	3.35	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.31	−0.5
	6-ring	CE1 PHE 269 (A)	pi-H	4.86	−0.2
	6-ring	CB HIS 278 (A)	pi-H	3.81	−0.2
4	N 11	SD CSS 111 (A)	H-donor	3.67	−0.2	−7.481
	N 17	O VAL 276 (A)	H-donor	2.86	−0.8
	5-ring	CB PRO 163 (A)	pi-H	3.32	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.31	−0.6
	6-ring	CE1 PHE 269 (A)	pi-H	4.83	−0.2
	6-ring	CB HIS 278 (A)	pi-H	3.63	−0.6
5	C 2	O VAL 276 (A)	H-donor	3.14	−0.2	−8.192
	N 11	SD CSS 111 (A)	H-donor	3.59	−0.2
	N 17	O VAL 276 (A)	H-donor	2.99	−0.7
	C 23	O THR 274 (A)	H-donor	3.43	−0.2
	5-ring	CB PRO 163 (A)	pi-H	3.34	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.3	−0.5
	5-ring	CB HIS 278 (A)	pi-H	3.93	−0.8
6	N 11	SD CSS 111 (A)	H-donor	3.66	−0.2	−8.051
	N 17	O VAL 276 (A)	H-donor	2.88	−0.8
	5-ring	CB PRO 163 (A)	pi-H	3.33	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.32	−0.6
	6-ring	CE1 PHE 269 (A)	pi-H	4.84	−0.2
	6-ring	CA GLY 277 (A)	pi-H	4.71	−0.3
	6-ring	CB HIS 278 (A)	pi-H	3.82	−0.2
7	C 2	O VAL 276 (A)	H-donor	3.14	−0.2	−9.32
	N 11	SD CyS 111 (A)	H-donor	3.62	−0.2
	N 17	O VAL 276 (A)	H-donor	2.97	−0.7
	5-ring	CB PRO 163 (A)	pi-H	3.33	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.31	−0.5
	6-ring	CB HIS 278 (A)	pi-H	3.74	−0.3
8	C 2	O VAL 276 (A)	H-donor	3.13	−0.2	−8.037
	N 11	SD CSS 111 (A)	H-donor	3.59	−0.2
	N 17	O VAL 276 (A)	H-donor	3	−0.6
	C 32	O THR 274 (A)	H-donor	3.44	−0.2
	5-ring	CB PRO 163 (A)	pi-H	3.35	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.32	−0.5
	6-ring	CE1 PHE 269 (A)	pi-H	4.87	−0.2
	6-ring	CB HIS 278 (A)	pi-H	3.8	−0.2
9	N 11	SD CYS 111 (A)	H-donor	3.39	−0.3	−7.65
	S 13	O ALA 275 (A)	H-donor	3.81	−0.4
	N 17	O VAL 276 (A)	H-donor	2.91	−0.4
	N 11	SD CYS 111 (A)	H-acceptor	3.39	−0.4
	5-ring	CB PRO 163 (A)	pi-H	3.4	−0.3
	6-ring	CE1 PHE 269 (A)	pi-H	4.81	−0.3
10	N 11	SD CSS 111 (A)	H-donor	3.63	−0.2	−8.7
	N 17	O VAL 276 (A)	H-donor	2.94	−0.7
	5-ring	CB PRO 163 (A)	pi-H	3.31	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.3	−0.6
	6-ring	CB HIS 278 (A)	pi-H	3.63	−0.6
11	N 11	SD CSS 111 (A)	H-donor	3.66	−0.2	−7.415
	N 17	O VAL 276 (A)	H-donor	2.94	−0.7
	5-ring	CB PRO 163 (A)	pi-H	3.33	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.32	−0.6
	6-ring	CB HIS 278 (A)	pi-H	3.68	−0.3
12	N 11	SD CSS 111 (A)	H-donor	3.63	−0.2	−8.656
	N 17	O VAL 276 (A)	H-donor	2.9	−0.7
	N 11	SD CSS 111 (A)	H-acceptor	3.63	−0.2
	5-ring	CB PRO 163 (A)	pi-H	3.33	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.3	−0.5
	6-ring	CE1 PHE 269 (A)	pi-H	4.82	−0.2
	6-ring	CB HIS 278 (A)	pi-H	3.5	−0.5
13	N 11	SD CSS 111 (A)	H-donor	3.73	−0.2	−6.914
	N 17	O VAL 276 (A)	H-donor	2.85	−0.8
