Impact of PmrB mutations on clinical Klebsiella pneumoniae with variable colistin-susceptibilities: Structural insights and potent therapeutic solutions.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections continue to impose high morbidity threats to hospitalized patients worldwide, limiting therapeutic options to last-resort antibiotics like colistin. However, the dynamic genomic landscape of colistin-resistant K. pneumoniae (COLR-Kp) invoked ardent exploration of underlying molecular signatures for therapeutic propositions/designs. We unveiled the structural impact of the widespread and emerging PmrB mutations involved in colistin resistance (COLR) in K. pneumoniae. In the present study, clinical isolates of K. pneumoniae expressed variable susceptibilities to colistin (>0.5 µg/mL for resistant and ≤0.25 µg/mL for susceptible) despite mutations such as T157P, G207D and T246A. The protein sequences extracted from in-house sequenced genomes were used to model mutant PmrB proteins and analyze the underlying structural alterations. The mutations were contrasted based on molecular dynamics simulation trajectories, free-energy landscapes and structural flexibility profiles. The altered backbone flexibilities can be an essential factor for mutant selection by COLR K. pneumoniae and can provide clues to deal with emerging mutants. Furthermore, PmrB having high druggability confidence (>0.99), was explored as a potential target for 1396 virtually screened FDA-approved drug candidates. Among the top-10 compounds (scores >70), amphotericin B was found to be potential candidate with high affinity (Binding energy <-8 kcal/mol) and stable interactions (RMSF <0.7 Å) against PmrB druggable pockets, despite the mutations, which encourages future adjunct therapeutic research against COLR-Kp.

Non-steroidal anti-inflammatory drugs ketorolac and etodolac can augment the treatment against pneumococcal meningitis by targeting penicillin-binding proteins.

BACKGROUND: Streptococcus pneumoniae is the principal etiological agent of acute bacterial meningitis (ABM) which has fatal outcome in children and elderly. Due to poor blood-brain barrier (BBB) permeation, conventional ß-lactam antibiotics fail to establish the requisite bactericidal concentration in central nervous system leading to resistance in meningeal infections. The present study intended to identify potential therapeutic alternatives against Streptococcal meningitis. METHODS: Virtual screening, pharmacokinetics/pharmacodynamics (PK/PD) and anti-bacterial evaluations were employed to screen potential drugs. Molecular docking and structural dynamics simulations were performed to analyze the binding affinity and interaction stability of the drugs against the conventional Penicillin binding protein (PBP) targets. Screened drugs were also checked for interactions with other possible Streptococcal targets and relevant host targets. RESULTS: Non-steroidal anti-inflammatory drugs (NSAIDs) ketorolac and etodolac exhibiting high BBB-permeation and anti-bacterial potency were identified. Ketorolac and etodolac possessed uniform binding affinities against PBP1A, PBP2X, PBP2B and PBP3 with low inhibition constants (<50 µM). Against PBP2B and PBP3, higher binding affinities were observed for ketorolac (-6.45 and -6Kcal/mol respectively) and etodolac (-6.36 and -6.55Kcal/mol respectively) than penicillin (-5.95 and -5.85Kcal/mol respectively) and cefotaxime (-5.08 and -5.07Kcal/mol respectively). The binding affinities were contributed by conventional H-bonds and non-canonical interactions with active site residues of PBPs. Structural dynamics simulations further indicated the overall stability of the drug-bound complexes through minimal overall average root-mean square fluctuations (RMSFs) (<1.0 Å). The average binding affinities of Ketorolac and Etodolac with PBPs were marginally higher than other Streptococcal targets and comparable to their conventional inflammatory targets. CONCLUSION: Pharmacological and structural profiles indicated that ketorolac and etodolac can potentially subdue the cause and effects of streptococcal meningitis and hence encourage experimental validations.

A comprehensive review on genomics, systems biology and structural biology approaches for combating antimicrobial resistance in ESKAPE pathogens: computational tools and recent advancements.

In recent decades, antimicrobial resistance has been augmented as a global concern to public health owing to the global spread of multidrug-resistant strains from different ESKAPE pathogens. This alarming trend and the lack of new antibiotics with novel modes of action in the pipeline necessitate the development of non-antibiotic ways to treat illnesses caused by these isolates. In molecular biology, computational approaches have become crucial tools, particularly in one of the most challenging areas of multidrug resistance. The rapid advancements in bioinformatics have led to a plethora of computational approaches involving genomics, systems biology, and structural biology currently gaining momentum among molecular biologists since they can be useful and provide valuable information on the complex mechanisms of AMR research in ESKAPE pathogens. These computational approaches would be helpful in elucidating the AMR mechanisms, identifying important hub genes/proteins, and their promising targets together with their interactions with important drug targets, which is a crucial step in drug discovery. Therefore, the present review aims to provide holistic information on currently employed bioinformatic tools and their application in the discovery of multifunctional novel therapeutic drugs to combat the current problem of AMR in ESKAPE pathogens. The review also summarizes the recent advancement in the AMR research in ESKAPE pathogens utilizing the in silico approaches.

