1,3,4-oxadiazole conjugates of capsaicin as potent NorA efflux pump inhibitors of Staphylococcus aureus.

NorA efflux pump pertaining to the major facilitator superfamily (MFS) is known to play a key role in antibiotic and biocide resistance in Staphylococcus aureus (S. aureus). It accounts for the extrusion of antibiotics like fluoroquinolones (e.g. ciprofloxacin). Several compounds including synthetic and natural products have been identified as potential NorA efflux pump inhibitors (EPIs) and found to restore the antibacterial activity of antibiotics. However, none of the reported EPIs have reached to clinical approval probably due to their high toxicity profiles. Considering the NorA efflux pump inhibitory potential of capsaicin, a series of capsaicin-based 1,3,4 oxadiazole conjugates were prepared and evaluated for ciprofloxacin activity potentiating effect. Among the new capsaicinoids tested, 17i displayed a minimum effective concentration (MEC) of 12.5 µg/mL against NorA overexpressing S. aureus strain (SA1199B), whereas capsaicin showed MEC of 50 µg/mL. The kill kinetics curve for the combination showed that ciprofloxacin at a sub-inhibitory concentration (0.25 × MIC) was equipotent in effect, to its MIC. 17i has significantly decreased the ethidium bromide efflux confirming NorA inhibition as the mode of action. Mutation prevention concentration of the ciprofloxacin was reduced in combination with 17i.In silico studies revealed the binding efficiency and binding affinity of 17i with NorA. This compound may serve as a template for the further drug discovery.

Benzothiazole Derivative as a Novel Mycobacterium tuberculosis Shikimate Kinase Inhibitor: Identification and Elucidation of Its Allosteric Mode of Inhibition.

Mycobacterium tuberculosis shikimate kinase (Mtb-SK) is a key enzyme involved in the biosynthesis of aromatic amino acids through the shikimate pathway. Since it is proven to be essential for the survival of the microbe and is absent from mammals, it is a promising target for anti-TB drug discovery. In this study, a combined approach of in silico similarity search and pharmacophore building using already reported inhibitors was used to screen a procured library of 20,000 compounds of the commercially available ChemBridge database. From the in silico screening, 15 hits were identified, and these hits were evaluated in vitro for Mtb-SK enzyme inhibition. Two compounds presented significant enzyme inhibition with IC50 values of 10.69 ± 0.9 and 46.22 ± 1.2 µM. The best hit was then evaluated for the in vitro mode of inhibition where it came out to be an uncompetitive and noncompetitive inhibitor with respect to shikimate (SKM) and ATP, respectively, suggesting its binding at an allosteric site. Potential binding sites of Mtb-SK were identified which confirmed the presence of an allosteric binding pocket apart from the ATP and SKM binding sites. The docking simulations were performed at this pocket in order to find the mode of binding of the best hit in the presence of substrates and the products of the enzymatic reaction. Molecular dynamics (MD) simulations elucidated the probability of inhibitor binding at the allosteric site in the presence of ADP and shikimate-3-phosphate (S-3-P), that is, after the formation of products of the reaction. The inhibitor binding may prevent the release of the product from Mtb-SK, thereby inhibiting its activity. The binding stability and the key residue interactions of the inhibitor to this product complex were also revealed by the MD simulations. Residues ARG43, ILE45, and PHE57 were identified as crucial that were involved in interactions with the best hit. This is the first report of an allosteric binding site of Mtb-SK, which could largely address the selectivity issue associated with kinase inhibitors.

Synthesis of new generation triazolyl- and isoxazolyl-containing 6-nitro-2,3-dihydroimidazooxazoles as anti-TB agents: in vitro, structure-activity relationship, pharmacokinetics and in vivo evaluation.