	5-ring	CB PRO 163 (A)	pi-H	3.33	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.34	−0.6
	6-ring	CE1 PHE 269 (A)	pi-H	4.85	−0.2
	6-ring	CB HIS 278 (A)	pi-H	3.67	−0.4
14	N 11	SD CSS 111 (A)	H-donor	3.74	−0.2	−7.292
	N 17	O VAL 276 (A)	H-donor	2.88	−0.9
	5-ring	CB PRO 163 (A)	pi-H	3.3	−0.2
	6-ring	N ASP 164 (A)	pi-H	4.33	−0.7
	6-ring	CB HIS 278 (A)	pi-H	3.92	−0.3
15	N 11	SD CSS 111 (A)	H-donor	3.63	−0.2	−9.067
	N 17	O VAL 276 (A)	H-donor	2.96	−0.7
	5-ring	CB PRO 163 (A)	pi-H	3.33	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.31	−0.5
	6-ring	CB HIS 278 (A)	pi-H	3.73	−0.3
16	C 2	O VAL 276 (A)	H-donor	3.13	−0.2	−8.291
	N 11	SD CSS 111 (A)	H-donor	3.59	−0.2
	N 17	O VAL 276 (A)	H-donor	2.99	−0.6
	5-ring	CB PRO 163 (A)	pi-H	3.35	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.31	−0.5
	6-ring	CB HIS 278 (A)	pi-H	3.76	−0.3
17	C 2	O VAL 276 (A)	H-donor	3.14	−0.2	−8.141
	N 11	SD CSS 111 (A)	H-donor	3.58	−0.2
	C 14	O GLY 277 (A)	H-donor	3.52	−0.2
	N 17	O VAL 276 (A)	H-donor	2.98	−0.6
	5-ring	CB PRO 163 (A)	pi-H	3.35	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.31	−0.5
	6-ring	CB HIS 278 (A)	pi-H	3.62	−0.3
18	C 2	O VAL 276 (A)	H-donor	3.14	−0.2	−7.35
	N 11	SD CSS 111 (A)	H-donor	3.58	−0.2
	C 14	O GLY 277 (A)	H-donor	3.52	−0.2
	N 17	O VAL 276 (A)	H-donor	2.98	−0.6
	5-ring	CB PRO 163 (A)	pi-H	3.34	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.3	−0.5
	6-ring	CB HIS 278 (A)	pi-H	3.62	−0.3
19	C 2	O VAL 276 (A)	H-donor	3.13	−0.2	−7.607
	N 11	SD CSS 111 (A)	H-donor	3.53	−0.2
	C 14	O GLY 277 (A)	H-donor	3.56	−0.3
	N 17	O VAL 276 (A)	H-donor	3.04	−0.5
	N 11	SD CSS 111 (A)	H-acceptor	3.53	−0.2
	5-ring	CB PRO 163 (A)	pi-H	3.38	−0.4
	6-ring	N ASP 164 (A)	pi-H	4.3	−0.4
	6-ring	CE1 PHE 269 (A)	pi-H	4.87	−0.2
20	N 11	SD CSS 111 (A)	H-donor	3.59	−0.2	−9.485
	N 17	O VAL 276 (A)	H-donor	2.89	−0.8
	C 25	O THR 274 (A)	H-donor	3.48	−0.2
	N 11	SD CSS 111 (A)	H-acceptor	3.59	−0.2
	5-ring	CB PRO 163 (A)	pi-H	3.33	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.29	−0.5
	6-ring	CE1 PHE 269 (A)	pi-H	4.81	−0.2
	6-ring	CB HIS 278 (A)	pi-H	3.84	−0.3
21	N 11	SD CSS 111 (A)	H-donor	3.63	−0.2	−8.424
	N 17	O VAL 276 (A)	H-donor	2.88	−0.8
	C 25	O THR 274 (A)	H-donor	3.53	−0.2
	N 11	SD CSS 111 (A)	H-acceptor	3.63	−0.2
	5-ring	CB PRO 163 (A)	pi-H	3.34	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.3	−0.5
	6-ring	CE1 PHE 269 (A)	pi-H	4.83	−0.2
	6-ring	CB HIS 278 (A)	pi-H	3.85	−0.3
22	C 2	O VAL 276 (A)	H-donor	3.14	−0.2	−9.479
	N 11	SD CSS 111 (A)	H-donor	3.61	−0.2
	N 17	O VAL 276 (A)	H-donor	2.97	−0.7
	5-ring	CB PRO 163 (A)	pi-H	3.34	−0.3
	6-ring	N ASP 164 (A)	pi-H	4.31	−0.5
	5-ring	CB HIS 278 (A)	pi-H	3.99	−0.2
	N 3	SD CYS 111 (A)	H-acceptor	3.2	−0.3

