Structural insight into the binding mode of cefotaxime and meropenem to TEM-1, SHV-1, KPC-2, and Amp-C type beta-lactamases.

Antimicrobial resistance is an emerging threat to public health around the world. The study employs computational and biophysical methods to investigate the properties of cefotaxime and meropenem's binding to various beta-lactamases like TEM-1, SHV-1, KPC-2, and Amp-C. The enzyme kinetics of purified proteins revealed an increase in Michaelis constant (Km) value in the presence of meropenem and cefotaxime, indicating a decrease in enzyme affinity for nitrocefin. Proteins interact with meropenem/cefotaxime, causing quenching through complex formation. All proteins have one binding site, and binding constant (Kb) values are 104, indicating strong interaction. The study found that meropenem and cefotaxime had high fitness scores for Amp-C, KPC-2,TEM-1 and SHV-1, with binding energy ranging from -7.4 to -7.8, and hydrogen bonds between them. Molecular Dynamic simulation of protein-ligand complexes revealed cefotaxime-binding proteins have slightly lower Root Mean Square Deviation(RMSD) than meropenem-binding proteins, indicating stable association antibiotics with these proteins.

Inhibitors against New Delhi metallo-betalactamase-1 (NDM-1) and its variants endemic in Indian settings along with the laboratory functional gain mutant of NDM-1.

PURPOSE: The emergence of NDM-1 producing bacteria has become common in both hospital and community settings, but no inhibitor has yet been available for clinical treatment. Hence, demanding the urgent need of NDM-1 inhibitors, we initiated to screen broad spectrum inhibitors against NDM natural variants and laboratory mutant. METHODS: We used docking and molecular dynamics simulations, in silico pharmacokinetic investigations, and density functional theory calculation to characterize molecules. Furthermore, an in vitro study, including MIC, kinetics, and fluorescence study were carried out to confirm the efficacies of the selected compounds. RESULTS: According to the findings of the computational studies, three compounds were effective against NDM variants. Fourfold reduction in MIC of imipenem and meropenem was observed when combined with inhibitors (D2573, D2148, and D63) against blaNDM-1, blaNDM-4, blaNDM-6, and blaNDM-1Q123A, while twofold reduction in MIC of imipenem and meropenem was observed against blaNDM-5 and blaNDM-7. Similarly in the presence of inhibitors (D2573, D2148, and D63) the efficiency of nitrocefin hydrolysis by NDM-4, NDM-6, and Q123A decreases to much more extent as compared to NDM-5 and NDM-7. These results showed that the efficacy of these broad spectrum inhibitors decreases with increasing resistance of NDM variants. CONCLUSION: This is the first time inhibitors were tested against different NDM natural variants which are endemic in Indian settings. Moreover, a functional gain laboratory mutant was also checked for their efficacies. We may propose these molecules for the pre-clinical trial to further translate.

Mechanistic Approach of Effective Combination of Antibiotics Against Clinical Bacterial Strains Having New Delhi Metallo-Beta-Lactamase Variants and Functional Gain Laboratory Mutant.

Antimicrobial resistance has emerged as a serious issue for physicians and health-care workers treating infections that could lead to the next pandemic. One of the key resistance mechanisms is beta-lactamases. Although several beta-lactamase inhibitors in combination with antibiotics have been created and are being utilized in clinical settings, resistance to these formulations has also been evolving in the bacterial population due to their distinct targets. In this study we used effective combination of antibiotic as an approach to inhibit multidrug resistance bacteria. We used four combinations and checked its efficacy against NDM (New Delhi Metallo-beta-lactamase) variants and functional gain laboratory mutant by employing FICI, enzyme kinetics, fluorescence and computational biology approaches (Docking and Molecular Dynamics Simulation). FICI values of all the combinations were either less than 0.5 or equal to 0.5. Binding features acquired by spectroscopic techniques showed important interaction and complex formation between drugs and enzymes with decreased ksv and kq values. In steady-state kinetics, a reduction in hydrolytic efficiency of enzymes was shown by cooperative binding behaviour when they were treated with different drugs. We have also tested functional gain laboratory mutant developed in our lab, keeping in view that if in future upcoming variants of this kind be emerged then these mutants could also be subsided by combinational therapy. This study identifies three other combinations better than fluoroquinolones effective against NDM variants and laboratory mutant.

Repurposing FDA approved drug molecules against A B C classes of ß-lactamases: a computational biology and molecular dynamics simulations study.

