Computational resources in the management of antibiotic resistance: Speeding up drug discovery.

This article reviews more than 50 computational resources developed in past two decades for forecasting of antibiotic resistance (AR)-associated mutations, genes and genomes. More than 30 databases have been developed for AR-associated information, but only a fraction of them are updated regularly. A large number of methods have been developed to find AR genes, mutations and genomes, with most of them based on similarity-search tools such as BLAST and HMMER. In addition, methods have been developed to predict the inhibition potential of antibiotics against a bacterial strain from the whole-genome data of bacteria. This review also discuss computational resources that can be used to manage the treatment of AR-associated diseases.

A Web-Based Platform on Coronavirus Disease-19 to Maintain Predicted Diagnostic, Drug, and Vaccine Candidates.

A web-based resource CoronaVIR (https://webs.iiitd.edu.in/raghava/coronavir/) has been developed to maintain the predicted and existing information on coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have integrated multiple modules, including "Genomics," "Diagnosis," "Immunotherapy," and "Drug Designing" to understand the holistic view of this pandemic medical disaster. The genomics module provides genomic information of different strains of this virus to understand genomic level alterations. The diagnosis module includes detailed information on currently-in-use diagnostics tests as well as five novel universal primer sets predicted using in silico tools. The Immunotherapy module provides information on epitope-based potential vaccine candidates (e.g., LQLPQGTTLPKGFYA, VILLNKHIDAYKTFPPTEPKKDKKKK, EITVATSRTLS, GKGQQQQGQTV, SELVIGAVILR) predicted using state-of-the-art software and resources in the field of immune informatics. These epitopes have the potential to activate both adaptive (e.g., B cell and T cell) and innate (e.g., vaccine adjuvants) immune systems as well as suitable for all strains of SARS-CoV-2. Besides, we have also predicted potential candidates for siRNA-based therapy and RNA-based vaccine adjuvants. The drug designing module maintains information about potential drug targets, tertiary structures, and potential drug molecules. These potential drug molecules were identified from FDA-approved drugs using the docking-based approach. We also compiled information from the literature and Internet on potential drugs, repurposing drugs, and monoclonal antibodies. To understand host-virus interaction, we identified cell-penetrating peptides in the virus. In this study, state-of-the-art techniques have been used for predicting the potential candidates for diagnostics and therapeutics.

A mechanistic approach to prove the efficacy of combination therapy against New Delhi metallo-ß-lactamases producing bacterial strain: a molecular and biochemical approach.

BACKGROUND: NDM-1 is a novel broad-spectrum metallo-ß-lactamase with the capability to grant resistance to almost all ß-lactam antibiotics. Its widespread dissemination made treatment options a major challenge to combat, causing threat to public health worldwide. Due to antibiotic resistance problems, development of effective therapeutics for infections caused by NDM-1 producing strains is urgently required. Since combination therapies are proved to be effective in many cases, this study was initiated to put forward novel effective antibiotics combinations for fighting infections caused by NDM-1 producing strains. METHODS: Streptomycin and amikacin combination and streptomycin and ciprofloxacin combination were tested by checkerboard assay. NDM-1 protein/enzyme was then expressed and purified to carry out enzyme kinetics study, CD and fluorescence spectroscopic studies. RESULTS: Streptomycin and amikacin combination and streptomycin and ciprofloxacin combination showed synergistic effect towards NDM-1 producing bacterial strains as shown by FICI results. NDM-1 producing bacterial cells were expressed and purified to obtain protein as the source of enzyme. When NDM-1 enzyme was treated with streptomycin along with amikacin, the efficiency of enzyme was decreased by 49.37% and when the enzyme was treated with streptomycin along with ciprofloxacin, the efficiency of enzyme was decreased by 29.66% as revealed by enzyme kinetic studies. Due to binding of streptomycin and amikacin in combination and streptomycin and ciprofloxacin in combination, conformational changes in the secondary structure of NDM-1 enzyme were observed by CD spectroscopic studies. Antibiotics streptomycin and ciprofloxacin bind with NDM-1 through exothermic processes, whereas amikacin binds through an endothermic process. All three antibiotics bind spontaneously with an association constant of the order of 104 M-1 as revealed by fluorescence spectroscopic studies. CONCLUSIONS: The therapeutic combination of streptomycin with amikacin and ciprofloxacin plays an important role in inhibiting NDM-1 producing bacterial strains. Therefore, these combinations can be used as effective future therapeutic candidates against NDM-1 producing bacterial cells.