3.5. Docking

The Docking program is widely employed in drug development strategies, and in the forecasting of the interaction between drugs and proteins or macromolecular in pharmaceutical research. The docking technique includes seeking for binding sites on the entire protein surface. The PLpro from MERS (4RNA), SARS (4OW0), SARS-CoV-2 (7JIW), and MHV (5WFI) are compared. Both of similarity and configuration or alignment calculations were executed using MOE2019.10 (Fig. 6).

Fig. 6.

Positions of residues forming catalytic triads are indicated by red triangles, while amino acids responsible of making ubiquitination and interferon simulating gene glycation binding sites are indicated by azure stars. The blocking loop2 (BL2) is enclosed in a yellow rectangle.

The secondary structure shown in (Fig. 7) was predicted online using JPred4 for MERS-CoV PLpro (4RNA)(JPred4 2015).

Fig. 7.

The secondary structure predicted by JPred4 for MERS-CoV PLpro (4RNA). Residue positions forming the catalytic triads are pointed out with red arrows.

As seen in the ligand interaction diagrams of the compounds [Fig. 8: (A) 2, (B) 7, and (C) 20], the compounds are the top biological results with fit pose into the binding site of the MERS-COV PLpro (4RNA) using MOE. Circles of varying shades of pink and green represent polar and non-polar amino acids, respectively; hydrogen bonding is specified by green dashed arrows (side-chain), blue dashed arrows (backbone), and red dashed arrows (arene). The dotted lines around the ligand represent proximity contours, in addition to indicating the shape of the binding site as well as the space available to the more exposed portions of the ligand. The violaceous areola denotes the region of the crystal structure where the ligands are exposed to the solvent. D, E, and F are the optimized docking models of compounds 2, 7, and 20 with MERS-COV PLpro, respectively. The 3D representation shows the selected amino acid residues. Hydrogen bonds are indicated by dashes. For clarity, backbone and non-polar hydrogen atoms are omitted.

Fig. 8.

Two-dimensional ligand interaction diagrams of the compounds.

