Development of Mupirocin-Impregnated Bacterial Cellulosic Transdermal Patches for the Management of Skin Infection.

The present study reports the production of bacterial cellulose (BC) membranes using Komagataeibacter hansenii for the development of transdermal wound healing patches. BC-based transdermal patches were developed by impregnating varied concentrations of antibiotic mupirocin and characterized by SEM, FTIR, TGA, and DSC to study the interaction of BC with antibiotic. Developed patches were evaluated for antimicrobial activity, in vitro drug release study, in vivo efficacy, and acute dermal toxicity studies. The antibacterial activity of mupirocin-impregnated patches (mup-BC) showed an inhibition zone from 26.16 ± 0.76 to 35.86 ± 0.61 mm. The in vivo efficacy of mup-BC patches on the superficial abrasion mouse model infected with MRSA 15187 was determined. A single application of the mup-BC (Batch-3) showed a significant decrease up to 2.5 log10 colony-forming units (CFUs) in the infected skin. Acute dermal toxicity study showed no notable sign of toxicity. Pharmacokinetic study indicated that an application of mup-BC (Batch-3) showed a peak plasma concentration of around 1.5 µg/mL mupirocin. The overall preparation, ease of application, and efficacy results clearly indicate that the patches developed in the present study find immense application in the healthcare sector, especially for the treatment of burn or dermal wound infections.

Exploring the Antibacterial Potential of Semisynthetic Phytocannabinoid: Tetrahydrocannabidiol (THCBD) as a Potential Antibacterial Agent against Sensitive and Resistant Strains of Staphylococcus aureus.

Antimicrobial resistance (AMR) is one of the most challenging problems and is responsible for millions of deaths every year. We therefore urgently require new chemical entities with novel mechanisms of action. Phytocannabinoids have been adequately reported for the antimicrobial effect but not seriously pursued because of either stringent regulatory issues or poor drug-like properties. In this regard, the current work demonstrated the antibacterial potential of tetrahydrocannabidiol (THCBD, 4), a semisynthetic phytocannabinoid, against Staphylococcus aureus, the second-most widespread bug recognized by the WHO. THCBD (4) was generated from cannabidiol and subjected to extensive antibacterial screening. In in vitro studies, THCBD (4) demonstrated a potent MIC of 0.25 µg/mL against Gram-positive bacteria, S. aureus ATCC-29213. It is interesting to note that THCBD (4) has demonstrated strong effectiveness against efflux pump-overexpressing (SA-1199B, SA-K2191, SA-K2192, and Mupr-1) and multidrug-resistant (MRSA-15187) S. aureus strains. THCBD (4) has also shown a good effect in kill kinetic assays against ATCC-29213 and MRSA-15187. In the checkerboard assay, THCBD (4) has shown additive/indifference effects with several well-known clinically used antibiotics, tetracycline, mupirocin, penicillin G, and ciprofloxacin. THCBD (4) also exhibited good permeability in the artificial skin model. Most importantly, THCBD (4) has significantly reduced CFU in mice's in vivo skin infection models and also demonstrated decent plasma exposure with 16-17% oral bioavailability. Acute dermal toxicity of THCBD (4) suggests no marked treatment-related impact on gross pathophysiology. This attractive in vitro and in vivo profile of plant-based compounds opens a new direction for new-generation antibiotics and warrants further detailed investigation.

Piperine analog PGP-41 treatment overcomes paclitaxel resistance in NCI/ADR-RES ovarian cells by inhibition of MDR1.