Identification of potential inhibitors for Klebsiella pneumoniae carbapenemase-3: a molecular docking and dynamics study.

Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative bacterium, which is a leading causal agent for nosocomial infections. Penicillin, cephalosporin and carbapenems along with the inhibitors such as tazobactam, sulbactam and clavulanic acid are prescribed for the treatment of K. pneumoniae infections. Prolonged exposure to ß-lactam antibiotics leads to the development of resistance. The major reason for the ß-lactam resistance in K. pneumoniae is the secretion of the enzyme K. pneumoniae carbapenemase (KPC). Secretion of KPC-2 and its variant KPC-3 by the K. pneumoniae strains causes resistance to both the substrate imipenem and the ß-lactamase inhibitors. Hence, molecular docking and dynamics studies were carried out to analyze the resistance mechanism of KPC-2-imipenem and KPC-3-imipenem at the structural level. It reveals that KPC-3-imipenem has the highest c-score value of 4.03 with greater stability than the KPC-2-imipenem c-score value of 2.36. Greater the interaction between the substrate and the ß-lactamase enzyme, higher the chances of hydrolysis of the substrate. Presently available ß-lactamase inhibitors are also ineffective against KPC-3-expressing strains. This situation necessitates the need for development of novel and effective inhibitors for KPC-3. We have carried out the virtual screening process to identify more effective inhibitors for KPC-3, and this has resulted in ZINC48682523, ZINC50209041 and ZINC50420049 as the best binding energy compounds, having greater binding affinity and stability than KPC-3-tazobactam interactions. Our study provides a clear understanding of the mechanism of drug resistance and provides valuable inputs for the development of inhibitors against KPC-3 expressing K. pneumoniae. Communicated by Ramaswamy H. Sarma.

MurB as a target in an alternative approach to tackle the Vibrio cholerae resistance using molecular docking and simulation study.

Cholera is a serious threat to a large population in the under developed countries. Though oral rehydration therapy is the preferred choice of treatment, the use of antibiotics could reduce the microbial load in the case of severity. The use of antibiotics is also sought in the scenarios where there is problem with access to clean water. However, Vibrio cholera (V. cholerae) strains have developed resistance to antibiotics such as amoxicillin, ampicillin, chloramphenicol, doxycycline, erythromycin, and tetracycline. In this work, we have addressed the resistance issue by targeting MurB protein which is essential for the cell wall biosynthesis in V. cholerae. 20 Phytochemical compounds were chosen to screen the potential inhibitors against V. cholerae to avoid the complications faced by synthesis of small molecules. The molecular docking and dynamics study indicates that quercetin is the most potential and stable inhibitor of Murb.

Clinical Evaluation of a Polyherbal Nutritional Supplement in Dyslipidemic Volunteers.

Ten important plant parts routinely used in South Indian ethnic food preparation as spices and condiments were investigated for their potential antidyslipidemic properties. The aim of the study was to characterize the biochemical properties of the polyherbal formulation (nutritional supplement) and evaluate its use to control dyslipidemia in patients. Phytochemical evaluation, in vitro α-amylase inhibitory assay, and high performance thin layer chromatography (HPTLC) fingerprinting were carried out with alcoholic extracts of all 10 individual plants and with the nutritional supplement. Investigation in human volunteers was conducted to evaluate the effect on dyslipidemia as measured by serum lipid biomarkers. Sixty-five volunteers were recruited for this study. Biomarker values at baseline and at 6th visit (end of review, 8/9 months) were compared to assess the usefulness of the nutritional supplement in the normalization of lipid biomarkers. In the qualitative analysis of metabolites, the results revealed the presence of various bioactive primary and secondary metabolites that might be responsible for their medicinal attributes. In human volunteers, after supplement intake along with standard therapy, we observed significant decrease in serum cholesterol, triglyceride, low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) levels. High-density lipoprotein (HDL) level did not change in test patient volunteers. Reductions in hemoglobin A1C (HBA1C) and postprandial blood sugar levels were observed; the difference was not statistically significant. We believe that the polyherbal formulation of 10 medicinal plants has potent antidyslipidemic activity. Our results contribute for the first time toward documentation of augmented dyslipidemia control by use of the formulation.

Ethyl 4-(4-methylphenyl)-4-pentenoate from Vetiveria zizanioides Inhibits Dengue NS2B-NS3 Protease and Prevents Viral Assembly: A Computational Molecular Dynamics and Docking Study.