In particular, with respect to the treatments that have been recommended for MERS-CoV, we have also completed molecular docking studies with MERS-CoV PLpro for selected compounds. Alignment of the amino acid sequence revealed that MERS PLpro and Betacoronavirus PLpro (96.9 %) were extremely similar, but there were noteworthy differences between MERS-CoV and SARS PLpro (30.7) and SARS-CoV-2 PLpro (29.8), and MHV PLpro (27.0) (Fig. 6). Consequently, MERS-CoV PLpro is more similar to Betacoronavirus than SARS PLpro, SARS-CoV-2 PLpro, and MHV PLpro. The catalytic trinity accepts Cys111 as a nucleophilic agent in the form of -SH, His272 as a general acid and alkali, and Asp286 help to promote Cys111 deprotonation. (Niemeyer et al., 2018, Su et al., 2021).

Compounds 2, 7, and 20, with significant biological activities, were selected for detailed docking studies. All compounds were docked into a crystal structure of MERS-PLpro (PDB ID: 4RNA), and 10 binding modes for each complex were identified. Furthermore, their interaction details are shown in Table 1 and Fig. 5B. The molecular docking shows that the benzimidazole moiety is involved in several interactions with residues Cys111 and His278 of the catalytic triad. The top binding mode had a predicted binding affinity of −9.65 kcal/mol and elaborate 10 key residues of MERS-CoV PLpro, including residues Cys111 and His278 of the catalytic triad and amino acids Val276, Pro163, and Asp162. The predicted binding modes of MERS-PLpro/2, 7, and 20 were found inside a cleft created between α7 and β8 of MERS-PLpro (Fig. 7, Fig. 5).

Fig. 5.

Ligand/PLpro binding interactions. (A) Domain with an illustration of the PLpro backbone (PDB ID: 4RNA), with an overlay of the investigated compounds (with different color sticks) at the enzyme active site [Cys111, Asn109 (blocking loop 2), and His278]. (B) An overlay of the investigated compounds (with different color sticks) at the substrate-binding site (S1) with green color; substrate (S2) is demonstrated by a red color.

The docking of compounds 2, 7, and 20 with MERS-CoV PLpro was assessed by the aforementioned technique in the Method part. The docking of 2 (cyan-colored carbons), 7 (brown-colored carbons), and 20 (green-colored carbons) with MERS-CoV PLpro (PDB: 4RNA) was completed using MOE (Fig. 8A–F). The docked models and those for all compounds with SARS-CoV PLpro were overlaid for comparison in Fig. 5A. The hydrophilic interactions that occur between PLpro and inhibitors are represented by the dashed lines. In addition, 2, 7, and 20 were able to be directed to the catalytic triad of MERS-CoV PLpro. These compounds are located close to the catalytic triad and show hydrogen bonding interactions with residues Val276 and Cys111. In addition, these compounds form several van der Waal’s Thr274, Gly277, Tyr279, Ala275, Tyr112, Asn109, Phe269, Gln270, and Gly271, three π-alkyl interactions with Pro163, His278, and Asp164. Taking into consideration the structure of MERS-CoV PLpro complexed with Ub, the docked site of the investigated compounds should be located close to the putative S1–S2 subsites. Moreover, it seems that the unsubstituted phenyl ring of the benzoyl group leads to compound 2, which showed the most potent inhibitory activity against the PLpro enzyme. In addition, the only substitution that may lead to an improved inhibitory activity against PLpro is disubstitution at the meta and para positions. However, other substitutions on the aforementioned phenyl ring are not tolerated and lead to inactive compounds.

3.6. Molecular dynamics simulations

In order to investigate the stability and interaction profile of the active inhibitor within the putative pocket of the MERS-CoV PLpro, MD simulations of PLpro-ligand complexes were conducted for 40 ns. In addition, MD were conducted on MERS-CoV- inhibitors (compound 2). As a function of time, structural parameters comprising RMSD, RMSF, H-bonds, Rg, per-residue, and binding energy computation were examined.