Synergistic effect of doripenem in combination with cefoxitin and tetracycline in inhibiting NDM-1 producing bacteria.

Aim: To propose newer combinations of antibiotics effective against NDM-1-producing bacterial strains. Materials & methods: Antibiotics combinations were tested by checkerboard assay. NDM-1 protein/enzyme was expressed and purified to perform enzyme kinetics, circular dichroism and fluorescence spectroscopic studies. Results: Doripenem-cefoxitin combination and doripenem-tetracycline combination showed synergistic effect toward NDM-1-producing strains. The catalytic efficiency of NDM-1 enzyme was decreased drastically by 96.6% upon doripenem-cefoxitin treatment and by 35.54% after doripenem-tetracycline treatment. Conformational changes were observed in NDM-1 upon combination treatment. Conclusion: NDM-1-producing bacterial strains show resistance to multiple antibiotics but the combination of doripenem-cefoxitin and doripenem-tetracycline are effective against them. The combination of a carbapenem and cephamycin antibiotic is proposed for future treatment options against bacteria-producing NDM-1.

Significant role of Asn-247 and Arg-64 residues in close proximity of the active site in maintaining the catalytic function of CTX-M-15 type ß-lactamase.

Members of Enterobacteriaceae cause antibiotic-resistant infections worldwide. One such marker, CTX-M-15, expressed by Enterobacteriaceae produces ß-lactamases, which hydrolyze the cephalosporin group of antibiotics, such as cefotaxime, used in the treatment of both Gram-positive and negative bacterial infections. Amino acid residues present in close proximity of the active site might also play a major role in the structure and function of CTX-M-15, hence the objective of this study was to investigate the significance of two amino acid residues, Asn-247 and Arg-64, present near to the active site in the hydrolysis of cefotaxime. bla CTX-M-15, cloned from the E. cloacae strain, and using Polymerase Chain Reaction (PCR)-based site-directed mutagenesis, Asn247Val and Arg64Leu mutations were introduced. The minimum inhibitory concentrations of cefotaxime for the CTX-M-15 (N247V) and CTX-M-15 (R64L) mutants were reduced by 512 and 128 fold, respectively. Proteins/enzymes of wild-type CTX-M-15, CTX-M-15 (N247V) and CTX-M-15 (R64L) mutants were expressed and purified. Kinetic studies showed that the catalytic efficiencies of the N247V mutant and R64L mutant enzymes in the hydrolysis of cefotaxime were reduced by 89.66% and 71.11%, respectively. Circular dichroism spectroscopic studies showed considerable changes in the α-helical content of the mutant enzymes. A fluorescence study showed that N247V mutant-cefotaxime and R64L mutant-cefotaxime underwent complex formation with strong interactions. The study provides an understanding of the crucial role of the amino acid residues asparagine 247 and arginine 64 present in close proximity of the active site in the hydrolytic mechanism of CTX-M-15 type ß-lactamases. Hence, Asn-247 and Arg-64 can be used as potential target sites for the design of inhibitory molecules against CTX-M-15-producing bacterial strains.

Combination of aztreonam and cefotaxime against CTX-M-15 type ß-lactamases: A mechanism based effective therapeutic approach.

