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. 2021 Jan 15;16(1):e0244967. doi: 10.1371/journal.pone.0244967

Discovery of beta-lactamase CMY-10 inhibitors for combination therapy against multi-drug resistant Enterobacteriaceae

Nousheen Parvaiz 1,‡, Faisal Ahmad 1,‡, Wenbo Yu 2,‡, Alexander D MacKerell Jr 2,*, Syed Sikander Azam 1,*
Editor: Massimiliano Galdiero3
PMCID: PMC7810305  PMID: 33449932

Abstract

β-lactam antibiotics are the most widely used antimicrobial agents since the discovery of benzylpenicillin in the 1920s. Unfortunately, these life-saving antibiotics are vulnerable to inactivation by continuously evolving β-lactamase enzymes that are primary resistance determinants in multi-drug resistant pathogens. The current study exploits the strategy of combination therapeutics and aims at identifying novel β-lactamase inhibitors that can inactivate the β-lactamase enzyme of the pathogen while allowing the β-lactam antibiotic to act against its penicillin-binding protein target. Inhibitor discovery applied the Site-Identification by Ligand Competitive Saturation (SILCS) technology to map the functional group requirements of the β-lactamase CMY-10 and generate pharmacophore models of active site. SILCS-MC, Ligand-grid Free Energy (LGFE) analysis and Machine-learning based random-forest (RF) scoring methods were then used to screen and filter a library of 700,000 compounds. From the computational screens 74 compounds were subjected to experimental validation in which β-lactamase activity assay, in vitro susceptibility testing, and Scanning Electron Microscope (SEM) analysis were conducted to explore their antibacterial potential. Eleven compounds were identified as enhancers while 7 compounds were recognized as inhibitors of CMY-10. Of these, compound 11 showed promising activity in β-lactamase activity assay, in vitro susceptibility testing against ATCC strains (E. coli, E. cloacae, E. agglomerans, E. alvei) and MDR clinical isolates (E. cloacae, E. alvei and E. agglomerans), with synergistic assay indicating its potential as a β-lactam enhancer and β-lactamase inhibitor. Structural similarity search against the active compound 11 yielded 28 more compounds. The majority of these compounds also exhibited β-lactamase inhibition potential and antibacterial activity. The non-β-lactam-based β-lactamase inhibitors identified in the current study have the potential to be used in combination therapy with lactam-based antibiotics against MDR clinical isolates that have been found resistant against last-line antibiotics.

Introduction

Despite the incredible initiatives taken in modern medicine to combat antibiotic-resistant microorganisms, emerging β-lactamase-mediated resistance remains a threat to the most prominent class of antibiotics, the β-lactams [1]. According to report by World Health Organization (WHO), urgent action is required to combat antimicrobial resistance expected to cause a global financial crisis by forcing 24 million people into extreme poverty by 2030 and causing 10 million deaths annually by 2050 [2]. The most problematic multi-drug resistant microorganisms are Gram-negative pathogens by acquiring mobile genetic elements linked with multiple resistance factors for most antibacterial agents [3]. Literature evidence strongly suggests that Enterobacter species such as Enterobacter cloacae, Enterobacter agglomerans, Enterobacter alvei, and Enterobacter aerogenes characterized by potential antimicrobial resistance mechanisms are one of the leading causes of fatal nosocomial infections worldwide [4].

The production of β-lactamases by Gram-negative pathogens which can hydrolytically inactivate β-lactam drugs is a prevalent resistance mechanism that renders even the most effective antibiotics ineffective. According to the molecular classification, β-lactamases are divided into class A, B, C, and D enzymes. β-lactamase enzymes of class A, C, and D use serine to hydrolyze β-lactam ring. However, β-lactamases of class B are metalloenzymes which use divalent zinc ions to hydrolyze the substrate. Research states that class C β-lactamases have conferred resistance against β-lactam containting β-lactamase inhibitors in combination with the clinically used β-lactamase inhibitor clavulanate [5]. Plasmid encoded class C β-lactamases are more problematic compared to other classes because of horizontal gene transfer [5]. The emergence of carbapenemase and Extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae, which are resistant even to third-generation antibiotics, pose a serious therapeutic challenge worldwide. The current study, therefore, targets the β-lactamase CMY-10 to design novel and effective inhibitors that can reestablish antibiotic activity against bacteria producing serine β-lactamases.

β-lactam antibiotics act as substrate mimics of the penultimate d-Ala-d-Ala on the peptidoglycan stem peptide targeting the final step in peptidoglycan synthesis and inhibiting the transpeptidation of adjacent peptidoglycan strands [1]. Inhibitors such as tazobactam, clavulanic acid, and sulbactam containing β-lactam ring were previously used to overcome serine β-lactamase-mediated resistance [1]. These inhibitors themselves contain s lactam ring and are structurally similar to β-lactam antibiotics. They target β-lactamase by creating stable acyl-enzyme intermediate in the active site with the catalytic serine. One of the downsides of using structurally similar β-lactamase inhibitors in combination therapy is that in most cases initial competitive binding between antibiotics and inhibitors to β-lactamase results in significant loss of antibiotics. Consequently, antibiotics are used in excess to overcome the loss thereby bacteria under increased evolutionary pressure leading to the development of MDR and XDR pathogenic strains [6].

The current research study uses Site Identification by Ligand Competitive Saturation (SILCS) based Computer Aided Drug Design (CADD) [7–9] to identify novel inhibitors against class C β-lactamase of multi-drug resistant Enterobacteriaceae. It proposes a non-β-lactam-based β-lactamase inhibitor that might potentially inhibit broad-spectrum plasmid-encoded Class C β-lactamases. Furthermore, the strategy of combinational therapeutics has been exploited by subjecting the computationally screened compounds to experimental validation against MDR clinical isolates. The potential for application of a combinatorial therapeutic approach in to treat MDR clinical isolates is indicated through our experimental studies.

Results

The complete protocol used in this study is shown in Fig 1.

Fig 1. The computational screening and experimental validation protocol followed to identify putative CMY-10 inhibitors for combination therapy against multi-drug resistant Enterobacteriaceae.

Fig 1

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Computational screening

SILCS simulations were undertaken to map the functional group requirements of the active site of CMY-10 including contributions from desolvation and accounting for local protein flexibility. SILCS FragMaps at the active site of CMY-10 show apolar FragMaps at both R1 and R2 sub-sites (Fig 2). Negatively charged FragMaps are seen at the R2 site as well as some hydrogen bonding donor and acceptor FragMaps. The crystal binding mode of IMP (inosine monophosphate) from the CMY-10-IMP complex (PDB entry 5K1F) aligns well with the generated FragMaps (Fig 2). Notable is the phosphate in the negative FragMaps, the location of a hydroxyl next to an acceptor FragMap and the presence of the base in the Apolar FragMaps. The functional group binding patterns encoded in the FragMaps were further utilized to build pharmacophore features for use in Virtual Screening (VS) (S1 Fig).

Fig 2.

Fig 2

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Apolar (green), hydrogen bonding donor (blue) and acceptor (red) and negatively (orange) charged SILCS FragMaps overlaid on the active site of CMY-10. The crystal binding mode of IMP in the CMY-10-IMP complex is shown. Consistencies between the crystal binding mode of IMP with FragMaps are shown by arrows.

The pharmacophore model that was developed for the R2 site is shown in Fig 3. Consistent with the SILCS FragMaps the pharmacophore features align well with the IMP binding mode. The model contains one hydrophobic feature (F1) corresponding to the base in IMP, an anionic feature for phosphate (F2), and hydrogen bonding acceptor feature (F3) for the sugar oxygen which forms a hydrogen bond with residue N340. In addition to features associated with the IMP binding mode, another hydrogen binding donor feature (F4) is defined that represents functional groups interacting with the N340 carbonyl group.

Fig 3. Pharmacophore model for R2 site.

Fig 3

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Hydrophobic, hydrogen bonding donor and acceptor, and anionic features are shown in cyan, blue, red and dark red colors, respectively. Crystal binding mode of IMP is also shown with surrounding protein residues labeled.

To encompass both the R1 and R2 sites, the pharmacophore model in Fig 4 was generated from the FragMaps. In the figure the CMY-10 structure is aligned with the crystal structure AmpC beta-lactamase from Escherichia coli in complex with ceftazidime as shown in pink color (PDB ID: 1IEL). CMY-10 has high sequence similarity with AmpC, and the R1 and R2 site definitions were originally defined from studies on AmpC [5]. Accordingly, the crystal binding mode of Ceftazidime bound to AmpC is useful to inform CMY-10 R1-R2 site inhibitor design. To cover both R1 and R2 sites, two hydrophobic features (F2 and F3) were selected, which align well with the crystal binding mode of the aromatic moieties in Ceftazidime, were considered. One acceptor feature (F1), which aligns well with the carbonyl group in the ligand was also included. This feature actually is at the same position of the anionic feature that was developed for the R2 site above. In this case it is set to be an acceptor feature to be more general and cover both charged and neutral acceptors as inspired by AmpC inhibitors. Though being highly similar, CMY-10 still shows some sequence difference with AmpC at the R1 site, e.g. polar residues N317 and N214 for CMY-10 while non-polar residues are at the same positions for AmpC. This motivated selection of feature F4 as an acceptor for CMY-10 at the R1 site that is not present in the AmpC binder Ceftazidime as shown.

Fig 4. Pharmacophore model for R1-R2 site.

Fig 4

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Hydrophobic and hydrogen bonding acceptor features are shown in cyan and red colors, respectively. Crystal binding mode of Ceftazidime with AmpC is also shown with surrounding protein residues labeled. CMY-10 residues are shown in green colored carbons and AmpC residues are represented in pink colored carbons.

Compounds that can match either of the two pharmacophore models are expected to have the potential to bind to the active binding site of CMY-10 at R2 sub-site in a manner similar to that of IMP or to occupy both the R1 and R2 sites, potentially with higher affinity. Such compounds would thus act as competitive inhibitors with respect to the lactam substrates of the protein.