Around 50 % of the world's population is at the risk of dengue, a viral infection. Presently, there are not many drugs and prophylactic measures available to control dengue viral infection, and hence, there is an urgent need to develop effective antidengue compound from natural sources. In the current study, we explored the antiviral properties of the medicinal plant Vetiveria zizanioides against dengue virus. Initially, the antiviral properties of active compounds were examined using docking analysis along with reference ligand. The enzyme-ligand complex which showed higher binding affinity than the reference ligand was employed for subsequent analysis. The stability of the top scoring enzyme-ligand complex was further validated using molecular simulation studies. On the whole, the study reveals that the compound Ethyl 4-(4-methylphenyl)-4-pentenoate has an effective antiviral property, which can serve as a potential lead molecule in drug discovery process.

Computational analysis reveal inhibitory action of nimbin against dengue viral envelope protein.

Dengue has emerged to be global health problem worldwide. Hence there is an immediate need to adopt new strategies in the development of effective anti-dengue drugs. Extracts from the leaves of Azadirachta indica has been traditionally used in folk medicine for viral infections. In the present study we report the anti-viral potency of nimbin, the active compound from the neem leaf extract against the envelope protein of dengue virus. Progression of viral entry into the host cell is facilitated by the envelope protein of dengue virus, suggesting; it as an effective anti-viral target. Nimbin is found to be effective against the envelope protein of all four types of dengue virus (dengue 1-4), which is evident from our in silico analysis. Our findings suggest the clinical importance of nimbin, which can serve as effective lead compound for further analysis.

A high-throughput cidality screen for Mycobacterium tuberculosis.

Exposure to Mycobacterium tuberculosis (Mtb) aerosols is a major threat to tuberculosis (TB) researchers, even in bio-safety level-3 (BSL-3) facilities. Automation and high-throughput screens (HTS) in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitating TB research. In the present study, we report the development and validation of a high-throughput, 24-well 'spot-assay' for selecting bactericidal compounds against Mtb. The bactericidal screen concept was first validated in the fast-growing surrogate Mycobacterium smegmatis (Msm) and subsequently confirmed in Mtb using the following reference anti-tubercular drugs: rifampicin, isoniazid, ofloxacin and ethambutol (RIOE, acting on different targets). The potential use of the spot-assay to select bactericidal compounds from a large library was confirmed by screening on Mtb, with parallel plating by the conventional gold standard method (correlation, r2 = 0.808). An automated spot-assay further enabled an MBC90 determination on resistant and sensitive Mtb clinical isolates. The implementation of the spot-assay in kinetic screens to enumerate residual Mtb after either genetic silencing (anti-sense RNA, AS-RNA) or chemical inhibition corroborated its ability to detect cidality. This relatively simple, economical and quantitative HTS considerably minimized the bio-hazard risk and enabled the selection of novel vulnerable Mtb targets and mycobactericidal compounds. Thus, spot-assays have great potential to impact the TB drug discovery process.

Evaluation and comparison of radical scavenging properties of solvent extracts from Justicia adhatoda leaf using DPPH assay.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) method is routinely practiced for the assessment of antioxidant activity of compounds and their mixtures. The method is based on the spectrophotometric measurement of DPPH(·) concentration that changes resulting from the DPPH radical reaction with an antioxidant. The amount of remaining DPPH(·) in the examined system is a measure of the antioxidant activity of compounds. Our study aims at exploring the antioxidant properties of Justicia adhatoda leaf extract and comparing the results in terms of effective concentration which scavenges 50 % radical (EC50). The correlation of the activities for both cold and Soxhlet methanolic extracts is reported with DPPH assay. The antioxidant capacity of the methanolic extract derived by two different methods is positively correlated. Correlation between antioxidant capacity and phenolic content of methanolic extract in both the cases indicates the efficiency of the extraction procedure. Positive correlation and p value <0.05 validate the efficiency of the procedures and results.

Detection and confirmation of alkaloids in leaves of Justicia adhatoda and bioinformatics approach to elicit its anti-tuberculosis activity.

The extraction and determination of alkaloids was performed and confirmed by phytochemical analysis. Six different quinazoline alkaloids (vasicoline, vasicolinone, vasicinone, vasicine, adhatodine and anisotine) were found in the leaf of Justicia adhatoda (J. adhatoda). The presence of the peaks obtained through HPLC indicated the diverse nature of alkaloid present in the leaf. The enzyme ß-ketoacyl-acyl-carrier protein synthase III that catalyses the initial step of fatty acid biosynthesis (FabH) via a type II fatty acid synthase has unique structural features and universal occurrence in Mycobacterium tuberculosis (M. tuberculosis). Thus, it was considered as a target for designing of anti-tuberculosis compounds. Docking simulations were conducted on the above alkaloids derived from J. adhatoda. The combination of docking/scoring provided interesting insights into the binding of different inhibitors and their activity. These results will be useful for designing inhibitors for M. tuberculosis and also will be a good starting point for natural plant-based pharmaceutical chemistry.