CTX-M-15 type ß-lactamases are a class of enzymes which hydrolyzes cefotaxime and aztreonam (a monobactam) antibiotics. The emergence of CTX-M-15 producing Enterobacteriaceae member is a major threat to public health. The objective of the study was to check the potency of aztreonam and cefotaxime in combination against ß-lactamase producing strains and to monitor the mechanism behind their interaction. FICI results showed the synergistic effect of aztreonam-cefotaxime against CTX-M-15 producing strain. The expressed and purified CTX-M-15 protein was used as the source of enzyme. Kinetic studies confirmed that the catalytic efficiency of the CTX-M-15 enzyme was decreased to about 78% when it was treated with aztreonam then with cefotaxime in 5 and 10 times molar ratio, respectively, in comparison to the studies where efficiency was enhanced by 33% when cefotaxime was taken alone. Fluorescence study showed that aztreonam binding with CTX-M-15 was an endothermic and spontaneous process with Ka of the order of 104 M-1. CD spectroscopic study showed conformational changes upon aztreonam/aztreonam-cefotaxime binding with CTX-M-15. The study concludes that aztreonam in combination with cefotaxime synergistically inhibits CTX-M-15 efficiency significantly. Hence the combination of a monobactam and cephalosporin can be used as the potential therapeutic candidates against ß-lactamase producing CTX-M-15 strains.

Understanding the mode of binding mechanism of doripenem to human serum albumin: Spectroscopic and molecular docking approaches.

The infections caused by multidrug resistant bacteria are widely treated with carabapenem antibiotics as a drug of choice, and human serum albumin (HSA) plays a vital role in binding with drugs and affecting its rate of delivery and efficacy. So, we have initiated this study to characterize the mechanism of doripenem binding and to locate its site of binding on HSA by using spectroscopic and docking approaches. The binding of doripenem leads to alteration of the environment surrounding Trp-214 residue of HSA as observed by UV spectroscopic study. Fluorescence spectroscopic study revealed considerable interaction and complex formation of doripenem and HSA as indicated by Ksv and Kq values of the order of 104  M-1 and 1012  M-1  s-1 , respectively. Furthermore, doripenem quenches the fluorescence of HSA spontaneously on a single binding site with binding constant of the order of 103  M-1 , through an exothermic process. Van der Waals forces and hydrogen bonding are the major forces operating to stabilize HSA-doripenem complex. Circular dichroism spectroscopic study showed changes in the structure of HSA upon doripenem binding. Drug displacement and molecular docking studies revealed that the binding site of doripenem on HSA is located on subdomain IB and III A. This study concludes that, due to significant interaction of doripenem on either subdomain IB or IIIA of HSA, the availability of doripenem on the target site may be compromised. Hence, there is a possibility of unavailability of threshold amount of drug to be reached to the target; consequently, resistance may develop in the bacterial population.

Designing of inhibitors against CTX-M-15 type ß-lactamase: potential drug candidate against ß-lactamases-producing multi-drug-resistant bacteria.

CTX-M-15 are the most prevalent types of ß-lactamases that hydrolyze almost all antibiotics of ß-lactam group lead to multiple-antibiotic resistance in bacteria. Three ß-lactam inhibitors are available for use in combination with different antibiotics of cephalosporine group against the CTX-M-15-producing strains. Therefore, strategies to identify novel anti ß-lactamase agents with specific mechanisms of action are the need of an hour. In this study, we screened three novel non-ß-lactam inhibitors against CTX-M-15 by multi-step virtual screening approach. The potential for virtually screened drugs was estimated through in vitro cell assays. Hence, we proposed a study to understand the binding mode of CTX-M-15 with inhibitors using bioinformatics and experimental approach. We calculated the dissociation constants (Kd), association constant (Ka), stoichiometry (n) and binding energies (ΔG) of compounds with the respective targets. Molecular dynamic simulation carried out for 25 ns, revealed that these complexes were found stable throughout the simulation with relative RMSD in acceptable range. Moreover, microbiological and kinetic studies further confirmed high efficacies of these inhibitors by reducing the minimum inhibitory concentration (MIC) and catalysis of antibiotics by ß-lactamases in the presence of inhibitors. Therefore, we conclude that these potential inhibitors may be used as a lead molecule for future drug candidates against ß-lactamases-producing bacteria.