VS were next performed using the two pharmacophores targeting over 750,000 compounds. From this pharmacophore-based VS, hit compounds were ranked using the Pharmer root mean square deviation (RMSD) score that measures the spatial similarity between the functional groups of the screened molecules and the query pharmacophore model. The top 10,000 scored compounds with the lowest RMSD were then selected from each pharmacophore search for further evaluation.

The selected 10,000 compounds from each pharmacophore screen were subsequently subjected to SILCS-MC (Site Identification by Ligand Competitive Saturation-Monte Carlo) pose refinement. Application of SILCS-MC allows the compounds to relax in the field of the FragMaps based on an initial orientation from the Pharmacophore screen. Ligand-grid Free Energy (LGFE) scores obtained from the SILCS-MC are then used to rank the compounds. In addition, Machine Learning Random Forest (MLRF) scoring was applied to the two sets of 10,000 compounds. From both the SILCS-MC and MLRF ranking the top 500 compounds were selected for each pharmacophore. For each pharmacophore common compounds in the SILCS-MC and MLRF top 500 ranked compounds list were then selected. This yielded 30 compounds for the R2 site and 53 compounds for the R1/R2 site. These compounds were then subjected to similarity clustering from which one compound was selected from each cluster, although most clusters only had one compound. As a result, 74 compounds were obtained for experimental testing.

Experimental analysis

β- lactamase activity assay

Beta-lactamase activity assay was performed spectrophotometrically using a chromogenic substrate Nitrocefin. The results of β-lactamase activity assay yielded compounds 1, 5, 11, 26, 36, and 47 exhibiting decreased BL-activity 0.005 u/mg, 0.3 u/mg, 0.08 u/mg, 0.0362 u/mg, 0.016 u/mg, and 0.014 u/mg, respectively, as β-lactamase inhibitors (BLA<Control) (Table 1). However, compounds 4, 5, 7, 9, 10, 12, 17, 20, 34, 69 and 71 exhibited enhanced β-lactamase activity (BLA>Control) (S1 Table, Fig 5). The LGFE distribution for top 10,000 ranked compounds from VS considering both R2 site and R1-R2 site has been shown in S2 Fig.

Table 1. BL-activity and LGFE scores of lead compounds identified as inhibitors.
S. No. Chembridge 1D BL-activity u/mg LGFE (kcal/mol)
1 6096429 0.005 -9.98
5 12728806 0.3 -8.49
11 5524250 0.08 -10.3
26 5241230 0.0362 -9.61
36 77764831 0.016 -9.66
47 7878453 0.014 -9.48
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Fig 5. BL-activity and LGFE scores of identified lead compounds.

Fig 5

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Blue bars showing BL activity with red color indicating LGFE along with positive control in black.

Molecular identification assay

The plasmid contents of three MDR clinical isolates of the bacteria were analyzed by Field Inversion Gel Electrophoresis (FIGE). Three large plasmids (130 kb) were detected in all the isolates. The plasmid-encoded β-lactamase genes of three isolates were constitutively encoded AmpC β-lactamase (CMY-10) with 1,179 bp PCR product obtained using agarose gel electrophoresis that shows resistance phenotypes of three clinical isolates for the CMY-10 genes (Fig 6).

Fig 6. Patterns of genomic plasmid from E. cloacae (lane 2), E. agglomerans (lane 3), E. alvei (lane 4), and lane 1 (100-bp stepwise ladder) show band patterns of ladder fragments (sizes in base pairs are indicated on the edge of the gel).

Fig 6

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In vitro susceptibility testing

Epsilometer test (E-test) was performed to quantify antimicrobial susceptibility of clinical isolates against advanced generation of macrolide and third and fourth generation antibiotics (S2 Table). Results from this assay showed that all the three clinical isolates were highly resistant except for E. agglomerans which was only found susceptible to fourth generation cefepime, imipenem, meropenem (S2 Table). According to the Clinical and Laboratory Standards Institute (CLSI) guidelines, the breakpoint ranges for Enterobacter species against cefixime suggest that Minimum Inhibitory Concentration (MIC) values with zone of inhibition ≤14 mm demonstrate resistant strains, between 17 mm to 14 mm are considered intermediate and ≥17 mm indicate sensitive strains [10]. The computationally selected compounds 1, 5, 11, 26, and 37 identified as β-lactamase inhibitors showed antibiotic activity against E. agglomerans (ATCC 31901), whereas compounds 5 and 11 were active against E. coli (ATCC 10536), compounds 5, 11, 26, and 47 were active against E. cloacae (ATCC 13047) and compounds 11, 26, 37, and 54 showed activity against E. alvei (ATCC 51815) (S3 Table). However, only 11 (Chembridge ID: 5524250) showed antibiotic activity against the three multi-drug resistant clinical isolates (S3 Fig). The zone of inhibition observed for 11 and β-lactam drug cefixime (cephalosporin) used as control was <14 mm demonstrating a fair extent of resistance in clinical isolates (Table 2). MIC of compound 11 was also checked along with control imipenem and cefixime against MDR clinical isolates. The minimum zone of inhibition was measured at 4mg/ml against all the MDR clinical isolates (Table 3).

Table 2. Antibacterial activity of lead compounds against β-lactamase producer clinical bacterial isolates.
S. No BACTERIAL ISOLATES (CLINICAL)
In house and Chembridge ID
Enterobacter Alvei Enterobacter Cloacae Enterobacter Agglomerans
Zone of inhibition (mm)
M ± SD M ± SD M ± SD
1 6096429 0 ± 0 0 ± 0 0 ± 0
5 12728806 0 ± 0 0 ± 0 0 ± 0
11 5524250 9.6 ±0.4 11.6 ±0.4 8.3 ±0.4
26 5241230 0 ± 0 0 ± 0 0 ± 0
36 77764831 0 ± 0 0 ± 0 0 ± 0
37 7989492 0 ± 0 0 ± 0 0 ± 0
47 7878453 0 ± 0 0 ± 0 0 ± 0
54 7960496 0 ± 0 0 ± 0 0 ± 0
Control Cefixime 10.6 ±0.4 10.6 ±0.4 9.6 ±0.4
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* M ± SD, Mean ± Standard Deviation, mm, millimeter.

Table 3. Minimum inhibitory concentration of compound 11 against clinical bacterial isolates.
MIC test for compound 11 against clinical strains
Bacterial strains MIC concentration (zone of inhibition mm) imipenem cefixime
2mg/ml 3mg/ml 4mg/ml 5mg/ml 6mg/ml 7mg/ml 0.01mg/ml 0.005mg/ml
E. cloacae 0 0 7 10 12 14 8 7
E. agglomerans 0 0 7 10 12 15 8 7
E. alvei 0 0 7 11 13 14 8 7
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Synergistic assay

The synergistic assay was performed to evaluate the antibacterial activity of computationally screened compounds in combination with cefixime against MDR clinical isolates. The results of the combination study highlighted the compound 11-cefixime combination exhibiting enhanced activity with mean zone of inhibition ≥19 mm against E. alvei, a clinical isolate that showed maximum resistance in susceptibility tests against computationally screened compounds and last line antibiotics. However, a breakthrough was achieved against E. agglomerans and E. cloacae with the compound 11-cefixime combination showing promising activity with mean zone of inhibition in between 15 and 18 mm (Fig 7). Synergistic effect of MIC of compound 11 with minimum inhibitory concentrations of cefixime, imipenem, and amoxicillin-clavulanic acid against clinical isolates was also evaluated (Table 4). Results inferred a maximum zone of inhibition for all the three clinical strains at minimum inhibitory concentration. Herein, average mean zone of inhibition was ±20 mm against E. cloacae, ±19 mm against E. agglumerans and ±21 mm in response to E. alvei with minimum inhibitory level of 5ug+1ug (Cefixime: Compound 11).This response seems very effective as compare to the positive control amoxicillin + clavulanic acid. While, on other hand the combination of Imipenem and Compound 11 used in 10ug+1ug of MIC showed a potential response with ±22 mm average mean inhibition zone.

Fig 7. Graphical representation of synergetic effect of the computationally screened compounds-cefixime with positive control.

Fig 7

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Table 4. Synergistic effect of compound 11 with different concentration against clinical isolates.
Synergistic effect of compound 11 against clinical antibiotic resistance strains
Cefixime: Compound 11 Imipenem: Compound 11 amoxicillin + clavulanic acid (+ve control) (20+10)μg/ml DMSO (- ve control)
Bacterial strains 5ug+1ug 10ug+1ug
Zone of inhibition (mm)
E. cloacae ±20 ±22 ±23 0
E. agglumerans ±19 ±22 ±20 0
E. alvei ±21 ±21 ±24 0
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SEM analysis

The SEM analysis was performed to investigate the difference of morphological changes in bacterial cell walls of untreated sample and control sample in comparison with the cells exposed to the β-lactam-β-lactamase inhibitor combination (11-cefixime) (Fig 8). Compound 11 was used in minimum inhibitory concentration in combination with cefixime. The morphological change was apparent in cells of the pathogens treated with β-lactam-β-lactamase inhibitor combination (11-cefixime), in which the destruction of the cells and formations of pores were observed in the cell wall. The surface of the cell wall of untreated sample and control sample was relatively smooth compared to the cell wall of the strain treated with the β-lactam-β-lactamase inhibitor combination. The observations were consistent with experimental findings suggesting that the combined use of 11 and cefixime has enhanced therapeutic efficacy against MDR clinical isolates.

Fig 8.

Fig 8

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Scanning electron microscopy (SEM) analysis (A) Effects of control on E. Cloacae (B) Effects of lead compound 11 in combination with antibiotic, cefixime against Enterobacter cloacae. (C) Effects of control on E. alvei, (D) Effects of lead compound 11 in combination with antibiotic, cefixime against E. alvei. (E) Effects of control on E. agglomerans (F) Effects of lead compound 11 in combination with antibiotic, cefixime against E. agglomerans. The red arrows indicate the morphological changes exhibited on bacterial cell wall after the use of combination therapy.