The nitroimidazole scaffold has attracted great interest in the last decade, which ultimately led to the discovery of the successful drug Delamanid for multi-drug resistant tuberculosis (MDR-TB). Herein, we report medicinal chemistry on a 6-nitro-2,3-dihydroimidazooxazole (NHIO) scaffold with SAR on the novel series of triazolyl- and isoxazolyl-based NHIO compounds. In the present study, 41 novel triazolyl- and isoxazolyl-based NHIO compounds were synthesized and evaluated against Mycobacterium tuberculosis (MTB) H37Rv. The active compounds with MIC of 0.57-0.13 µM were further screened against dormant, as well as against resistant strains of MTB. Based on the overall in vitro profile, five compounds were studied for in vivo oral pharmacokinetics, wherein two compounds: 1g and 2e showed a good PK profile. In in vivo efficacy studies in the intra-nasal model of acute infection, 1g showed 1.8 and 1 log CFU reduction with respect to the untreated and early control, respectively. The lead compound 1g also showed an additive to synergistic effect in combination studies with first line-TB drugs and no CYP inhibition. From the present studies, the compound 1g represents another alternative lead candidate in this class and needs further detailed investigation.

Anti-tubercular agents. Part 8: synthesis, antibacterial and antitubercular activity of 5-nitrofuran based 1,2,3-triazoles.

A series of 5-nitrofuran-triazole conjugates were synthesized and evaluated for their antimicrobial activity against both Gram-positive and Gram-negative bacterial strains. All the compounds exhibited promising inhibition towards Gram-positive pathogenic strains, while mild inhibitory effects were observed towards Gram-negative bacterial strains. Some of the compounds 8a, 8b, 8e, 8f, 8h are most active among the series exhibiting MIC value of 1.17 µg/ml against different bacterial strains. The bactericidal activity is found to be in accordance with the bacterial growth inhibition data. Compound 8e was found to be equipotent to the standard drug Ciprofloxacin displaying MBC value of 1.17 µg/ml against the bacterial strain Bacillus subtilis. The compounds have also demonstrated promising antibacterial activity against the resistant strain MRSA and were found to be effective inhibitors of biofilm formation. The compound 8b exhibited excellent anti-biofilm activity with IC50 value as low as 0.8 µg/ml. These conjugates were also screened for antitubercular activity against Mycobacterium tuberculosis H37Rv strain. Compound 8e showed promising antitubercular activity with MIC value of 0.25 µg/ml. Most of these compounds are less toxic to normal mammalian cells than the widely used antibacterial drug Ciprofloxacin.

Shikimate Kinase Inhibitors: An Update on Promising Strategy against Mycobacterium tuberculosis.

Humanity has been battling with tuberculosis (TB) for a long period, and despite the availability of drugs well-known to act against the deadly microbe, the menace is still very far from reaching its end. Moreover, problems related to TB chemotherapy, such as lengthy treatment periods leading to poor patient compliance, increasing drug resistance, and association with another deadlier disease HIV-AIDS, make the situation alarming, thereby pressing the need for the discovery of new potent drugs urgently. Therefore, a drug target that is essential for survival and exclusive to M. tuberculosis presents a promising platform to explore novel molecules against the microorganism for better pathogen clearance with minimal toxicity. The shikimate pathway that leads to the synthesis of essential aromatic amino acids is one such attractive target. Shikimate kinase, the fifth enzyme of this pathway, converts shikimate to shikimate-3-phosphate by using ATP as a cosubstrate. Targeting shikimate kinase could be an effective strategy in light of its essentiality and absence of any homologue in mammals. This review discusses different strategies adopted for discovering novel compounds or scaffolds targeting M. tuberculosis shikimate kinase (MtSK) in vitro. The application of substrate analogues, their structure, and ligand-based approach for screening a library of anti-mycobacterial compounds, marine-derived molecules, and commercially available libraries have yielded promising MtSK inhibitors exhibiting micro-molar activities. To develop these leads into future drugs with minimum off-target effects on the host microenvironment, the molecules need to be structurally optimized for improved activities against enzymes and whole-cell organisms.

Antimicrobial, antimycobacterial and antibiofilm properties of Couroupita guianensis Aubl. fruit extract.