3.7. RMS-deviation and RMS-Fluctuation

Cα RMSD studies were performed on docked complexes 2 complexed with MERS-CoV PLpro to determine the flexibility of PLpro residues. Compound 2 reached equilibrium in 10 ns, and demonstrated RMSD value of 3 Å (Fig. 9). These results demonstrated that compound 2 stabilize MERS-CoV PLpro protein. But, the RMSD value of compound 2 was to some extent higher than the RMSD of the free-ligand protein, However, it remained steady over the course of simulations, showing a robust binding of compound 2 to the PLpro binding site.

Fig. 9.

The RMSD values; (A) lead2-protein complex (blue), and protein backbone (red).

The RMSF assessment is an essential criterion that provides data on the structural flexibility of each and every residue in the system. As presented in Fig. 10, the RMSF values for the residues in the PLpro only and PLpro-ligand complex were studied. In comparison to PLpro on its own, the RMS fluctuation outcomes of the conserved amino acids loops, Cys158, Thr159, Leu162, Asp164; Ser258, Thr259, Ala260, Por261, and the high fluctuation of the (Cys226, Gln227, Cys228, Gly229, Gly230) then the BL2 loop (267–271) region. PLpro-compound 2 were low flexibility, indicating that this molecule could make significant interactions with these conserved amino acids. Based upon the previous scrutiny, we are able to draw the conclusion that this particular compound, which was chosen as hits, is able to bind firmly to the binding pocket of PLpro.

Fig. 10.

The RMSF values; (A) Compound2–protein complex (blue), and protein backbone (red).

3.8. Radius of gyration (Rg)

For the purpose of determining whether or not the PLpro-hit complex configurations are stable, we observed the alterations of secondary structures that occurred as a result of binding to hits through MD simulations utilizing the DSSP technique (Fig. 11). During the entire simulation time, no noteworthy modifications in structural components including α-helical and β-sheet content were noticed, which further proved the stability of our examined complex. We used RMSD-based cluster analysis to obtain the structural representative from simulated systems. The typical photo of the PLpro-hit complex was superimposed with the PLpro only using the RMSD (red) (Fig. 11) shows clearly that the PLpro complex superposes well with the PLpro alone with a Cα RMSD, demonstrating that all systems preserved the identical conformation and structural integrity with only insignificant alterations in the PLpro of MERS-Cov.

Fig. 11.

(A) Radius of gyration (R_g) of compound 2 (blue) complexed with the protein and the nonligand protein (red).

3.9. H-Bonds and distance monitoring

In MD simulation of compound 2, Asn109 was highly correlated with H-bond stability for over 90 %, and the salt bridge contribution was noticed by residues Cys111, His278, Asp164, Gly277, and Pro163 with occupancy (100 %, 72.54 %, 64.39 %, 49.12 % and 1. 83 %). In order to determine the influence of hot-spot residues to the PLpro protein–ligand interaction, H-bond studies of the complex were performed over a 40-ns simulation. From the MD of compound 2, it was revealed that it formed 4 continuous H-bonds with occupancy 98.1 % (Fig. 12A); F269 (BL2 loop residues) formed H-bond with compound2 in occupancy 13.3 %. Protein–ligand contacts were observed throughout the simulation. The traced contacts comprised hydrogen bonds, hydrophobic interactions, and ionic and water bridges (Fig. 12A&B). Compound 2 formed hydrogen bonds, or hydrophobic interactions with MERS-CoV PLpro. The main interactions were detected with the active site residues Asn109, Cys111, and Asp164 (H-bond). According to the H-bond analysis, we could conclude that this compound fits perfectly into the binding pocket of PLpro.

Fig. 12.

(A) number of H-bonds of compound2 with MERS-CoV PLpro putative pocket. (B) Percentage of H-bonds, side and main chain interaction occupations of the MERS-CoV PLpro residues contributed to compound 2.