ß-lactamase are the main resistance factor for ß-lactam antibiotics in Gram-negative bacteria. Since ß-lactam antibiotics are being utilised as an antimicrobial agents extensively for the past 70 years, a large number of ß-lactam-inactivating ß-lactamases have been produced by bacteria. Here, we employed a structure-based drug discovery approach to identify and assess the efficacy of a potential medication that might block the ß-lactamases which hydrolyse antibiotics. The FDA-approved medications were subjected to virtual screening, molecular docking, molecular dynamics simulations, density functional theory, and covalent docking against the ß-lactamases. We identified diosmin, hidrosmin, monoxuritin and solasulfone as ß-lactamase inhibitors which are authorised for therapeutic use in humans. These medications interact in a remarkable variety of non-covalent ways with the conserved residues in the substrate-binding pocket of the ß-lactamases. Diosmin has been identified as an inhibitor that binds covalently to the NDM-1 a class B metallo-betalactamase. After experimental validation and clinical demonstration, this study offers adequate evidence for the therapeutic use of these drugs for controlling multidrug resistance.Communicated by Ramaswamy H. Sarma.

Studies on the interaction of 2,4-dibromophenol with human hemoglobin using multi-spectroscopic, molecular docking and molecular dynamics techniques.

2,4-Dibromophenol (DBP) has several industrial applications, including as a wood preservative and flame retardant. This study investigated the interaction between DBP and human hemoglobin (Hb) using spectroscopic, molecular docking and molecular dynamic techniques. The UV-visible spectra showed ground-state complex formation between DBP and Hb. Fluorescence studies revealed that DBP binding caused significant quenching of Hb fluorescence by the static quenching mechanism. The binding of DBP to Hb is a spontaneous process that involves van der Waals forces and hydrogen bonds. There is one DBP binding site on each Hb molecule that is located at the α1ß2 interface of Hb. DBP binding did not alter the microenvironment of tyrosine and tryptophan residues in Hb. Circular dichroism studies revealed that DBP increased the α-helical content of Hb. The intrinsic esterase activity of Hb was inhibited by DBP in a concentration-dependent manner. Molecular docking showed that DBP binds to Hb via hydrogen bonds, hydrophobic, van der Waals and π-π interactions. Molecular dynamics simulation confirmed that the Hb-DBP complex is stable. Overall, the results of this study clearly show that DBP induces structural changes and interferes with the function of Hb. This can have important implications for human health.Communicated by Ramaswamy H. Sarma.

Chemically synthesised flavone and coumarin based isoxazole derivatives as broad spectrum inhibitors of serine ß-lactamases and metallo-ß-lactamases: a computational, biophysical and biochemical study.

The ß-lactam antibiotics are the most effective medicines for treating bacterial infections. Resistance to them, particularly through the production of ß-lactamases, which can hydrolyse all kinds of ß-lactams, poses a threat to their continued use. The synthesised flavone and coumarin based isoxazole derivatives have the potential to be used as broad-spectrum inhibitors of the mechanistically different serine-(SBL) and metallo-ß-lactamases (MBL). The synthesised compounds were discovered as potent ß-lactamase inhibitors using molecular docking and in silico pharmacokinetic analysis. We studied the binding of chemically synthesised inhibitors to clinically significant ß-lactamases of class A, B, and C using biophysical and biochemical approaches, and computational analyses. These molecules follow Lipinski's rule of five and have acceptable solubility, permeability, and oral bioavailability. These molecules were found to be non-toxic and non-carcinogenic. MIC results suggest that these molecules restore the antibiotic efficacy against class A, B, and C ß-lactamases. Kinetics data showed that these molecules reduce the catalytic efficiency of clinically relevant class A, B, and C ß-lactamases. Fluorescence study showed significant interaction between these flavone-/coumarin-based isoxazole derivatives and class A/B/ C ß-lactamases. This study showed promising effect of these new generation compounds as broad spectrum ß-lactamase inhibitors of both SBLs and MBLs.Communicated by Ramaswamy H. Sarma.

Therapeutic approaches to combat the global antibiotic resistance challenge.

Antimicrobial resistance (AMR) has become a major concern for healthcare workers due to the emergence of new variants of resistant markers, especially carbapenemases. Combinational antibiotic therapy is one of the best and easiest approaches to handle the current situation of AMR. Although some antibiotic combinations are already in clinical use, they remain to be studied in detail. This review focuses on therapeutic options for AMR mechanisms of resistance in bacteria that can be overcome by combinational therapy and testing methods for synergy. The integration of diverse approaches may provide information that is imperative in mitigating the threat of AMR.

Broad-Spectrum Inhibitors against Class A, B, and C Type ß-Lactamases to Block the Hydrolysis against Antibiotics: Kinetics and Structural Characterization.