Activity of compounds similar to compound 11

To determine if 11 is a viable lead compound for further drug development efforts, similar compounds in the database were identified using chemical fingerprint screening [11]. Results obtained from the β- lactamase assay suggest that 28 compounds (1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25 and 29) having structural similarity with the active compound exhibited β- lactamase inhibition potential (Fig 9, S4 Table). However, three compounds, 19, 26, and 27 showed higher BL activity suggesting that these compounds might act as potential enhancers of the enzyme. Values can be inferred from S5 Table, while the activity can be visualized in Fig 9.

Fig 9. BL activity of further screened compounds with negative control (Chembridge ID: 5524250) in green color and positive control in red color.

Fig 9

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The similar compounds were also subjected to susceptibility testing (S6 Table). Majority of compounds exhibiting β- lactamase inhibition showed antibiotic activity against E. agglomerans (ATCC 31901), E. coli (ATCC 10536), E. cloacae (ATCC 13047) and E. alvei (ATCC 51815). The exceptions were compounds 15 and 26 that were inactive against both the strains. The high zone of inhibition among these compounds was observed for compound 20 (13mm) (S6 Table). However, the results of the antibiotic assay against the MDR clinical isolates namely, E. alvei, E. cloacae, and E. agglomerans suggests that only compounds 5,7, 11, 14, 20, 22, and 24 exhibited the degree of inhibition in coherence with their values obtained for respective zones of inhibition (S7 Table).

Discussion and conclusions

ESBLs have evolved as a result of continuous use of third generation antibiotics mainly characterized by bulky oxyimino groups which were poor substrates for narrow substrate spectrum class C β-lactamases. ESBLs such as CMY-10 exhibit extended catalytic activity due to noticeable conformational changes in the active site caused by the deletion of three amino acids in the R2 loop leading to significant widening of the R2 site. The localized mutations in the R2 site of CMY-10 have played a major role in changing and extending the substrate spectrum of the enzyme contributing to its catalytic versatility. The current study has therefore employed a receptor-based CADD approach, termed SILCS, to explore the functional group binding patterns of CMY-10. This information in the form of FragMaps was then used to identify ligands with similar functional groups based on pharmacophore screening. The computational search was focused on the active site, which covers the R2-loop (residues 289–307), the flexible part of the Ω-loop (residues 212–226), α11, β11, Tyr151 loop (residues 149–152) and Gln121 loop (residues 118–128) [5]. The active site of CMY-10 can be divided into two sub-sites: R1 site (red ellipse) and R2 site (blue ellipse) (S4 Fig). The R1 side-chain of the β-lactam nucleus in β-lactam antibiotics is accommodated by the R1 site of the protein, whereas the right part of the β-lactam ring interacts with the R2 site of the receptor [5]. The R1 site is surrounded by the Ω-loop, Gln121 loop and β11, and the R2 site by Tyr151 loop, α10 in the R2-loop and α11 [5].

The active site FragMaps of CMY-10 were compared with the crystal binding mode of IMP from the CMY-10-IMP complex (Fig 2) [12]. The comparison suggests that the aromatic hypoxanthine group in IMP aligns well with apolar FragMaps (green arrow). The binding mode of the phosphate group in IMP is captured by negatively charged FragMap (orange arrow) and a hydrogen bonding acceptor FragMap is found near the sugar hydroxyl group (red arrow) (Fig 2). These observations indicate that the SILCS FragMaps can correctly capture the binding mode of ligands interacting with CMY-10 at the active site. The information obtained from GFE FragMaps was used to generate pharmacophore models for VS of 777,605 compounds to identify potential β-lactamase inhibitors. Computational screening, including both SILCS-MC and MLRF secondary screening and similarity clustering yielded 74 non-β-lactam-based compounds which showed potential affinity to the binding pocket of the protein.

The β-lactamase activity assay results suggested that among the 74 computationally screened hits, seven compounds are β-lactamase inhibitors while eleven compounds are β-lactamase enhancers. The results of in vitro susceptibility testing against ATCC strains were also in accordance with β-lactamase activity assay. The β-lactamase inhibitors exhibited zone of inhibition whereas the enhancers showed no activity except compound 5 which was active against ATCC strains. The assay results revealed compounds 5, and 11 as potential inhibitors showing zones of inhibition 11.6 ± 0.4 and 18.3 ± 0.4 mm, respectively, compared with positive control cefixime exhibiting zone of inhibition of 19.6 mm against E. coli (ATCC 10536). However, compounds 5, 11, 26, and 47 were found active exhibiting zones of inhibition of 13.3 ± 0.4, 18.3 ± 0.4, 18.3 ± 0.4, and 18.3 ± 0.4 mm, respectively, with positive control cefixime displaying zone of inhibition of 19.3 ± 0.4 mm against E. cloacae (ATCC 13047). Compounds 1, 5, 11, 26, and 37 were active against E. agglomerans (ATCC 31901) showing zones of inhibition 13.3 ± 0.4, 8.3 ± 0.4, 18.3 ± 0.4, 11.6 ± 0.4, and 18.3 ± 0.4 mm, respectively, with positive control cefixime showing zone of inhibition of 19.3 mm. Compounds 11, 26, 37, and 54 were active against E. alvei (ATCC 51815) showing zones of inhibition 11.6 ± 0.4, 13.3 ± 0.4, 11.6 ± 0.4, and 11.6 ± 0.4 mm, respectively.

The predicted binding mode of compound 11 using SILCS pharmacophore screening followed by SILCS-MC is shown in S5 Fig. The compound is predicted to bind to both R1 and R2 sites. At the R2 site, 11 adopts a similar binding orientation as the crystal IMP as shown in Fig 2 with the central phenyl ring in 11 occupping the hydrophobic region of R2 site and carboxyl group reproduces the crystal binding mode of phosphate group in IMP. In addition, the chlorophenyl group in 11 occupies the hydrophobic region at R1 site. Research studies exploring structural basis of ESBLs have revealed a wider active site which decreases steric hindrance and allows the hydrolysis of third generation antibiotics [5, 13, 14]. For instance, imipenem has a long R2 side-chain which helps in forming a strained conformation of acyl-enzyme complex, preventing hydrolysis of the drug by non-extended spectrum class C β-lactamases. However, the wider R2 site of the ESBL CMY-10 allows the rapid hydrolyzation of impinem exhibiting a higher kcat value [5].

In a previous study by King and co-workers (2015), molecular mechanism of Avibactam-mediated β-lactamase inhibition has been discussed. Avibactam is a non-β-lactam-based β-lactamase inhibitor which has shown promising results against multidrug resistant bacterial strains such as Pseudomonas aeruginosa [1]. Molecular mechanism insights into the inhibitory activity of avibactam suggest that the drug forms a carbamyl linkage with nucleophilic serine in the active site of serine β-lactamases (SBLs). Similar to β-lactam-based serine β-lactamase inhibitors, the carbamyl linkage of avibactam with catalytic serine does not decompose through hydrolytic mechanism. Instead, the drug is decarbamylated by the recyclization of the diazobicyclooctane (DBO) ring reforming the inhibitor which is either released into the solution to inhibit other β-lactamases or recarbamylate in the active site of the same enzyme [1]. The possible molecular mechanism of inhibition of non-β-lactam-based inhibitors such as compound 11 in the current findings might be similar to that of avibactam-mediated reversible SBL inhibition. The identified β-lactamase inhibitors interact with the active site of β-lactamase and might form an intermediate resistant to decomposition via hydrolytic mechanism thus affecting the catalytic activity of the enzyme.

Antibacterial activity assay was also performed on clinical isolates E. agglomerans, E. cloacae, E. alvei to investigate the potential of computationally screened compounds against clinical pathogens. The results of E-test confirmed the increased prevalence of MDR strains of Enterobacter species in Pakistan which are resistant to the fourth generation antibiotic including cefixime, and β-lactam antibiotics, reported as a preferred choice against ESBL producing pathogens [15]. The findings of the study by Abrar et al. also suggested an alarming increase in multi-drug resistance of Enterobacteriaceae in Pakistan which can have negative impact on healthcare costs, infection rates and clinical outcomes [16]. In addition to this, previous research studies have also reported the prevalence of MDR clinical isolates of Shigella species, Campylobacter species, Salmonella typhi, and Neisseria gonorrhoeae in Pakistan highlighting the need for potential interventions and development of novel therapeutics to control and reduce antimicrobial resistance [17–21]. Compound 11 which was highly active against ATCC strains also showed antibacterial activity against all three clinically isolated MDR Enterobacteriaceae strains while all the other compounds including those identified as enhancers and inhibitors remained inactive against the clinical isolates. The MIC of compound 11 exhibited mean zone of inhibition with diameters of 9.6 ± 0.4 mm, 11.6 ± 0.4 mm, and 8.3 ± 0.4 mm, while control (cefixime) having diameters of 10.6 ± 0.4 mm, 10.6 ± 0.4 mm, and 9.6 ± 0.4 mm against E. alvei, E. cloacae and E. agglomerans, respectively. These observations suggest that compound 11 may have high binding affinity for the PBPs of Enterobacteriaceae nearly equal to that of third generation antibiotic cefixime. This does not indicate that other computationally screened compounds identified as inhibitors do not have any activity against clinical isolates of diverse bacterial species. They may be active against other multidrug resistant strains if subjected to further experimentation. Therefore, in the future studies, the activity of the rest of the computationally screened leads should be tested against different bacterial strains to explore their antibacterial potential.