Synergistic Effect of Doripenem and Cefotaxime to Inhibit CTX-M-15 Type ß-Lactamases: Biophysical and Microbiological Views.

CTX-M-15 type ß-lactamase has the ability to hydrolyse cefotaxime, a third generation cephalosporin. The infections caused by multidrug resistant strains, especially CTX-M-15 producing strains are being treated with carbapenem group of ß-lactam antibiotics. The objective of the study was to know if cefotaxime in combination with doripenem (carbapemen antibiotic) at very low concentration, inhibits the CTX-M-15 producing bacterial strains. blaCTX-M-15 gene was cloned to express CTX-M-15 enzyme and construct CTX-M-15 producing strain. The clone carrying CTX-M-15 was found susceptible to doripenem. Doripenem and CTX-M-15 binding was an endothermic and spontaneous process leading to change in polarity in the micro-environment and conformational changes of enzyme as shown by fluorescence, UV and CD spectroscopic study. The catalytic efficiency of CTX-M-15 enzyme was reduced to about 15.86% when it was treated with doripenem along with cefotaxime (in 5 times molar ratio each of doripenem and cefotaxime w.r.t CTX-M-15), as compared to the studies where enzyme's efficiency was increased by 33% when treated with cefotaxime alone. Hence, doripenem in combination with cefotaxime reduces enzyme's efficiency to hydrolyse cefotaxime by about 48%. FIC study showed that doripenem paired with cefotaxime showed synergistic effect against CTX-M-15 producing bacterial strain. The study concludes that doripenem at very low concentration (25 nM), induces such a structural changes in CTX-M-15 which reduced enzyme's activity to hydrolyse cefotaxime. Hence, the synergistic use of doripenem and cefotaxime plays a significant role in inhibiting the efficiency of CTX-M-15 type ß-lactamase, and may provide an alternative approach to reduce the resistance against the cephalosporin type antibiotics.

Structure, Genetics and Worldwide Spread of New Delhi Metallo-ß-lactamase (NDM): a threat to public health.

BACKGROUND: The emergence of carbapenemase producing bacteria, especially New Delhi metallo-ß-lactamase (NDM-1) and its variants, worldwide, has raised amajor public health concern. NDM-1 hydrolyzes a wide range of ß-lactam antibiotics, including carbapenems, which are the last resort of antibiotics for the treatment of infections caused by resistant strain of bacteria. MAIN BODY: In this review, we have discussed bla NDM-1variants, its genetic analysis including type of specific mutation, origin of country and spread among several type of bacterial species. Wide members of enterobacteriaceae, most commonly Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and gram-negative non-fermenters Pseudomonas spp. and Acinetobacter baumannii were found to carry these markers. Moreover, at least seventeen variants of bla NDM-type gene differing into one or two residues of amino acids at distinct positions have been reported so far among different species of bacteria from different countries. The genetic and structural studies of these variants are important to understand the mechanism of antibiotic hydrolysis as well as to design new molecules with inhibitory activity against antibiotics. CONCLUSION: This review provides a comprehensive view of structural differences among NDM-1 variants, which are a driving force behind their spread across the globe.

Structural insight into mode of binding of Meropenem to CTX-M-15 type ß-lactamase.

Among Enterobacteriaceae, CTX-M type extended spectrum beta lactamase confers potent hydrolytic activity against cephalosporin group of antibiotics. Strains producing CTX-M type beta lactamase enzymes, show high level of resistance against cefotaxime. Therefore carbapenem antibiotics are used against beta lactamase producing strains. Hence, this study was designed to understand an insight of molecular basis of CTX-M-15 interaction with meropenem, and its effect on CTX-M-15 efficiency. Clinical strain of Enterobacter cloacae (EC-15) was used to clone blaCTX-M-15 gene in E.coli BL21cells. The protein was then expressed and purified. Results showed that CTX-M-15 producing strains are susceptible to meropenem. It quenches the fluorescence of CTX-M-15 spontaneously with binding constant of the order of 103M-1. Meropenem binds on the active site of CTX-M-15, hydrogen bonded with four common amino acid residues of cefotaxime binding site, as revealed by molecular docking studies. Conformational change in the structure of CTX-M-15 was observed upon meropenem binding by CD spectroscopy. The catalytic efficiency of CTX-M-15 was decreased up to 4 times upon meropenem binding. Docking study shows that few amino acids of active site of enzyme are also involved in meropenem binding, hence substrate is difficult to bind on active site properly and does not get hydrolysed. Moreover, meropenem binding induces structural changes in CTX-M-15, making the enzyme less efficient.