Chemoresistance is one of the leading causes of the failure of chemotherapy. Overexpression of P-glycoprotein (P-gp) in cancer cells is one of the most important contributing factors toward the development of chemoresistance. This study was designed to synthesize the derivatives of dihydronaphthyl and to evaluate the P-gp inhibition activity of these compounds. Among all the compounds, PGP-41 showed the most potent P-gp inhibition activity in colorectal adenocarcinoma LS-180 cells. This compound showed potent P-gp inhibition activity in chemoresistant ovarian cell line NCI/ADR-RES. Paclitaxel is one of the first lines of drugs for treating ovarian cancer and is a substrate of P-gp; therefore, NCI/ADR-RES cells are highly resistant to treatment with paclitaxel. Based on this information, we evaluated PGP-41 to overcome the paclitaxel resistance of NCI/ADR-RES cells. PGP-41 was able to sensitize the NCI/ADR-RES cells to the treatment of paclitaxel, which was evident by the reduced IC50 value of paclitaxel from 6.64 µM to 0.12 µM. The sensitization of NCI/ADR-RES cells by PGP-41 was comparable to that of elacridar and Zosuquidar. Further studies revealed that the PGP-41 exerts its effect by downregulating the expression of P-gp. Reduction of P-gp activity leads to the accumulation of higher intracellular concentration of paclitaxel, and thus allowing it to interact with its targets, which further helps in its increased efficacy. Paclitaxel was able to arrest the sensitized NCI/ADR-RES cells into G2M phase, which ultimately led to the induction of apoptotic proteins and the death of cancer cells. Being a different scaffold from zosuquidar and elacridar, further studies are required to develop PGP-41 into a potential drug to overcome chemoresistance in cancer cells.

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.

Identification of novel MurA inhibitors using in silico approach, their validation and elucidation of mode of inhibition.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an important enzyme involved in the first cytosolic step of bacterial cell wall synthesis. In this study a combination of ligand based and structure based in silico virtual screening methods were utilised for screening of more than 50,000 drug-like compounds from CSIR-IIIM in-house compound library in order to identify potent inhibitors of MurA. The identified hits were validated in vitro under various incubation conditions using Malachite green phosphate assay, and two potent hits viz 3772-9534 and D396-0012 were identified. Among these hits, compound 3772-9534 showed significant changes in the activity values in different assay conditions. The MD simulation study of 3772-9534 suggested a novel binding site in MurA enzyme, independent of the two-substrate binding sites. Binding of inhibitors at the allosteric site induces conformational changes in the enzyme, which leads to inhibition of enzymatic activity. Overall, the study offers new insight for targeting MurA, which may promote the discovery of novel MurA allosteric site inhibitors.

Boswellic acids, as novel inhibitor targeting peptidoglycan biosynthetic enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) in Escherichia coli.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an essential cytosolic enzyme in the biosynthesis of peptidoglycan. It becomes a potential bacterial target for screening promising antibacterial compounds as it is associated with the early phases of peptidoglycan production. MurA enzyme is conserved and necessary for bacterial viability with no mammalian homolog, which is a well-proven therapeutic research target. The present study reports the natural compounds from Boswellia serrata targeting the MurA enzyme. The identified inhibitors against MurA Escherichia coli (E. coli): ß-boswellic acid (IC50 33.65 µM), Acetyl-ß-boswellic acid (IC50 30.17 µM), and Acetyl-11-keto-ß-boswellic acid (IC50 37.67 µM). Inhibitors showed a fourfold decrease in IC50 values on pre-incubation with substrate-UDP-N-acetyl-glucosamine (UDP-GlcNAc). Mode-of-inhibition studies revealed their uncompetitive nature with both the substrates. Although these boswellic acids have been explored for their pharmacological potential, this is the first study reporting these compounds' E. coli MurA inhibiting potential.

Potential Inhibitors Targeting Escherichia coli UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA): An Overview.

Antibiotic resistance is one of the biggest challenges that is escalating and affecting humanity across the globe. To overcome this increasing burden of resistance, discovering novel hits by targeting the enzymes involved in peptidoglycan (murein) biosynthesis has always been considered better in antimicrobial drug discovery. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme has been identified as essential for Escherichia coli survival and catalyzes the early-stage step in bacterial cell wall synthesis. The present article gives a brief overview of the role of enzymes in peptidoglycan synthesis and MurA enzyme (previously known as MurZ in E. coli), in particular, including its structural and active site features. This review also provides an insight into the current knowledge of the reported MurA inhibitors, their mechanism of action and drawbacks of these hits that hinder their clinical trials, which would be helpful for synthesis and discovering potent molecules. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00988-6.