Various research studies have reported the efficacy of using a combination of β-lactam antibiotic with β-lactamase inhibitor in enhancing the potency of antibacterial agents [1, 22, 23]. In the current study, MICs were also determined using computationally screened inhibitors in combination with cefixime against MDR clinical isolates to exploit the strategy of combination therapeutics. The results of the synergistic assay were dominated by the antibacterial activity of compound 11 against MDR clinical strains. The activity of cefixime was notably improved in the presence of compound 11 against E. agglomerans, E. cloacae, and E. alvei. In addition, minor synergistic effect was recorded for compound 5 against E. agglomerans with a zone of inhibition 11 mm while the MDR clinical isolates remained resistant to the combination of cefixime with all the other computationally screened inhibitors. The effect of compound 11 along cefixime and imipenem with commercially available positive control amoxicillin + clavulanic acid was also checked. This minimum inhibitory concentration was applied according to the results obtained via microdilution disc diffusion assay. Results inferred the maximum zone of inhibition against all three clinical strains at minimum inhibitory concentration.

The morphological changes induced by using the combination of cefixime and compound 11 evaluated using SEM suggest that the use of β-lactam-β-lactamase inhibitor has negatively affected the synthesis of cell wall in all three clinical isolates. Extensive structural damage can be seen in E. Alvei which was higher than that observed as compared to the E. agglomerans and E. cloacae (Fig 8). Cefixime has been found ineffective against MDR clinical isolates used in this study. However, the clinical isolates have exhibited susceptibility when exposed to the combination of compound 11 and cefixime. These findings are consistent with the results of the synergistic assay suggesting that 11 might have the potential to act as β-lactam enhancer and β-lactamase inhibitor, significantly contributing to overcome antibiotic resistance.

The current study identifies computationally screened compound 11 exhibiting enhanced β-lactamase inhibitory activity in the experimental assays as a potent lead compound against MDR pathogens. The chemical structure of compound 11 (N-(2-{[(4-chlorophenyl) amino] carbonyl}-4, 6-dinitrophenyl)) contains two nitro groups and a halogen, chlorine (S3 Fig). Nitro groups are reported as best leaving groups that are smoothly displaced by nucleophiles [24]. Halogens mediate hydrogen bond donor interactions and can also act as nucleophilic acceptors widely contributing to receptor-ligand interactions. Halogens have always received significant attention in drug design due to their crucial role in enhancing selectivity and binding affinity. In addition, research studies have explicitly reported that the presence of halogen Cl facilitates interaction with hydrogen bond donor group in protein resulting in enhanced ligand binding affinity [25–27]. Research studies exploring the structure and mechanism of β-lactamase enzymes have reported the role of conserved active site serine which acts as a nucleophile to facilitate the interaction with β-lactam antibiotics and β-lactamase inhibitors [1, 5]. The presence of two nitro groups and a halogen Cl in compound 11 may have contributed to its promising activity against MDR clinical isolates in susceptibility testing and synergistic assays. In contrast to compound 11, all the other screened compounds either do not have nitro groups or have only one nitro group in their structure. However, the combination of two nitro groups along with one halogen is only present in compound 11 which may play a crucial role in the enhanced antimicrobial and β-lactamase inhibitory activity reflected in the experimental assays. This property of having two nitro groups and one halogen can be considered as a unique property determination yielded as an outcome of the current investigation. However, extensive structural, kinetic and mutagenesis studies are required to understand the molecular details and active site features responsible of compound 11 mediated inhibition.

The potential β-lactamase inhibitor, compound 11, proposed in the current study is not based on a β-lactam core structure which reduces its chances of getting hydrolyzed by wild and mutant β-lactamase enzymes. Non-β-lactam-based β-lactamase inhibitors are capable of escaping different pathogen resistance mechanisms mobilized against β-lactams thus reducing the chance of overexpression of β-lactamase protein [28–30]. Therefore, in order to refine the quest for potential non-β-lactam-based β-lactamase inhibitors, compound 11 was further subjected to a next round of similarity searching. As an outcome of the search, 28 compounds were found to be similar to 11. Of these compounds, compound 20, which keeps higher zone of inhibition against MDR clinical, also inherits two nitro groups and one halogen which may have contributed to its activity in susceptibility testing. Values obtained from β-lactamase activity assay, and susceptibility testing suggested that majority of the similarity searched compounds are also active against the enzyme (Fig 9, S5 and S6 Tables). Meanwhile, MDR clinical isolates exhibited susceptibility against compound 5, 7, 11, 14, 20, 22, and 24 in total. The availability of multiple similar compounds with the desired antimicrobial activity indicates that 11 is a viable lead compound for further optimization and evaluation towards clinical trials. It is concurred that the use of combination therapy with non-β-lactam-based β-lactamase inhibitors can be effective against decreased susceptibility pathogens that use ESBLs as their primary resistance mechanisms. Accordingly, the compounds reported here have the potential to be effective clinical agents with the ability to circumvent the antimicrobial resistance caused by the species of Enterobacteriaceae.

Material and methods

Computational screening

The SILCS [9] based CADD protocol starts by using the CMY-10 protein structure to initialize SILCS simulation from which functional group requirements of the protein in terms of free energies are obtained. The FragMaps are then used with SILCS-Pharm [31, 32] to build pharmacophore models for virtual database screening. Subsequent SILCS-MC docking [7–9] was conducted to refine the screening results, with final ranking using SILCS based ligand-grid free energy (LGFE) scores and energy scores based on a Machine Learning Random Forest (MLRF) virtual screening model [33]. Final compounds selected for the experimental assay are those common to both the LGFE and MLRF selected compounds supplemented with similarity clustering to allow for the selection of compounds of maximal diversity for testing. The details of each step of CADD protocol are addressed in the following sections.

SILCS simulations

The SILCS [9] simulations involves combined Grand Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) [34] simulations of the target protein immersed in an aqueous solution that contains organic solutes of different chemical classes. The solutes and water then compete for binding sites on the protein surface and in pockets in the protein during the simulation, yielding a free energy fragment competition assay from which the 3D fragment probability distributions of the solutes and water are used to define affinity patterns, termed FragMaps, encompassing a dynamic protein surface.

The current SILCS run was performed using the Grand Canonical Monte Carlo (GCMC)/MD protocol for SILCS [34]. The target protein was solvated in a water box, the size of which is determined to have the protein extrema separated from the box edge by 12 Å on all sides. Eight representative solutes with different chemical properties (benzene, propane, acetaldehyde, methanol, formamide, imidazole, acetate, and methylammonium) were added into the system at ~0.25 M concentration, to probe the functional group requirements of the protein.

Ten such systems with different fragment positions and with the side chain chi1 dihedrals of solvent-exposed residues randomized were prepared to expedite the convergence of the simulations [8]. Each system was minimized for 5000 steps with the steepest descent (SD) algorithm [35] in the presence of periodic boundary conditions (PBC) [36] and was followed by a 250 ps MD equilibration. During SILCS simulations, weak restraints were applied on the backbone Cα carbon atoms with a force constant (k in 1/2 kδx 2) of 0.12 kcal/mol/Å2 to limit large conformational changes in the protein and to prevent the rotation of the protein in the simulation box. Ten GCMC/MD simulations were run for 125 cycles where each cycle has 200,000 steps of GCMC and 1 ns of MD. The first 25 cycles included only the GCMC steps were treated as equilibration and discarded yielding a cumulative 200 million steps of GCMC and 1 microsecond of MD over the 10 simulation systems. During GCMC, solutes and water are exchanged between their gas-phase reservoirs; the excess chemical potential used to drive such exchange is varied every 3 cycles to yield an average concentration corresponding to 0.25 M of each fragment. The configuration at the end of each GCMC run was used as the starting configuration for the following MD. During MD, the Nosé−Hoover method (Hoover, 1985; Nosé, 1984) was used to maintain the temperature at 298 K and pressure was maintained at 1 bar using the Parrinello−Rahman barostat [37–39]. CHARMM36 protein force field [40], CHARMM General Force Field (CGenFF) [41, 42] and modified TIP3P water model [43] were used to describe protein fragments, and water during the simulation, respectively. GCMC was performed by an in-house code and MD was conducted using GROMACS program [42, 44].

3D probability distributions of the selected atoms from the solutes from the SILCS simulations were constructed and combined to obtain both specific and generic FragMap types as previously described [45]. Atoms from snapshots output every 10 ps from each SILCS simulation trajectory were binned into 1 Å × 1 Å × 1 Å cubic volume elements (voxels) of a grid spanning the entire system to acquire the voxel occupancy for each FragMap atom type being counted. The voxel occupancies computed in the presence of the protein were divided by the value in bulk to obtain a normalized probability. Normalized distributions were then converted to grid-free energies (GFE) based on a Boltzmann transformation for visualization and quantitative use [45].

SILCS-pharmacophore for CMY-10

The SILCS-Pharmacophore (SILCS-Pharm) protocol was used to prepare pharmacophore models for virtual screenings (VS). SILCS-Pharm can generate receptor-based pharmacophore models using information from the SILCS FragMaps. This protocol includes conversion of SILCS FragMaps into pharmacophore features followed by pharmacophore hypotheses generation and ranking, with the resulting pharmacophores suitable for a range of VS tools [31, 32]. FragMaps in the active site of CMY-10 were used to find all possible pharmacophore features using the SILCS-Pharm program. Two pharmacophore models were developed for VS. One model is focused on the R2 site, which is the IMP/GMP binding site with the second pharmacophore encompassing both the R1 and R2 sites. Two four-feature pharmacophore models were developed for VS for the 1) R2 binding site alone and the 2) combined R1 and R2 sites [12]. Four-feature models were found to give the best performance as shown in previous tests [31, 32]. Compounds with patterns of functional groups that match the two pharmacophore models are expected to have the potential to bind to the active site of CMY-10 and disrupt it catalytic activity.

VS for CMY-10

VS was carried out to screen our in-house database of commercially available compounds using the developed two four-feature pharmacophore models. The in-house database contains 721,368 compounds (1,695,786 molecules considering different protonation states and tautomers) from the vendor Chembridge and 56,237 compounds (126,575 molecules) from the vendor Maybridge. In addition to the four features in each model, the SILCS exclusion map was also used in the model to represent the forbidden region that ligands cannot occupy. Pharmer [46] was used to carry out the pharmacophore-based VS.