3.10. ADMET study

Toxicity and pharmacokinetics effects are critical in selecting potential compounds and designing safe and effective drugs. As a result, the ADMET properties of the compounds were examined. In order to identify the drug-like properties of the compounds, The rule of five proposed by Lipinski and the set of rules proposed by Veber (Lipinski et al., 2001, Veber et al., 2002) were employed (Table 2, Table 3). The ADMET parameters were obtained using BIOVIA Discovery Studio 4.5. Additionally, an ADMET plot was generated utilizing the calculated AlogP98 vs 2D PSA. The graph demonstrated the accuracy of the HIA and BBB penetration model predictions. As seen in Fig. 13, the plot corresponding to the HIA and BBB penetration models revealed 99 % confidence for all 22 compounds. PSA value was inversely proportional to HIA value, indicating an inverse relationship between PSA and cell wall permeability. (Egan and Lauri 2002). AlogP98 has been revealed to be lipophilic; furthermore, as the value is a ratio, it can be utilized to evaluate the hydrophilicity and hydrophobicity of a chemical. In addition to the AlogP98 calculation, the hydrogen bonding characteristics revealed by the PSA calculation may end up playing a significant part (Egan et al. 2000). The ellipsoid with 95 % confidence showed a chemical boundary that comprised compounds with high absorption (90 %). While, the ellipsoid with 99 % confidence indicated the region of a chemical boundary with compounds that exhibited exceptional absorption through the cellular membrane. Any chemical with optimal cell permeability must satisfy the conditions of PSA less than 140 Å2 and AlogP98 less than 5.

Table 2.

ADME values of the 22 compounds.

Comp.	Absorption level	CYP2D6	Hepatotoxicity	PPB	Solubility level	Solubility	BBB level	AlogP98	2D PSA
1	0	false	true	true	3	−3.032	1	2.247	26.316
2	0	false	true	true	2	−4.83	1	3.684	43.616
3	0	true	true	true	2	−5.152	1	3.89	43.616
4	0	false	true	true	2	−5.094	2	3.453	61.477
5	0	false	true	false	2	−4.348	2	3.08	56.171
6	0	false	true	true	1	−6.216	1	4.593	43.616
7	0	false	true	true	2	−4.486	2	3.426	73.362
8	0	false	true	true	2	−4.887	1	3.668	52.547
9	0	false	true	true	2	−4.949	1	3.652	61.477
10	0	false	true	true	2	−5.001	2	3.635	70.407
11	0	false	true	true	2	−4.501	2	3.426	73.362
12	0	false	true	true	2	−4.899	1	3.668	52.547
13	0	false	true	true	2	−4.911	1	3.668	52.547
14	0	false	true	true	2	−4.355	2	3.442	64.432
15	0	false	true	true	2	−4.363	2	3.442	64.432
16	0	false	true	true	2	−4.307	2	2.938	70.156
17	0	false	true	true	2	−5.158	1	3.89	43.616
18	0	false	true	true	2	−5.478	1	4.095	43.616
19	0	false	true	true	2	−5.485	1	4.095	43.616
20	0	false	true	true	2	−5.636	1	4.433	43.616
21	0	false	true	true	2	−5.56	1	4.349	43.616
22	0	false	true	true	2	−4.643	1	3.41	43.616

For optimal BBB uptake, AlogP98 needs to be less than 5. Absorption values range from 0 to 4, with 0 meaning good, 1 meaning moderate, 2 meaning low, 3 meaning very low, and 4 meaning undefined. The toxicity of a drug can be determined by hepatotoxicity, with false indicating that the drug is nontoxic and true indicating that it is toxic. The inhibitory effect is categorized by CYP2D6 according to the following known categories: False indicates that there is no inhibitor, and True indicates that there is an inhibitor. PPB: true = binding and false = nonbinding. The molar solubility of drugs can be predicted based on their solubility: level 0 or log(Sw) −8.0 means the solubility is very low; level 1 or −8.0 log(Sw) −6.0 means the solubility is very low but not impossible; level 2 or −6.0 log(Sw) −4.1 means the solubility is low; level 3 or −4.1 log(Sw) −2.0 means the solubility is good; level 4 or −2.0 log(Sw) means optimal solubility, and level 5 or 0.0 < log(Sw) = no, too soluble.