Regulation of polyphosphate glucokinase gene expression through cotranscriptional processing in Mycobacterium tuberculosis H37Rv.

Transcription is a molecular process that involves the synthesis of RNA chain into the 5'-3' direction, and simultaneously nascent RNA chain tends to form geometric structures, known as cotranscriptional folding. This folding determines the functional properties of RNA molecules and possibly has a critical role during the synthesis. This functioning includes the characterized properties of riboswitches and ribozymes, which are significant when the transcription rate is comparable to the cellular environment. This study reports a novel noncoding region important in the genetic expression of polyphosphate glucokinase (ppgk) in Mycobacterium tuberculosis. This noncoding element of ppgk gene undergoes cleavage activity during the transcriptional process in M.tuberculosis. We revealed that cleavage occurs within the nascent RNA, and the resultant cleaved 3'RNA fragment carries the Shine-Dalgarno (SD) sequence and expression platform. We concluded cotranscriptional processing at the noncoding region as the required mechanism for ppgk expression that remains constitutive within the bacterial environment. This study defines the molecular mechanism dependent on the transient but highly active structural features of the nascent RNA.

Screening of compound library identifies novel inhibitors against the MurA enzyme of Escherichia coli.

Bacterial cell has always been an attractive target for anti-infective drug discovery. MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) enzyme of Escherichia coli (E.coli) is crucial for peptidoglycan biosynthetic pathway, as it is involved in the early stages of bacterial cell wall biosynthesis. In the present study we aim to identify novel chemical structures targeting the MurA enzyme. For screening purpose, we used in silico approach (pharmacophore based strategy) for 52,026 library compounds (Chembridge, Chemdiv and in house synthetics) which resulted in identification of 50 compounds. These compounds were screened in vitro against MurA enzyme and release of inorganic phosphate (Pi) was estimated. Two compounds (IN00152 and IN00156) were found to inhibit MurA enzyme > 70% in primary screening and IC50 of 14.03 to 32.30 µM respectively. These two hits were further evaluated for their mode of inhibition studies and whole-cell activity where we observed 2-4 folds increase in activity in presence of Permeabilizer EDTA (Ethylenediaminetetraacetic acid). Combination studies were also performed with known antibiotics in presence of EDTA. Hits are reported for the first time against this target and our report also support the use of OM permeabilizer in combination with antibacterial compounds to address the permeability and efficacy issue. These lead hits can be further optimized for drug discovery. KEY POINTS: • Emerging Gram negative resistant strains is a matter of concern. • Need for new screening strategies to cope with drying up antibiotics pipeline. • Outer membrane permeabilizers could be useful to improve potency of molecules to reach its target.

High-throughput screening of compounds library to identify novel inhibitors against latent Mycobacterium tuberculosis using streptomycin-dependent Mycobacterium tuberculosis 18b strain as a model.

One of the significant challenges to treat tuberculosis is the phenotypic resistance adapted by the latent or dormant Mycobacterium tuberculosis (M. tuberculosis) cells against most of the available drugs. Different in-vitro assay such as oxygen depletion model and nutrient starvation models have contributed to unravelling the pathogen phenotypic resistance but are too cumbersome for application to high-throughput screening (HTS) assays. In this context, non-replicating streptomycin-starved 18b (SS18b) mutant strain of M. tuberculosis provided a simple and reproducible model. This model mimics latent tuberculosis and is best suited for screening medicinally appropriate libraries. Using SS18b strain in a resazurin reduction microplate assay (REMA), high-throughput screening of ChemDiv library constituting of 30,000 compounds resulted in the identification of 470 active compounds. Clustering and scaffolding based medicinal chemistry analysis characterized these hits into 15 scaffolds. Seven most potent compounds exhibiting an MIC ≤ 1 µg/ml against SS18b were non-toxic in HepG2 cell line (selective Index ≥ 160). Our screening revealed seven novel compounds exhibiting activity against the non-replicating form of M tuberculosis. 8002-7516 was the most promising compound showing intracellular killing and could be optimized to develop a lead drug candidate.