SILCS-MC for hit compounds

Top compounds selected from the SILCS-Pharm screen were rescored using Monte Carlo (MC) sampling using SILCS FragMaps (SILCS-MC) [45]. SILCS-MC allows the docking pose of hit compounds from pharmacophore VS to relax in the field of the FragMaps using MC sampling. SILCS-MC is based on the ligand grid free energy (LGFE) score of the ligands, which is the sum of the atomic GFE contributions of the SILCS-classified non-hydrogen atoms in each ligand. Local MC sampling leads to better matching with the energetic details in the binding pocket as defined by the SILCS FragMaps. SILCS-MC was conducted in local sampling mode with 100 steps of MC followed by 1,000 simulated annealing (SA) steps to refine docking poses locally. The Metropolis criteris is based on the ligand LGFE score along with the intramolecular energy based on the CGenFF energy function along with a 1/4r effective dielectric constant. After SILCS-MC, each hit compound was scored based on the LGFE and on the ligand efficiency (LE), which is the LGFE divided by the number of non-hydrogen atoms. Details of the screening protocol may be found in Ustach et al. (2019).

MLRF-score

A machine-learning-based random-forest (MLRF) scoring scheme (RF-Score-VS) was shown to yield results similar to or better than traditional VS methods [33]. As it represents a knowledge-based alternate scoring function developed based on an approach significantly different then SILCS method, it was used to score all docked poses from SILCS-MC run with the goal of reducing false positives.

Common-compounds selection

Common compounds selection of the top-ranked compounds based on the individual LGFE ranked and RF-Score ranked list was performed for both the R2 and R1/R2 searches. This type of consensus scoring scheme has been shown to decrease the false positive rate [47].

Similarity clustering

Since all hit compounds were selected by matching the pharmacophore features, chemical structure redundancy was a concern. Similarity clustering was conducted to maximize the chemical diversity of the compounds selected for assay by clustering hit compounds. BIT-MACCS fingerprints [48] were calculated to index all selected compounds, and Tanimoto similarity coefficients were calculated between all compound pairs and similarity clustering was performed to put chemically similar compounds into clusters. The final compounds were ranked by LGFE with cluster numbers and were further subjected to experimental validation.

Experimental analysis

β-lactamase activity assay

The top selected compounds obtained from computational analysis were purchased (1mg each) from ChemBridge (https://www.chembridge.com/screening_libraries/). β-lactamase activity was detected spectrophotometrically using nitrocefin as substrate. Positive control solution along with reaction mix was prepared following the protocol provided by abcam® β-lactamase kit (ab197003). Stock solution of the selected compounds was prepared by adding 0.001 g of compound in 100 μl. The final concentration of test compounds was set to 1 μM, which were then added in 96 well microplate and screened under a multiscan spectrophotometer for absorbance at 490 nm. The samples were kept in the dark and the optical density (OD490 nm) was measured in a kinetic mode at room temperature for 30 mins. The complete absorbance upon different time intervals was then calculated i.e. initial time and the final time were then correlated. The data was further analyzed by plotting the correct absorbance values for each standard as a function of the final concentration of hydrolyzed Nitrocefin.

Molecular identification assay

The clinical bacterial strains E. cloacae, E. alvei, and E. agglomerans used in this study were obtained from the Pakistan Institute of Medical Sciences (PIMS), Islamabad, Pakistan. Molecular assay was performed to analyze whether the MDR clinical bacterial isolates are β-lactamase CMY-10 producers.

Plasmid DNA from three clinical isolates was isolated according to guidelines of Sambrook and Russell [49]. These plasmids were then separated in 1.0% agarose using a FIGE Mapper Electrophoresis System (Bio-Rad, Hercules, CA). They were purified with a Gel Extraction Kit (Genomid, Research Triangle Park, NC). The purified plasmids were utilized as a source of template DNA for PCR amplification.

The primers were obtained from the literature and utilized against the desired plasmid DNA template to identify a gene of interest. Herein, 4 mM MgCl250 pM of each primer (5´-GTAGACCATATGCAACAACGACAATCC-3´) and C-XhoI (5´-GAATGTCTCGAGCTCTTTCTTTCAACC-3´) were used as forward and reverse primers, respectively. Amplification was carried followed by the analysis of products which was performed in 2% Seakem LE agarose (BMA, Rockland, ME). This further was followed by PCR amplifications on a thermal cycler.

In vitro susceptibility testing

The bacterial strains E. agglomerans (ATCC 31901), E. alvei (ATCC 51815), E. cloacae (ATCC 13047) and E. coli (ATCC 10536) were obtained from ATCC. Epsilometer test was performed against clinical isolates to check the culture sensitivity against different classes of antibiotics with minimum inhibitory dose. In order to determine a MIC with the E-test, the surface of an agar plate was swab inoculated with an adjusted bacterial suspension in the same manner as a disk diffusion test. One or more E test strips were then placed on the inoculated agar surface containing the clinical strains. The plates were incubated for 24 hrs at 37°C.

The antibacterial assays were performed to find out the inhibitory effect of the selected compounds against ATCC strains and β-lactamase producing clinical isolates. The agar disc diffusion method on Mueller Hington Agar (MHA, Oxoid, England) as defined by Lalitha [50] was applied to test the antibacterial potential of selected 74 compounds. Fresh bacterial colonies were prepared and inoculated in sterilized 1 ml of normal saline and were compared to the turbidity standard of 0.5 McFarland (1% BaCl2 and 1% H2SO4). ATCC strains and β-lactamase producing clinical isolates were used to prepare homogenous bacterial lawn. The test inoculum was ~ 1×104 cfu/ml. Test compounds (1 μM) with positive control cefixime (cephalosporin) at the same concentration were added on disks placed on Muller Hington Agar media. The tested strains were incubated for 24 hrs at 37°C. The minimum zone of inhibition of each compound was determined according to CLSI guidelines [10].

Synergistic assay

The parameters for the optimization of the antibacterial assay were set by following the protocol as discussed above. In order to check the inhibitory activity for the inhibitor molecules, the efficacy was examined in a synergistic assay against three clinical bacterial strains: E. cloacae, E. alvei and E. agglomerans. This technique is set to achieve the antagonistic effect of inhibitor molecules. The parameters were set to perform the assay by obtaining fresh culture of strains and preparing a bacterial lawn upon Muller Hington Agar media in petri dishes. The concentration of inhibitory molecules with cefixime drug is achieved with 1:1 μg/μl along with positive control of cefixime only in 1 μg/μl concentration. The tested strains were incubated for 24 hrs at 37°C.

Scanning Electron Microscopy (SEM) analysis

The sample was prepared following the protocol used by Murtey and Ramasamy (2016) and was centrifuged to obtain solid pallet of bacterial cells of three clinical strains [51]. The pallet was washed twice and dehydrated with a gradient solvent of ethanol for 20 minutes. This examination was done to check the potency of compound 11 with cefixime at 1:1 μg/μl and control with blank sample.

Similarity searching

BIT-MACCS fingerprint-based similarity search was conducted for lead compound 1011 to identify structurally similar compounds with improved biological activity. Compound 11 was searched against 5.04 million compound database and 28 compounds were selected for further assay tests (S4 Table). All previous assays mentioned above were repeated on these compounds.

Supporting information

S1 Table. BL-activity and LGFE scores of lead compounds identified as enhancers.

(DOCX)

Click here for additional data file. (30.3KB, docx)
S2 Table. Epsilometer test results for commercially available beta-lactam antibiotics against MDR clinical isolates.

(DOCX)

Click here for additional data file. (31.2KB, docx)
S3 Table. Antibacterial activity of lead compounds against ATCC bacterial isolates with zone of inhibition (mm).

(DOCX)

Click here for additional data file. (28.2KB, docx)
S4 Table. Two dimensional chemical structures of the similarity searched compounds including active compound 11.

(DOCX)

Click here for additional data file. (300.8KB, docx)
S5 Table. BL-activity of further screened compounds.

(DOCX)

Click here for additional data file. (26.5KB, docx)
S6 Table. Antibacterial activity of similarity searched compounds against ATCC bacterial isolates with zone of inhibition (mm).

(DOCX)

Click here for additional data file. (30.4KB, docx)
S7 Table. Antibacterial activity of similarity searched compounds against β lactamase producer’s clinical bacterial isolates.

(DOCX)

Click here for additional data file. (28.9KB, docx)
S1 Fig. All pharmacophore features generated from SILCS FragMaps at the R1 and R2 site region.

Hydrophobic, hydrogen bond acceptor, hydrogen bond donor and negatively charged features are colored in cyan, red, blue, and dark red, respectively.

(TIF)

Click here for additional data file. (8MB, tif)
S2 Fig

LGFE distributions for top 10,000 ranked compounds from VS for both R2 model (a) and R1-R2 (b) model. The vertical dashed line indicates the LGFE cutoff for selecting the top 500 ranked compounds. The LGFE values for the seven compounds identified as inhibitors are indicated by blue arrows with their compound ID labeled. 5 hits are from the R2 model VS and 2 hits are from the R1-R2 model VS.

(TIF)

Click here for additional data file. (4.4MB, tif)
S3 Fig. 2D chemical structure of compound 11 (Chembridge ID: 5524250).

(TIF)

Click here for additional data file. (195.1KB, tif)
S4 Fig

(A) The 3D structure of CMY-10 in ribbon representation. The residues of R1 site (Ω-loop, Gln121 loop and β11) and R2 site (Tyr151 loop, α10 and α11) are in yellow and green, respectively. The binding cavity of R1 site is represented by red ellipse and R2 site is represented by blue ellipse. The nucleophile Ser65 is shown in magenta using ball and stick representation within the blue ellipse.

(TIF)

Click here for additional data file. (4.3MB, tif)
S5 Fig. Predicted binding orientation of lead compound 11.

The binding site is shown in the same orientation as in the Fig 2. FragMaps are shown at GFE cutoff -1.0 kcal/mol for apolar (green), hydrogen bonding donor (blue) and acceptor (red) maps and at -1.5 kcal/mol for negatively (orange) charged map.