ADMET: Absorption, distribution, metabolism, excretion and toxicity; BBB: Blood–brain barrier; BBBP: Blood–brain barrier permeability; CYP2D6: Cytochrome P2D6; PPB: Plasma protein binding; PSA: Polar surface area.

Table 3.

Lipinski's rule of five was used to determine the physicochemical characteristics of all 22 of the compounds.

Compound	logP	Molecular weight	Num_H acceptor	Num_H _donor	Molecular fractional PSA	Num_aromatic ring	Num_rotatable bond
1	2.247	150.201	2	2	26.316	2	0
2	3.684	268.334	3	1	43.616	3	4
3	3.89	286.324	3	1	43.616	3	4
4	3.453	312.343	5	1	61.477	3	4
5	3.08	258.296	3	1	56.171	3	4
6	4.593	318.392	3	1	43.616	4	4
7	3.426	314.359	5	2	73.362	3	5
8	3.668	298.36	4	1	52.547	3	5
9	3.652	328.386	5	1	61.477	3	6
10	3.635	358.412	6	1	70.407	3	7
11	3.426	314.359	5	2	73.362	3	5
12	3.668	298.36	4	1	52.547	3	5
13	3.668	298.36	4	1	52.547	3	5
14	3.442	284.333	4	2	64.432	3	4
15	3.442	284.333	4	2	64.432	3	4
16	2.938	283.348	4	2	70.156	3	4
17	3.89	286.324	3	1	43.616	3	4
18	4.095	304.315	3	1	43.616	3	4
19	4.095	304.315	3	1	43.616	3	4
20	4.433	347.23	3	1	43.616	3	4
21	4.349	302.779	3	1	43.616	3	4
22	3.638	274.361	3	1	43.616	3	4

Fig. 13.

AlogP versus Polar Area for all compounds within 95 and 99% confidence threshold ellipses integrated with the absorption across the intestinal and blood–brain barriers.

All 22 Compounds were subjected to the ADMET study and exhibited good HIA (absorption level 0). Compound 6 showed extremely low aqueous solubility. Compound 1 showed good aqueous solubility. The rest of the compounds showed low aqueous solubility. Unfortunately, all compounds showed probable hepatotoxicity. Compound 3 seems to be metabolized by cytochrome P2D6. All compounds exhibited plasma protein binding except compound 5. All selected compounds were within the AlogP98 vs that of 2D PSA confidence ellipsoid. None of the designated compounds fell outside of the ellipse filter used in the ADMET model, which indicates a high intestinal absorption and BBB penetration ability. Fig. 13 presents a plot of PSA and AlogP for the designated compounds.

4. Conclusion

MERS-CoV PLpro, as a target for drug discovery, was expressed in E. coli as a host system and then purified to a homogeneous condition via two-step chromatography, i.e., Ni-NTA and gel filtration columns. We optimized a fast, reliable, and sensitive in-vitro assay to screen 22 PLpro inhibitors and identified 3 promising compounds with significant biological activity against PLpro. To analyze PLpro conformation and design better molecules with higher affinity to our drug target and stronger inhibitory effects, all 22 compounds were utilized for molecular docking simulations. Finally, computational studies demonstrated that three compounds, 2, 7, and 20, were anti-MERS-CoV PLpro agents. The unsubstituted phenyl ring of the benzoyl group appears to lead to compound 2 (2-((benzimidazol-2-yl) thio)-1-arylethan-1-ones) which had the strongest inhibitory effect against the PLpro enzyme. Furthermore, the only change that may boost inhibitory action against PLpro is disubstitution at the meta and para locations. Other substitutions on the aforementioned phenyl ring, on the other hand, are not tolerated and result in inactive molecules. Additional experimental research is required to corroborate these findings.

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.