Potential Inhibitors Against NDM-1 Type Metallo-ß-Lactamases: An Overview.

A new member of the class metallo-ß-lactamase (MBL), New Delhi metallo-beta-lactamase 1 (NDM-1) has emerged recently as a leading threat to the treatment of infections that have spread in all major Gram-negative pathogens. The enzyme inactivates antibiotics of the carbapenem family, which are a mainstay for the treatment of antibiotic-resistant bacterial infections. This review provides information about NDM-1 spatial structure, potential features of the active site, and its mechanism of action. It also enlists the inhibitors/compounds/drugs against NDM-1 in various development phases. Understanding their mode of inhibition and the structure-activity relationship would be beneficial for development, synthesis, and even increasing biological efficacy of inhibitors, making them more promising drug candidates.

Screening of Antitubercular Compound Library Identifies Inhibitors of Mur Enzymes in Mycobacterium tuberculosis.

The rapid rise in the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) mandates the discovery of novel tuberculosis (TB) drugs. Mur enzymes, which are identified as essential proteins in Mtb and catalyze the cytoplasmic steps in the peptidoglycan biosynthetic pathway, are considered potential drug targets. However, none of the clinical drugs have yet been developed against these enzymes. Hence, the aim of this study was to identify novel inhibitors of Mur enzymes in Mycobacterium tuberculosis. We screened an antitubercular compound library of 684 compounds, using MurB and MurE enzymes of the Mtb Mur pathway as drug targets. For experimental validation, the top hits obtained on in silico screening were screened in vitro, using Mtb Mur enzyme-specific assays. In all, seven compounds were found to show greater than 50% inhibition, with the highest inhibition observed at 77%, and the IC50 for these compounds was found to be in the range of 28-50 µM. Compound 5175112 showed the lowest IC50 (28.69 ± 1.17 µM), and on the basis of (1) the binding affinity, (2) the stability of interaction noted on molecular dynamics simulation, and (3) an in vitro assay, MurE appeared to be its target enzyme. We believe that the overall strategy followed in this study and the results obtained are a good starting point for developing Mur enzyme-specific Mtb inhibitors.

Screening of antitubercular compound library identifies novel ATP synthase inhibitors of Mycobacterium tuberculosis.

A limited number of anti-tuberculosis drug candidates with novel mode of action have entered clinical trials in recent years. ATP synthase is one such validated drug target which has yielded a drug recently. The aim of this study was to identify the novel chemical scaffolds targeting the Mycobacterium tuberculosis (M. tuberculosis) ATP synthase. In this study, inverted membrane vesicles of Mycobacterium smegmatis were prepared to establish luciferin based ATP estimation assay. This assay was used to screen 700 compounds which were earlier found to be active on the whole cell of M. tuberculosis. Antibacterial activity of hits against various susceptible and drug-resistant strains of M. tuberculosis was evaluated using the microplate alamar blue assay and their cytotoxicity was also determined to select the safe compounds for further study. Screening of 700 compounds resulted in the identification of two compounds (5228485 and 5220632) exhibiting an IC50 of 0.32 and 4.0 µg/ml respectively. Both compounds showed excellent anti-TB activity (MIC of 0.5-2.0 µg/ml against Mtb H37Rv) and low cytotoxicity in human cell line and sub-mitochondrial particles. The three-dimensional structure of M. tuberculosis ATPase was predicted using in-silico approach and docking studies were performed with the active compounds. The interaction between compounds and bacterial ATP synthase was confirmed by molecular docking analysis. In conclusion screening of compound library has resulted in the identification of two novel chemical scaffolds targeting mycobacterial ATP synthase.

Physicochemical, pharmacokinetic, efficacy and toxicity profiling of a potential nitrofuranyl methyl piperazine derivative IIIM-MCD-211 for oral tuberculosis therapy via in-silico-in-vitro-in-vivo approach.