(TIF)

Click here for additional data file. (4MB, tif)

Acknowledgments

We thank Pakistan–United States Science and Technology Cooperation Program and Higher Education Commission (HEC) Pakistan.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was funded by the Pakistan-United States Science and Technology Cooperation Program SSA and ADM (US/2017/360) and the USA National Institutes of Health to ADM (GM131710). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Massimiliano Galdiero

11 Sep 2020

PONE-D-20-14041

Discovery of beta-lactamase CMY-10 inhibitors for combination therapy against multi-drug resistant Enterobacteriaceae

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

**********

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Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is an interesting paper highlighting an innovative approach at antimicrobial resistance of Enterobacteriaceae spp.

The paper is well written and detailed.

However, I have some questions about article:

Authors should explain a potential action mechanism of compound 11.

Can author clarify the correlation between lab tests and potential clinical applications?

Reviewer #2: In the present study, the Authors describe an experimental model to discover beta-lactamase inhibitors, focusing on the characterization of the antimicrobial properties of the identified compounds. The work is very interesting but in my opinion some aspects need to be improved to make the study more clear and comprehensible to the reader.

Introduction

In the nomenclature of bacterial species of Enterobacter it is sufficient to write the Genus in full for the first time, then the dotted Genus can be used (for example E. agglomerans);

In the last paragraph of the introduction, write in full "site identification by ligand competitive saturation" and put the acronym SILCS in brackets, as it is mentioned for the first time in this section.

Results

When a bacterial species is named for the first time, the name must be written in full (Escherichia coli);

To make the comprehension clearer for a non-expert reader, Authors should write in full and clarify the meaning of the acronyms "VS", "MC", "LGFE" and "MLRF";

Experimental analysis

It would be clearer if the Authors already specified in the section Results that the Beta-lactamase activity assay was performed spectrophotometrically using a chromogenic substrate such as Nitrocefin;

The legend of Figure 4 should be described more clearly, it is not clear;

Add the sentence "in vitro" in the title "Susceptibility testing";

In vitro susceptibility tests were performed on clinical isolates of Enterobacter spp. the authors should have used ATCC reference strains of the same Genus. This aspect needs to be clarified, the sensitivity towards the molecule could be different between bacterial strains of different genera;

The first time the acronym MIC is used, the meaning must be written in full;

It would be interesting and would been strengthened the data if the in vitro susceptibility tests were also carried out in broth dilution in order to verify a bacteriostatic or bactericidal effect of the compounds;

In the SEM analysis paragraph the authors should clarify the observations, the paragraph is not clear, should be rewritten. Furthermore, the resolution of Figure 6 is very poor, it is not possible to appreciate the “extensive structural damage” reported.

In the legend of Figure 6, the nomenclature of the bacterial species must be corrected, the species must be always written in lower case;

Also in the paragraph "Activity of compounds similar to compound 11" the authors should clarify that the reference strains used do not belong to the same genus as the clinical isolates, and the test should be carried out with an Enterobacter reference strain. In my opinion is wrong to use reference bacterial strains belonging to different genera, indeed the behavior of the compounds selected between ATCC strains and clinical isolates is not comparable (see compound 20).

From the main text it would seem that it has not been verified whether the analyzed bacterial strains are β-lactamase CMY-10 producers. This should be clearly demonstrated for example with a molecular assay;

The resolution of Figures 4, 5, 6 and 7 is too poor, these are not clear. In Figure 5 the written are not legible;

In my opinion, to make the tables (Table 2; S4; S6 and S7) more streamlined, the Authors could directly report the average value without reporting the values of the individual batches, and Figure 8 should be moved as the first figure, to make the experimental design clearer.

Discussion

In the discussion paragraph the Authors indicate "disc diffusion assay", but in the Results section the Authors indicate "epsilometric test". However, the diameters of the inhibition zones are commented in the section Results. What kind of test was done? This aspect is confusing and should be clarified;

In my opinion, the results of the agar diffusion tests should be commented in the Results section, making the discussion more streamlined.

Material and Methods

The bacterial suspension must be indicated with “CFU/ml” and not “CFU/spot”. Replace the sentence;

E-test strips containing the tested compounds were produced by the authors? This aspect should be clarified and the specific MIC values obtained should be reported in the Results;

In the Synergistic assays the molecules were only tested in a 1: 1 ratio? Different ratio should also be checked.

Table S3, which reports a partial antibiogram of clinical strains, should be completed and improved, so it is not clear.

Reviewer #3: The article “Discovery of beta-lactamase CMY-10 inhibitors for combination therapy against multidrug resistant Enterobacteriaceae” by Parvaiz and coworkers deals with the identification of a novel β-lactamase inhibitors. The study is of interest because is crucial to support investigation to counter the antimicrobial resistance development in pathogens. However, the study is overall preliminary and lacks some crucial experimental work to drive proper conclusion. The title mentions beta-lactamase CMY-10 but the paper lacks an experimental demonstration that substance 11 specifically inhibits this enzyme. How do the authors explain this?

ABSTRACT

The abstract needs to be rewritten to reflect the aim, methods, results, and conclusions. For example, the sentence “Of these, one compound shows promising activity in β-lactamase activity assay, susceptibility testing against ATCC strains and MDR clinical isolates, with synergistic assay indicating its potential as a β-lactam enhancer and β-lactamase inhibitor” is not clear: what are the strains analyzed? what do the authors mean by “susceptibility testing”? Please rephrase.

P. 8, Line 14: Please change “11 compounds” with “Eleven compounds”.

P. 8, Line 18-20: “Structural similarity search against the active compound yielded 28 more compounds, the majority of which also showed β-lactamase inhibition potential and antibacterial activity”. This sentence is not clear, please rephrase.

AUTHOR SUMMARY

P. 9, Line 10-12: , please rephrase the sentence “Compounds screened as potential inhibitors of CMY-10 in the current study have the potential to be used in combination therapy as non-β-lactam-based β-lactamase inhibitors against MDR clinical isolates that have been found resistant against last-line antibiotics”, because the same sentence is already written in the abstract. Some sentences are repetitions of what previously said.

INTRODUCTION

Please, explain briefly the classification of �-lactamases and the reason for the choice of identify new inhibitors only against class C �-lactamase.

RESULTS

P.12: IMP should be spelled out

P.13: The names of the bacterial species in the all text must be written in italics (E. coli)

P.14, line 20: The experimental strategy used in β-lactamase activity assay is only briefly explained in the text. Clarification on this point may help. Which strains were used in this assay?

P.15, line 1: what is the Control?

P.15, line 2: The order of Supplementary Table is wrong…. Table S2 appears first in the text while Table S1 is at the end of the paper in the materials and methods section

P.15, line 8-10: “Epsilometer test (E-test) was performed to quantify antimicrobial susceptibility of clinical isolates against advanced generation of macrolide and third and fourth generation antibiotics”: Please indicate antibiotics or refer to the supplementary table

P.15, line 12: Table S3 is not clear and incomplete, the header is wrong, some of the data refer to Epsiolmeter test results (the bottom), the other to the diffusion test…. Please explain. Why are imipenem and meropenem antibiotics not also tested?

P.16, line 1-2: Why do the authors test the substances against ATCC reference strains of S. aureus and E. coli? why don't they test the substances against a reference ATCC strain of Enterobacter? Please, perform these experiments

P16, line 3: “three multi-drug resistant clinical isolates”: only 3 isolates of Enterobacter spp were tested. Please, increase the number of clinical isolates tested.

P.16, line 6: Table 2: indicate the method used in the antibacterial activity assays. What are the 3 batches? three replicated experiments? if yes, please report only the average with DS in the table (this also applies to the other tables in supplementary).

What is the MIC of compound 11? It is preferable to determine MIC of this compound by using the broth microdilution method. Please, perform these experiments.

P.17, line 1: the synergy assay should be performed using the microbroth checkerboard method. The authors should perform this experiment for at least compound 11 and discuss the results.

P.17, line 2: Why have you chosen this antimicrobial (cefixime)? Please explain.

P.17, line 12: Fig. 6: the resolution of the figure is too low and this does not allow to verify the data. What is the control of experiment? the untreated strains? the images of the strains treated only with cefixime are missing.

P.18, line 3: “Results obtained from the β- lactamase assay suggest that”…. Please indicate the total number of compounds similar to compound 11 found (28)

P.18, line 4: “having structural similarity”: is the chemical structure of the compounds shown in Table S1? if yes, please refer to the above table.

DISCUSSION

The Discussion section is a bit long and it would also benefit from some condensation.

MATERIAL AND METHODS

P.24, line 20: In my opinion, the sentence “The complete protocol used in this study is shown in Fig 8“ should be moved to the beginning of the results, in this way the experimental procedure is immediately clear to the reader of the manuscript.

P.29, line 4: The method described for the β-lactamase activity assay is not clear. The authors should describe in detail the method used. Which sample is used in the assay? bacteria? which bacterial strains? what are the conditions used to grow bacteria?

P.29, line 15: describe punctually the protocol

P.29, line 16: did the authors characterize (at the genomic level) the clinical strains of Enterobacter used? do these strains contain the gene that encodes for CMY-10? why did the authors not test Enterobacter ATCC reference strains? Please, perform these experiments.

P.29, line 22: Change the sentence: “The test was run for 24 hrs at 37 °C” with the sentence: “The plates were incubated for 24 hrs at 37 °C”

P.31, line 4: Table S1?????? The order of Supplementary Table is wrong.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Attachment

Submitted filename: PONE-D-20-14041.pdf

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PLoS One. 2021 Jan 15;16(1):e0244967. doi: 10.1371/journal.pone.0244967.r002

Author response to Decision Letter 0

2 Dec 2020

Response to Reviewer Comments

We thank the Referees for spending their time and interest in our work. We have checked all the comments and have made necessary changes accordingly.

-Reviewer 1

1) Authors should explain a potential action mechanism of compound 11.