BACKGROUND: Couroupita guianensis Aubl. (Lecythidaceae) is commonly called Ayahuma and the Cannonball tree. It is distributed in the tropical regions of northern South America and Southern Caribbean. It has several medicinal properties. It is used to treat hypertension, tumours, pain, inflammatory processes, cold, stomach ache, skin diseases, malaria, wounds and toothache. METHODS: The fruits of Couroupita guianensis were extracted with chloroform. Antimicrobial, antimycobacterial and antibiofilm forming activities of the chloroform extract were investigated. Quantitative estimation of Indirubin, one of the major constituent, was identified by HPLC. RESULTS: Chloroform extract showed good antimicrobial and antibiofilm forming activities; however it showed low antimycobacterial activity. The zones of inhibition by chloroform extract ranged from 0 to 26 mm. Chloroform extract showed effective antibiofilm activity against Pseudomonas aeruginosa starting from 2 mg/mL BIC, with 52% inhibition of biofilm formation. When the chloroform extract was subjected to HPLC-DAD analysis, along with Indirubin standard, in the same chromatographic conditions, it was found that Indirubin was one of the major compounds in this plant (0.0918% dry weight basis). CONCLUSIONS: The chloroform extract showed good antimicrobial and antibiofilm properties. Chloroform extract can be evaluated further in drug development programmes.

Antimicrobial and Antimycobacterial Activities of Methyl Caffeate Isolated from Solanum torvum Swartz. Fruit.

ABSTRACT: Solanum torvum Swartz. (Solanaceae) fruit is traditionally used for the treatment of bacterial and fungal infections. The methanolic extract was subjected to activity guided fractionation by column chromatography over silica gel. The structure of the compound was elucidated using physical and spectroscopic data. The antimicrobial activity was screened using five Gram-positive bacteria, six Gram-negative bacteria, seven clinical isolates and four fungi. Antimycobacterial activity was screened against two Mycobacterium strains. The zone of inhibition by methyl caffeate ranged from 0 to 22 mm. The lowest minimum inhibitory concentration (MIC) values of methyl caffeate were: 50 µg/ml against P. vulgaris, 25 µg/ml against K. pneumoniae (ESBL-3971), 8 µg/ml against M. tuberculosis (H(37)Rv) and 8 µg/ml against M. tuberculosis (Rif(R)). Methyl caffeate showed moderate antimicrobial and prominent antimycobacterial activities. Methyl caffeate can be evaluated further for drug development.

Dual inhibition of SARS-CoV-2 spike and main protease through a repurposed drug, rutin.

The global health emergency caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to alarming numbers of fatalities across the world. So far the researchers worldwide have not been able to discover a breakthrough in the form of a potent drug or an effective vaccine. Therefore, it is imperative to discover drugs to curb the ongoing menace. In silico approaches using FDA approved drugs can expedite the drug discovery process by providing leads that can be pursued. In this report, two drug targets, namely the spike protein and main protease, belonging to structural and non-structural class of proteins respectively, were utilized to carry out drug repurposing based screening. The exposed nature of the spike protein on the viral surface along with its instrumental role in host infection and the involvement of main protease in processing of polyproteins along with no human homologue make these proteins attractive drug targets. Interestingly, the screening identified a common high efficiency binding molecule named rutin. Further, molecular dynamics simulations in explicit solvent affirmed the stable and sturdy binding of rutin with these proteins. The decreased Rg value (4 nm for spike-rutin and 2.23 nm for main protease-rutin) and stagnant SASA analysis (485 nm/S2/N in spike-rutin and 152 nm/S2/N in main protease-rutin) for protein surface and its orientation in the exposed and buried regions suggests a strong binding interaction of the drug. Further, cluster analysis and secondary structure analysis of complex trajectories validated the conformational changes due to binding of rutin.

Engineering a multi epitope vaccine against SARS-CoV-2 by exploiting its non structural and structural proteins.