Recent tuberculosis (TB) drug discovery programme involve continuous pursuit for new chemical entity (NCE) which can be not only effective against both susceptible and resistant strains of Mycobacterium tuberculosis (Mtb) but also safe and faster acting with the target, thereby shortening the prolonged TB treatments. We have identified a potential nitrofuranyl methyl piperazine derivative, IIIM-MCD-211 as new antitubercular agent with minimum inhibitory concentration (MIC) value of 0.0072 µM against H37Rv strain. Objective of the present study is to investigate physicochemical, pharmacokinetic, efficacy and toxicity profile using in-silico, in-vitro and in-vivo model in comprehensive manner to assess the likelihood of developing IIIM-MCD-211 as a clinical candidate. Results of computational prediction reveal that compound does not violate Lipinski's, Veber's and Jorgensen's rule linked with drug like properties and oral bioavailability. Experimentally, IIIM-MCD-211 exhibits excellent lipophilicity that is optimal for oral administration. IIIM-MCD-211 displays evidence of P-glycoprotein (P-gp) induction but no inhibition ability in rhodamine cell exclusion assay. IIIM-MCD-211 shows high permeability and plasma protein binding based on parallel artificial membrane permeability assay (PAMPA) and rapid equilibrium dialysis (RED) assay model, respectively. IIIM-MCD-211 has adequate metabolic stability in rat liver microsomes (RLM) and favourable pharmacokinetics with admirable correlation during dose escalation study in Swiss mice. IIIM-MCD-211 has capability to appear into highly perfusable tissues. IIIM-MCD-211 is able to actively prevent progression of TB infection in chronic infection mice model. IIIM-MCD-211 shows no substantial cytotoxicity in HepG2 cell line. In acute toxicity study, significant increase of total white blood cell (WBC) count in treatment group as compared to control group is observed. Overall, amenable preclinical data make IIIM-MCD-211 ideal candidate for further development of oral anti-TB agent.

Antimicrobial investigation of selected soil actinomycetes isolated from unexplored regions of Kashmir Himalayas, India.

The aim of the present study was to isolate and evaluate the antimicrobial potential of soil actinomycetes of Kashmir Himalayas. The secondary metabolites of actinomycetes are the prominent source of antibiotics. A total of 121 morphologically different actinomycete strains were isolated and screened for antimicrobial activity against various human pathogens. The ethyl acetate extract of fermented broth an actinomycete strain, identified as Streptomyces pratensis exhibited significant antimicrobial activity against Staphylococcus aureus ATCC 29213 with MIC 0.25 µg/ml and Mycobacterium tuberculosis Strain H37Rv with MIC 0.062 µg/ml. The strain S. pratensis IIIM06 was grown on large scale and their broth was extracted with ethyl acetate. The extract was subjected to various chromatography techniques which led to the isolation of four compounds whose structures were established as actinomycin C1, actinomycin C2, actinomycin C3 and actiphenol on the basis of spectral data analysis. Actinomycin C1, C2 and C3 exhibited potent antimicrobial activity against S. aureus as well as M. tuberculosis. The isolated indigenous actinomycetes exhibited good antibacterial activity and the study reveals that IIIM06 is a promising strain and could be of great potential for industrial applications.

Anti-tubercular drug discovery: in silico implications and challenges.