The emergence of antibiotic resistance has led to a global crisis for the physician to handle infection control issues. All antibiotics, including colistin, have lost efficiency against emerging drug-resistant bacterial strains due to the production of metallo-ß-lactamases (MBLs) and serine-ß-lactamases (SBLs). Therefore, it is of the utmost importance to design inhibitors against these enzymes to block the hydrolytic action against antibiotics being used. Although various novel ß-lactamase inhibitors are being authorized or are under clinical studies, the coverage of their activity spectrum does not include MDR organisms expressing multiple classes of ß-lactamases at a single time. This study reports three novel broad-spectrum inhibitors effective against both SBLs and MBLs. Virtual screening, molecular docking, molecular dynamics simulations, and an in silico pharmacokinetic study were performed to identify the lead molecules with broad-spectrum ability to inhibit the hydrolysis of ß-lactam. The selected compounds were further assessed by in vitro cell assays (MIC, 50% inhibitory concentration [IC50], kinetics, and fluorescence against class A, B, and C type ß-lactamases) to confirm their efficacies. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to check the toxicity of screened lead molecules. All three selected inhibitors were found to reduce MIC and showed good affinity against all the SBLs and MBLs produced by class A, B, and C type ß-lactamases. These nontoxic novel non-ß-lactam broad-spectrum inhibitors bind to the active site residues of selected ß-lactamases, which are crucial for ß-lactam antibiotic hydrolysis. These inhibitors may be proposed as a future drug candidate in combination with antibiotics as a single formulation to control infection caused by resistant strains. Hence, this study plays a significant role in the cure of infections caused by antibiotic-resistant bacteria. IMPORTANCE Several inhibitors for usage in conjunction with antibiotics have been developed. However, to date, there is no commercially available broad-spectrum ß-lactamase inhibitor that targets both MBLs and SBLs. Here, we showed three novel broad-spectrum inhibitors with promising results through computational techniques and in vitro studies. These inhibitors are effective against both SBLs and MBLs and hence could be used as future drug candidates to treat infections caused by multidrug-resistant bacteria producing both types of enzymes (SBLs and MBLs).

Repurposing drug molecule against SARS-Cov-2 (COVID-19) through molecular docking and dynamics: a quick approach to pick FDA-approved drugs.

A novel coronavirus known as severe acute respiratory syndrome is rapidly spreading worldwide. The international health authorities are putting all their efforts on quick diagnosis and placing the patients in quarantine. Although different vaccines have come for quick use as prophylactics, drug repurposing seems to be of paramount importance because of inefficient therapeutic options and clinical trial limitations. Here, we used structure-based drug designing approach to find and check the efficacy of the possible drug that can inhibit coronavirus main protease which is involved in polypeptide processing to functional protein. We performed virtual screening, molecular docking and molecular dynamics simulations of the FDA-approved drugs against the main protease of SARS-CoV-2. Using well-defined computational methods, we identified amprenavir, cefoperazone, riboflavin, diosmin, nadide and troxerutin approved for human therapeutic uses, as COVID-19 main protease inhibitors. These drugs bind to the SARS-CoV-2 main protease conserved residues of substrate-binding pocket and formed a remarkable number of non-covalent interactions. We have found diosmin as an inhibitor which binds covalently to the COVID-19 main protease. This study provides enough evidences for therapeutic use of these drugs in controlling COVID-19 after experimental validation and clinical demonstration.

Designing multi-epitope vaccine candidates against functional amyloids in Pseudomonas aeruginosa through immunoinformatic and structural bioinformatics approach.

Pseudomonas aeruginosa (P. aeruginosa) displays high drug resistance and biofilm-mediated adaptability, which makes its infections difficult to treat. Alternative intervention methods and targets have made such infections treatment manageable. One of the biofilm components, functional amyloids of Pseudomonas (Fap) is correlated positively with virulence and mucoidy phenotype found in infection in cystic fibrosis (CF) patients. Extracellular accessibility, conservation across P. aeruginosa isolates and linkage with lung infections phenotype in CF patients, makes Fap a promising intervention target. Furthermore, the reported effect of bacterial amyloid on neuronal function and immune response makes it a targetable candidate. In the current study, Fap C protein and its immediate interactions were explored to extract antigenic T-cell and B-cell epitopes. A combination of epitopes and peptide adjuvants has been linked to derive vaccine candidate structures. The vaccine candidates were validated for antigenicity, allergenicity, physiochemical properties, stability and interactions with TLRs and MHC alleles. Immunosimulation studies have demonstrated that vaccines elicit Th1 dominated response, which can assist in good prognosis of infection in CF patients.

Efflux pumps as interventions to control infection caused by drug-resistance bacteria.

Antibiotic resistance has become a global concern for healthcare workers and physicians. Efflux pumps are one of the major mechanisms of resistance. Hence, we describe examples of natural efflux pump inhibitors used to combat antibiotic resistance.

Evolving trends of New Delhi Metallo-betalactamse (NDM) variants: A threat to antimicrobial resistance.

The rapid emergence of carbapenemase producing Gram-negative bacterial strains exhibit broad-spectrum ß-lactam resistance, especially New Delhi metallo-ß-lactamase (NDM-1). It is a major public health threat as it catalyses the hydrolysis of a vast variety of ß-lactam antibiotics, including carbapenems, which is the last choice for physicians to treat infections. NDM-1 and its variants are continuously spreading worldwide, in spite of constant efforts to control. Its clinical treatment remains challenging due to continuous evolution of new variants. A thorough structural study of all variants is required to develop new and effective inhibitors. This review focuses on the dissemination, position of substitution and carbapenemases activity of all the 28 NDM variants so far reported.