SARS-CoV-2, the causative agent behind the ongoing pandemic exhibits an enhanced potential for infection when compared to its related family members- the SARS-CoV and MERS-CoV; which have caused similar disease outbreaks in the past. The severity of the global health burden, increasing mortality rate and the emergent economic crisis urgently demands the development of next generation vaccines. Amongst such emergent next generation vaccines are the multi-epitope subunit vaccines, which hold promise in combating deadly pathogens. In this study we have exploited immunoinformatics applications to delineate a vaccine candidate possessing multiple B and T cells epitopes by utilizing the SARS-CoV-2 non structural and structural proteins. The antigenicity potential, safety, structural stability and the production feasibility of the designed construct was evaluated computationally. Furthermore, due to the known role of human TLR-3 immune receptor in viral sensing, which facilitates host cells activation for an immune response, the vaccine construct was examined for its binding efficiency using molecular docking and molecular dynamics simulation studies, which resulted in strong and stable interactions. Finally, the immune simulation studies suggested an effective immune response on vaccine administration. Overall, the immunoinformatics analysis advocates that the proposed vaccine candidate is safe and immunogenic and therefore can be pushed as a lead for in vitro and in vivo investigations.Communicated by Ramaswamy H. Sarma.

Computational and Synthetic Biology Approaches for the Biosynthesis of Antiviral and Anticancer Terpenoids from Bacillus subtilis.

Recent advancements in medicinal research have identified several antiviral and anticancer terpenoids that are usually deployed as a source of flavor, fragrances and pharmaceuticals. Under the current COVID-19 pandemic conditions, natural therapeutics with the least side effects are the need of the hour to save the patients, especially, which are pre-affected with other medical complications. Although plants are the major sources of terpenoids; however, for the environmental concerns, the global interest has shifted to the biocatalytic production of molecules from microbial sources. The gram-positive bacterium Bacillus subtilis is a suitable host in this regard due to its GRAS (generally regarded as safe) status, ease in genetic manipulations and wide industrial acceptability. The B. subtilis synthesizes its terpenoid molecules from 1-deoxy-d-xylulose-5-phosphate (DXP) pathway, a common route in almost all microbial strains. Here, we summarize the computational and synthetic biology approaches to improve the production of terpenoid-based therapeutics from B. subtilis by utilizing DXP pathway. We focus on the in-silico approaches for screening the functionally improved enzyme-variants of the two crucial enzymes namely, the DXP synthase (DXS) and Farnesyl Pyrophosphate Synthase (FPPS). The approaches for engineering the active sites are subsequently explained. It will be helpful to construct the functionally improved enzymes for the high-yield production of terpenoid-based anticancer and antiviral metabolites, which would help to reduce the cost and improve the availability of such therapeutics for the humankind.

An immunoinformatics approach to design a multi-epitope vaccine against Mycobacterium tuberculosis exploiting secreted exosome proteins.

Tuberculosis is one the oldest known affliction of mankind caused by the pathogen Mycobacterium tuberculosis. Till date, there is no absolute single treatment available to deal with the pathogen, which has acquired a great potential to develop drug resistance rapidly. BCG is the only anti-tuberculosis vaccine available till date which displays limited global efficacy due to genetic variation and concurrent pathogen infections. Extracellular vesicles or exosomes vesicle (EVs) lie at the frontier cellular talk between pathogen and the host, and therefore play a significant role in establishing pathogenesis. In the present study, an in-silico approach has been adopted to construct a multi-epitope vaccine from selected immunogenic EVs proteins to elicit a cellular as well as a humoral immune response. Our designed vaccine has wide population coverage and can effectively compensate for the genetic variation among different populations. For maximum efficacy and minimum adverse effects possibilities the antigenic, non-allergenic and non-toxic B-cell, HTL and CTL epitopes from experimentally proven EVs proteins were selected for the vaccine construct. TLR4 agonist RpfE served as an adjuvant for the vaccine construct. The vaccine construct structure was modelled, refined and docked on TLR4 immune receptor. The designed vaccine construct displayed safe usage and exhibits a high probability to elicit the critical immune regulators, like B cells, T-cells and memory cells as displayed by the in-silico immunization assays. Therefore, it can be further corroborated using in vitro and in vivo assays to fulfil the global need for a more efficacious anti-tuberculosis vaccine.

Emodin inhibited NADPH-quinone reductase competitively and induced cytotoxicity in rat primary hepatocytes.