Author Response: Compound 11 is a non-β-lactam-based inhibitor. The possible action mechanism has been discussed on P. 17 from Line 333-337.

2) Can author clarify the correlation between lab tests and potential clinical applications?

Author Response: As an evaluation of the potential clinical utility of the inhibitor identified in our study, we evaluated if known antibiotics in the presence of this inhibitor proved to be more effective based on maximum zone of inhibition as compared to the antibiotics given alone in resistant clinical isolates. The data from these lab experiments indicates the potential clinical utility of the identified inhibitor in combination therapy for the treatment of MDR pathogens. This is discussed on P.20-21 Line 415-421 of the revised manuscript.

-Reviewer 2

1) In the nomenclature of bacterial species of Enterobacter, it is sufficient to write the Genus in full for the first time, then the dotted Genus can be used (for example E. agglomerans);

Author Response: The Genus has been written in full for the first time on P.4 line 63-64.

2) In the last paragraph of the introduction, write in full "site identification by ligand competitive saturation" and put the acronym SILCS in brackets, as it is mentioned for the first time in this section.

Author Response: It has been corrected in the revised manuscript.

3) When a bacterial species is named for the first time, the name must be written in full (Escherichia coli)

Author Response: It has been modified in the revised manuscript.

4) To make the comprehension clearer for a non-expert reader, Authors should write in full and clarify the meaning of the acronyms "VS", "MC", "LGFE" and "MLRF"

Author Response: The acronyms have been written in full to clarify the meaning in the revised form of the manuscript.

5) Experimental analysis

It would be clearer if the Authors already specified in the section Results that the Beta-lactamase activity assay was performed spectrophotometrically using a chromogenic substrate such as Nitrocefin;

Author Response: Thank you for your valuable comment. The complete sentence has been added to the results section for clarity on P. 9 line 164-170 of the revised manuscript.

6) The legend of Figure 4 should be described more clearly; it is not clear;

Author Response: Now legend of Figure 4 has been modified for clarity.

7) Add the sentence "in vitro" in the title "Susceptibility testing";

Author Response: ‘In vitro’ has been added to the title ‘Susceptibility testing’ in overall test of the revised manuscript.

8) In vitro susceptibility tests were performed on clinical isolates of Enterobacter spp. the authors should have used ATCC reference strains of the same Genus. This aspect needs to be clarified, the sensitivity towards the molecule could be different between bacterial strains of different genera

Author Response: Thank you for your valuable comment. In vitro susceptibility testing was performed again using only the ATCC reference strains of Enterobacter spp. The results section and analysis has been revised accordingly on P.10 line 194-196 and P.16 line 308-315.

9) The first time the acronym MIC is used, the meaning must be written in full;

Author Response: The acronym MIC has been written in full, the first time it is used to clarify its meaning on P.10 line 192-193 in the revised manuscript.

10) It would be interesting and would been strengthened the data if the in vitro susceptibility tests were also carried out in broth dilution in order to verify a bacteriostatic or bactericidal effect of the compounds

Author Response: The disc diffusion assay was performed again specifically for compound 11 against MDR clinical isolates with different dosage concentration to check the Minimum inhibitory effect of compound 11 with respect to broad spectrum imipenem and cefixime antibiotics. The results have been added on P.10 line 197-203 in the revised manuscript.

11) In the SEM analysis paragraph, the authors should clarify the observations, the paragraph is not clear, should be rewritten. Furthermore, the resolution of Figure 6 is very poor, it is not possible to appreciate the “extensive structural damage” reported.

Author Response: Thank you for your valuable comment. The written SEM analysis paragraph has been improved. Furthermore, the resolution of Figure 6 has also been enhanced.

12) In the legend of Figure 6, the nomenclature of the bacterial species must be corrected, the species must be always written in lower case

Author Response: The nomenclature of bacterial species has been corrected in the legend of Figure 7.

13) In the paragraph "Activity of compounds similar to compound 11" the authors should clarify that the reference strains used do not belong to the same genus as the clinical isolates, and the test should be carried out with an Enterobacter reference strain. In my opinion is wrong to use reference bacterial strains belonging to different genera, indeed the behavior of the compounds selected between ATCC strains and clinical isolates is not comparable (see compound 20).

Author Response: The experiments have been rerun with the ATCC reference strains of the same genus as the clinical isolates and results are added in the line 264-271 on page 14 of the revised manuscript.

14) From the main text it would seem that it has not been verified whether the analyzed bacterial strains are β-lactamase CMY-10 producers. This should be clearly demonstrated for example with a molecular assay

Author Response: Molecular assay was performed which verified that clinical bacterial isolates are β-lactamase CMY-10 producers. The methods and results of this genome characterization experiment have been added in the corresponding sections on P.9 line 176-180 and P.26 line 527-541.

15) The resolution of Figures 4, 5, 6 and 7 is too poor, these are not clear. In Figure 5 the written are not legible

Author Response: The resolution of the figures has been improved and figure 5 has been modified in the revised manuscript.

16) In my opinion, to make the tables (Table 2; S4; S6 and S7) more streamlined, the Authors could directly report the average value without reporting the values of the individual batches

Author Response: Thank you for your comment. The tables have been revised accordingly in revisions.

17) Figure 8 should be moved as the first figure, to make the experimental design clearer.

Author Response: Thank you for your valuable comment. The figure 8 has been moved as first figure in the revised manuscript.

18) In the discussion paragraph the Authors indicate "disc diffusion assay", but in the Results section the Authors indicate "epsilometric test". However, the diameters of the inhibition zones are commented in the section Results. What kind of test was done? This aspect is confusing and should be clarified

Author Response: Thank you for highlighting this point. The term ‘disc diffusion assay’ has been replaced with ‘in vitro susceptibility testing’ for clarification.

19) The results of the agar diffusion tests should be commented in the Results section, making the discussion more streamlined.

Author Response: The results have been discussed on P.15-16 line 304-313 of the revised manuscript.

20) The bacterial suspension must be indicated with “CFU/ml” and not “CFU/spot”. Replace the sentence

Author Response: Thank you for highlighting the mistake. It has been corrected.

21) E-test strips containing the tested compounds were produced by the authors? This aspect should be clarified and the specific MIC values obtained should be reported in the Results

Author Response: E-test was performed for evaluating the antibiotic resistance of the clinical isolates used in the study against commercially available antibiotics. E-test was not performed for the compounds reported in the study. On other hand we have also confirmed the MIC value for the compound to be reported against clinical isolates in the revised manuscript.

22) In the Synergistic assays the molecules were only tested in a 1: 1 ratio? Different ratio should also be checked.

Author Response: Thank you for your valuable comment. The synergistic assay has been run again at minimum inhibitory concentration level against all the three clinical strains. We have checked the effect of compound 11 along Cefixime and Imipenem with commercially available positive control amoxicillin + clavulanic acid P.12 line 225-231. This minimum inhibitory concentration was applied according the results obtained via micro dilution disc diffusion assay (P.18 line 373) in the revised manuscript.

23) Table S3, which reports a partial antibiogram of clinical strains, should be completed and improved, so it is not clear.

Author Response: The table has been revised accordingly.

-Reviewer 3

1) The title mentions beta-lactamase CMY-10 but the paper lacks an experimental demonstration that substance 11 specifically inhibits this enzyme. How do the authors explain this?

Author Response: Thank you for your comment. Yes, we have done an experimental demonstration of CMY-10 target. Molecular identification assay was performed to check if the clinical isolates are CMY-10 producers or not. The results of the assay suggested that clinical isolates selected in the study are CMY-10 producers, therefore, further to address revisions experimental studies have been carried on selectel clinical isolates and inhibition results have been reported accordingly.

2) The abstract needs to be rewritten to reflect the aim, methods, results, and conclusions. For example, the sentence “Of these, one compound shows promising activity in β-lactamase activity assay, susceptibility testing against ATCC strains and MDR clinical isolates, with synergistic assay indicating its potential as a β-lactam enhancer and β-lactamase inhibitor” is not clear: what are the strains analyzed? what do the authors mean by “susceptibility testing”? Please rephrase.

Author Response: The abstract is divided into sections to reflect aim (P.2 line 24-26), methods (P.2 line 26-33), results (P.2 line 33-39), and conclusions (P.2 line 39-41). The sentence has been rephrased and the names of the ATCC and MDR clinical isolates have been added.

3) P. 8, Line 14: Please change “11 compounds” with “Eleven compounds”.

Author Response: The correction has been done on P.2 line 33.

4) P. 8, Line 18-20: “Structural similarity search against the active compound yielded 28 more compounds, the majority of which also showed β-lactamase inhibition potential and antibacterial activity”. This sentence is not clear, please rephrase.

Author Response: The sentence has been rephrased for clarity.

5) P. 9, Line 10-12: , please rephrase the sentence “Compounds screened as potential inhibitors of CMY-10 in the current study have the potential to be used in combination therapy as non-β-lactam-based β-lactamase inhibitors against MDR clinical isolates that have been found resistant against last-line antibiotics”, because the same sentence is already written in the abstract. Some sentences are repetitions of what previously said.

Author Response: The sentence has been rephrased in revised manuscript.

6) Please, explain briefly the classification of β-lactamases and the reason for the choice of identify new inhibitors only against class C β-lactamase.

Author Response: The classification has been explained briefly on Pg. 4 lines 68-72

7) P.12: IMP should be spelled out

Author Response: Thank you for highlighting this mistake. IMP has been spelled out the first time it has been written on P. 6 line 105

8) P.13: The names of the bacterial species in the all text must be written in italics (E. coli)

Author Response: Thank you for highlighting this mistake. It has been corrected accordingly.

9) P.14, line 20: The experimental strategy used in β-lactamase activity assay is only briefly explained in the text. Clarification on this point may help. Which strains were used in this assay?

Author Response: We have used β-lactamase enzyme instead of β-lactamase producer strains in inhibition assay.

10) P.15, line 1: what is the Control?

Author Response: The control used is cefixime and have been added on P.18 line 352.