Activation of Human Salivary Aldehyde Dehydrogenase by Sulforaphane: Mechanism and Significance.

Cruciferous vegetables contain the bio-active compound sulforaphane (SF) which has been reported to protect individuals against various diseases by a number of mechanisms, including activation of the phase II detoxification enzymes. In this study, we show that the extracts of five cruciferous vegetables that we commonly consume and SF activate human salivary aldehyde dehydrogenase (hsALDH), which is a very important detoxifying enzyme in the mouth. Maximum activation was observed at 1 µg/ml of cabbage extract with 2.6 fold increase in the activity. There was a ~1.9 fold increase in the activity of hsALDH at SF concentration of ≥ 100 nM. The concentration of SF at half the maximum response (EC50 value) was determined to be 52 ± 2 nM. There was an increase in the Vmax and a decrease in the Km of the enzyme in the presence of SF. Hence, SF interacts with the enzyme and increases its affinity for the substrate. UV absorbance, fluorescence and CD studies revealed that SF binds to hsALDH and does not disrupt its native structure. SF binds with the enzyme with a binding constant of 1.23 x 107 M-1. There is one binding site on hsALDH for SF, and the thermodynamic parameters indicate the formation of a spontaneous strong complex between the two. Molecular docking analysis depicted that SF fits into the active site of ALDH3A1, and facilitates the catalytic mechanism of the enzyme. SF being an antioxidant, is very likely to protect the catalytic Cys 243 residue from oxidation, which leads to the increase in the catalytic efficiency and hence the activation of the enzyme. Further, hsALDH which is virtually inactive towards acetaldehyde exhibited significant activity towards it in the presence of SF. It is therefore very likely that consumption of large quantities of cruciferous vegetables or SF supplements, through their activating effect on hsALDH can protect individuals who are alcohol intolerant against acetaldehyde toxicity and also lower the risk of oral cancer development.

A Mechanism of Synergistic Effect of Streptomycin and Cefotaxime on CTX-M-15 Type ß-lactamase Producing Strain of E. cloacae: A First Report.

A blaCTX-M-15 gene is one of the most prevalent resistant marker found in member of enterobacteriaceae. It encodes cefotaxime hydrolysing ß-lactamase-15 (CTX-M-15) causing resistance against beta lactam antibiotics. Since single antibiotic therapy fails to control infection caused by multidrug resistance strain, therefore combination therapy was came into practice as an effective treatment. We have first time explained the mechanism where two antibiotics of different classes work against resistant strains. Binding parameters obtained by spectroscopic approach showed significant interaction and complex formation between drugs and CTX-M-15 enzyme with decreased ksv and kq values. CD analysis showed altered conformation and significant changes in alpha helical content of CTX-M-15 enzyme on interaction with streptomycin in combination with cephalosporin. Steady state kinetics revealed decrease in hydrolytic efficiency of enzyme to about 27% by cooperative binding behavior upon sequential treatment of enzyme with streptomycin and cefotaxime. Therefore, the study concludes that combination therapy against CTX-M-15 producing strain with Cefotaxime/Streptomycin in 1:10 molar ratio, decreases CTX-M-15 efficiency significantly because of the fact that streptomycin induced structural changes in CTX-M-15 hence cefotaxime was not properly bound on its active site for hydrolysis rather available for the target to inhibit bacterial cells.