Consumption of Cassia occidentalis (CO) seeds, a ubiquitously distributed weed plant, is responsible for a pathological condition known as hepato-myo-encephalopathy (HME). The toxicity of CO seeds is largely attributed to the presence of anthraquinones (AQs). Here, we report that Emodin, a CO anthraquinone, inhibits the enzymatic activity of NADPH-Quinone reductase, which is an intracellular enzyme fundamentally involved in the detoxification of quinone containing compounds. Emodin binds to the active site of the enzyme and acts as a competitive inhibitor with respect to 2, 6-Dichlorophenolindophenol, a known substrate of NADPH-Quinone reductase. Moreover, our in-vitro study further revealed that Emodin was cytotoxic to primary rat hepatocytes.

Emodin inhibited NADPH-quinone reductase via competitive mode of inhibition and induced cytotoxicity in rat primary hepatocytes.

Consumption of Cassia occidentalis (CO) seeds, a ubiquitously distributed weed plant, is responsible for a pathological condition known as hepato-myo-encephalopathy (HME). The toxicity of CO seeds is largely attributed to the presence of anthraquinones. Here, we report that Emodin, a CO anthraquinone, inhibits the enzymatic activity of NADPH-Quinone reductase, which is an intracellular enzyme fundamentally involved in the detoxification of quinone containing compounds. Emodin binds to the active site of the enzyme and acts as a competitive inhibitor with respect to 2, 6-Dichlorophenolindophenol, a known substrate of NADPH-Quinone reductase. Moreover, our in-vitro study further revealed that Emodin was cytotoxic to primary rat hepatocytes.

Excavating the functionally crucial active-site residues of the DXS protein of Bacillus subtilis by exploring its closest homologues.

BACKGROUND: To achieve a high yield of terpenoid-based therapeutics, 1-deoxy-d-xylulose-5-phosphate (DXP) pathway has been significantly exploited for the production of downstream enzymes. The DXP synthase (DXS) enzyme, the initiator of this pathway, is pivotal for the convergence of carbon flux, and is computationally studied well for the industrially utilized generally regarded as safe (GRAS) bacterium Bacillus subtilis to decode its vital regions for aiding the construction of a functionally improved mutant library. RESULTS: For the 546 sequence dataset of DXS sequences, a representative set of 108 sequences is created, and it shows a significant evolutionary divergence across different species clubbed into 37 clades, whereas three clades are observed for the 76 sequence dataset of Bacillus subtilis. The DXS enzyme, sharing a statistically significant homology to transketolase, is shown to be evolutionarily too distant. By the mutual information-based co-evolutionary network and hotspot analysis, the most crucial loci within the active site are deciphered. The 650-residue representative structure displays a complete conservation of 114 loci, and only two co-evolving residues ASP154 and ILE371 are found to be the conserved ones. Lastly, P318D is predicted to be the top-ranked mutation causing the increase in the thermodynamic stability of 6OUW. CONCLUSION: The study excavates the vital functional, phylogenetic, and conserved residues across the active site of the DXS protein, the key rate-limiting controller of the entire pathway. It would aid to computationally understand the evolutionary landscape of this industrially useful enzyme and would allow us to widen its substrate repertoire to increase the enzymatic yield of unnatural molecules for in vivo and in vitro applications.

Nitrofuranyl Methyl Piperazines as New Anti-TB Agents: Identification, Validation, Medicinal Chemistry, and PK Studies.

Whole-cell screening of 20,000 drug-like small molecules led to the identification of nitrofuranyl methylpiperazines as potent anti-TB agents. In the present study, validation followed by medicinal chemistry has been used to explore the structure-activity relationship. Ten compounds demonstrated potent MIC in the range of 0.17-0.0072 µM against H37Rv Mycobacterium tuberculosis (MTB) and were further investigated against nonreplicating and resistant (Rif(R) and MDR) strains of MTB. These compounds were also tested for cytotoxicity. Among the 10 tested compounds, five showed submicromolar to nanomolar potency against nonreplicating and resistant (Rif(R) and MDR) strains of MTB along with a good safety index. Based on their overall in vitro profiles, the solubility and pharmacokinetic properties of five potent compounds were studied, and two analogues, 14f and 16g, were found to have comparatively better solubility than others tested and acceptable pharmacokinetic properties. This study presents the rediscovery of a nitrofuranyl class of compounds with improved aqueous solubility and acceptable oral PK properties, opening a new direction for further development.