11) P.15, line 2: The order of Supplementary Table is wrong…. Table S2 appears first in the text while Table S1 is at the end of the paper in the materials and methods section

Author Response: Thank you highlighting this mistake. The order of all the supplementary tables has been revised.

12) P.15, line 8-10: “Epsilometer test (E-test) was performed to quantify antimicrobial susceptibility of clinical isolates against advanced generation of macrolide and third and fourth generation antibiotics”: Please indicate antibiotics or refer to the supplementary table

Author Response: The supplementary table has been referred in the text on P.10 line 190-191 by adding fourth generation antibiotics imipenem, meropenem in the revised manuscript.

13) P.15, line 12: Table S3 is not clear and incomplete, the header is wrong, some of the data refer to Epsiolmeter test results (the bottom), the other to the diffusion test…. Please explain. Why are imipenem and meropenem antibiotics not also tested?

Author Response: Table S3 has been revised accordingly. Antibiotics imipenem and meropenem have been added in the experimental testing while adding revisions.

14) P.16, line 1-2: Why do the authors test the substances against ATCC reference strains of S. aureus and E. coli? why don't they test the substances against a reference ATCC strain of Enterobacter? Please, perform these experiments

Author Response: The experiments have been re-run to add reference ATCC strains of Enterobacter while other are excluded in instructed.

15) P16, line 3: “three multi-drug resistant clinical isolates”: only 3 isolates of Enterobacter spp were tested. Please, increase the number of clinical isolates tested.

Author Response: The data has been collected from Pakistan Hospital. Therefore, the available three clinical isolates of Enterobacter that are prevalent in Pakistan were selected in the study.

16) P.16, line 6: Table 2: indicate the method used in the antibacterial activity assays. What are the 3 batches? three replicated experiments? if yes, please report only the average with DS in the table (this also applies to the other tables in supplementary).

Author Response: Yes, these were 3 batches indicating three replicated experiments. The table has been modified for further clarification in the revised manuscript.

17) What is the MIC of compound 11? It is preferable to determine MIC of this compound by using the broth microdilution method. Please, perform these experiments.

Author Response: MIC was evaluated using disc diffusion assay. Please see table 3 and 4 for reference.

18) P.17, line 1: the synergy assay should be performed using the microbroth checkerboard method. The authors should perform this experiment for at least compound 11 and discuss the results.

Author Response: Synergistic effect of minimum inhibitory concentration of compound 11 with minimum inhibitory concentrations of cefixime, imipenem, and positive control amoxicillin-clavulanic acid against clinical isolates was also evaluated in the revised manuscript in page 12 line 225-231.

19) P.17, line 2: Why have you chosen this antimicrobial (cefixime)? Please explain.

Author Response: Imipenem has also been added now. Cefixime has been selected because it is third generation antibiotic whereas Imipenem is a fourth generation antibiotic, both of which are used to treat multi-drug resistant strains.

20) P.17, line 12: Fig. 6: the resolution of the figure is too low and this does not allow to verify the data. What is the control of experiment? the untreated strains? the images of the strains treated only with cefixime are missing.

Author Response: The experiment has been revised as per instruction of the reviewers. We have performed the experiment along control as well to check the efficacy of our compound in treated and untreated way against the clinical isolates in the revised manuscript.

21) P.18, line 3: “Results obtained from the β- lactamase assay suggest that”…. Please indicate the total number of compounds similar to compound 11 found (28)

Author Response: The total numbers of compounds similar to compound 11 have been incorporated in the text (P.13 line 257).

22) P.18, line 4: “having structural similarity”: is the chemical structure of the compounds shown in Table S1? if yes, please refer to the above table.

Author Response: Thank you for pointing out this. The corresponding table has been referred in the text.

23) The Discussion section is a bit long and it would also benefit from some condensation.

Author Response: A condensation has been tried but some new experiments have also been performed to address comments of Reviewer 1 and Reviewer 2. So the addition of those results prevented the reflection of possible condensation.

24) P.24, line 20: In my opinion, the sentence “The complete protocol used in this study is shown in Fig 8“ should be moved to the beginning of the results, in this way the experimental procedure is immediately clear to the reader of the manuscript.

Author Response: The figure 8 has been replaced as figure 1 in the text of the revised manuscript.

25) P.29, line 4: The method described for the β-lactamase activity assay is not clear. The authors should describe in detail the method used. Which sample is used in the assay? bacteria? which bacterial strains? what are the conditions used to grow bacteria?

Author Response: The authors have provided the brief explanation of the protocol regarding beta-lactamase activity assay in the revised manuscript on P.25 line 514-524. Sample in the assay have been mentioned (Chembridge compounds). No bacterial samples were used in this assay.

26) P.29, line 15: describe punctually the protocol

Author Response: The schematic flow of complete protocol has been given in Figure 1.

27) P.29, line 16: did the authors characterize (at the genomic level) the clinical strains of Enterobacter used? do these strains contain the gene that encodes for CMY-10? why did the authors not test Enterobacter ATCC reference strains? Please, perform these experiments.

Author Response: The experiments have been performed again as instructed by the reviewers in order to test Enterobacter ATCC reference strains page 26 line 540-541. Molecular identification assay has additionally been performed to characterize clinical strains of Enterobacter at genomic level while addressing the overall revisions at page 26 line 525-539.

28) P.29, line 22: Change the sentence: “The test was run for 24 hrs at 37 °C” with the sentence: “The plates were incubated for 24 hrs at 37 °C”

Author Response: The sentence has been revised accordingly.

29) P.31, line 4: Table S1?????? The order of Supplementary Table is wrong.

Author Response: The order of all the supplementary tables has been revised.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (36KB, docx)

Decision Letter 1

Massimiliano Galdiero

21 Dec 2020

Discovery of beta-lactamase CMY-10 inhibitors for combination therapy against multi-drug resistant Enterobacteriaceae

PONE-D-20-14041R1

Dear Dr. Syed Sikander Azam,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Massimiliano Galdiero, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: THIS PAPER IS CLEARLY REVISIONED FOLLOWING REVIEWER' SUGGESTIONS. I BELIEVE THAT AFTER THIS REVISION THE PAPER IS SUITABLE FOR PUBBLICATION.

Reviewer #2: Authors should pay attention to the nomenclature of bacteria throughout the text including the tables (see Table 2). The bacterial species must always be written in lowercase italics.

Furthermore, the units of measurement reported throughout the text must be checked (see Table 4 and related text).

Finally, in the Molecular assay, as in the electrophoresis there is no control it would be useful to insert in the text a reference on the high molecular weight plasmid.

Reviewer #3: (No Response)

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Reviewer #3: No

Acceptance letter

Massimiliano Galdiero

7 Jan 2021

PONE-D-20-14041R1

Discovery of beta-lactamase CMY-10 inhibitors for combination therapy against multi-drug resistant Enterobacteriaceae

Dear Dr. Azam:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Prof. Massimiliano Galdiero

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PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. BL-activity and LGFE scores of lead compounds identified as enhancers.

    (DOCX)

    Click here for additional data file. (30.3KB, docx)
    S2 Table. Epsilometer test results for commercially available beta-lactam antibiotics against MDR clinical isolates.

    (DOCX)

    Click here for additional data file. (31.2KB, docx)
    S3 Table. Antibacterial activity of lead compounds against ATCC bacterial isolates with zone of inhibition (mm).

    (DOCX)

    Click here for additional data file. (28.2KB, docx)
    S4 Table. Two dimensional chemical structures of the similarity searched compounds including active compound 11.

    (DOCX)

    Click here for additional data file. (300.8KB, docx)
    S5 Table. BL-activity of further screened compounds.

    (DOCX)

    Click here for additional data file. (26.5KB, docx)
    S6 Table. Antibacterial activity of similarity searched compounds against ATCC bacterial isolates with zone of inhibition (mm).

    (DOCX)

    Click here for additional data file. (30.4KB, docx)
    S7 Table. Antibacterial activity of similarity searched compounds against β lactamase producer’s clinical bacterial isolates.

    (DOCX)

    Click here for additional data file. (28.9KB, docx)
    S1 Fig. All pharmacophore features generated from SILCS FragMaps at the R1 and R2 site region.

    Hydrophobic, hydrogen bond acceptor, hydrogen bond donor and negatively charged features are colored in cyan, red, blue, and dark red, respectively.

    (TIF)

    Click here for additional data file. (8MB, tif)
    S2 Fig

    LGFE distributions for top 10,000 ranked compounds from VS for both R2 model (a) and R1-R2 (b) model. The vertical dashed line indicates the LGFE cutoff for selecting the top 500 ranked compounds. The LGFE values for the seven compounds identified as inhibitors are indicated by blue arrows with their compound ID labeled. 5 hits are from the R2 model VS and 2 hits are from the R1-R2 model VS.

    (TIF)

    Click here for additional data file. (4.4MB, tif)
    S3 Fig. 2D chemical structure of compound 11 (Chembridge ID: 5524250).

    (TIF)

    Click here for additional data file. (195.1KB, tif)
    S4 Fig

    (A) The 3D structure of CMY-10 in ribbon representation. The residues of R1 site (Ω-loop, Gln121 loop and β11) and R2 site (Tyr151 loop, α10 and α11) are in yellow and green, respectively. The binding cavity of R1 site is represented by red ellipse and R2 site is represented by blue ellipse. The nucleophile Ser65 is shown in magenta using ball and stick representation within the blue ellipse.

    (TIF)

    Click here for additional data file. (4.3MB, tif)
    S5 Fig. Predicted binding orientation of lead compound 11.

    The binding site is shown in the same orientation as in the Fig 2. FragMaps are shown at GFE cutoff -1.0 kcal/mol for apolar (green), hydrogen bonding donor (blue) and acceptor (red) maps and at -1.5 kcal/mol for negatively (orange) charged map.

    (TIF)

    Click here for additional data file. (4MB, tif)
    Attachment

    Submitted filename: PONE-D-20-14041.pdf

    Click here for additional data file. (200.5KB, pdf)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (36KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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