Tuberculosis (TB) has been reported as a major public health concern, especially in the developing countries. WHO report on tuberculosis 2016 shows a high mortality rate caused by TB leading to 1.8 million deaths worldwide (including deaths due to TB in HIV positive individuals), which is one of the top 10 causes of mortality in 2015. However, the main therapy used for the treatment of TB is still the Direct Observed Therapy Short-course (DOTS) that consists of four main first-line drugs. Due to the prolonged and unorganized use of these drugs, Mycobacterium tuberculosis (Mtb) has developed drug-resistance against them. To overcome this drug-resistance, efforts are continuously being made to develop new therapeutics. New drug-targets of Mtb are pursued by the researchers to develop their inhibitors. For this, new methodologies that comprise of the computational drug designing techniques are vigorously applied. A major limitation that is found with these techniques is the inability of the newly identified target-based inhibitors to inhibit the whole cell bacteria. A foremost factor for this limitation is the inability of these inhibitors to penetrate the bacterial cell wall. In this regard, various strategies to overcome this limitation have been discussed in detail in this review, along with new targets and new methodologies. A bunch of in silico tools available for the prediction of physicochemical properties that need to be explored to deal with the permeability issue of the Mtb inhibitors has also been discussed.

Mechanism and Potential Inhibitors of GlmU: A Novel Target for Antimicrobial Drug Discovery.

BACKGROUND: Multidrug resistant Gram negative pathogens pose a persistent threat to the health care system and require investigation of new targets and molecules for the development of antibiotics to treat infections caused by MDR bacterial pathogens. OBJECTIVE: It is essential to work on multidisciplinary approaches and diverse strategies for developing new compounds acting on novel antibacterial targets. N-acetylglucosamine-1-phosphateuridyltransferase/ glucosamine-1-phosphate-acetyltransferase (GlmU) is one such target which is involved in the synthesis of both peptidoglycan and Lipopolysaccharide in Gram negative and Gram positive bacteria making GlmU an attractive target for developing antibacterials. RESULTS: GlmU, as revealed by X- ray crystallographic studies, is made up of two domains connected by α helical arm; the N-terminal uridyltransferase domain resembles a dinucleotide Rossmann fold and the C- terminal acetyltransferase domain adopts a left handed parallel ß helix structure. The GlmU molecules are arranged in a trimeric array, the acetyltransferase active site being formed at the junction of adjacent LßH domain. Many potent inhibitors of both the acetyltransferase and uridyltransferase activity of GlmU have been identified. Inhibitors of the acetyltransferase activity of GlmU include nonspecific thiol reactive agents, 2-phenylbenzofurans, arylamines and arylsulfonamides. A aminopiperidine based inhibitor has also been reported to inhibit uridyltransferase activity of hiGlmU. CONCLUSION: The present review provides an insight of the structure of GlmU and its reported inhibitors which make GlmU a potential target for drug designing. The GlmU is a promising target for drug discovery against Gram negative pathogens and future studies should focus on GlmU for the development of more potent compounds for treating Gram negative infections.

Hydroxychavicol: A phytochemical targeting cutaneous fungal infections.

The present study was designed to investigate the potency of hydroxychavicol on selected cutaneous human pathogenic fungi by the use of in vitro and in vivo assays and mechanistic characterization along with toxicological effects. Hydroxychavicol consistently displayed a fungicidal effect against all fungal species tested. Inoculum concentrations over the range of 104 to 107 CFU/ml did not significantly alter its antifungal potential and time-kill curve results revealed concentration-dependent killing. It also inhibited the growth of biofilm generated by Trichophyton mentagrophytes and Candida parapsilosis and reduced the preformed biofilms. Hydroxychavicol was highly effective in the treatment, and mycological eradication of an experimentally induced topical infection model of dermatophytosis (tinea corporis) and cutaneous candidiasis in guinea pigs, respectively. The mode of action of hydroxychavicol appears to originate from the disruption of cell membrane integrity. Administration of hydroxychavicol in mice at 500 mg per kg of body weight by orally produced no overt toxicity. The retention capacity of hydroxychavicol in vitro, in the presence of keratin has attributed to its in vivo effectiveness in the guinea pig model of topical infections. Furthermore, it is suggestive of its potential use as phytochemical for topical use in cutaneous fungal infections.

Amino acid amides of piperic acid (PA) and 4-ethylpiperic acid (EPA) as NorA efflux pump inhibitors of Staphylococcus aureus.

A total of eighteen piperic acid (PA) and 4-ethylpiperic acid (EPA) amides (C1-C18) with α-, ß- and γ-amino acids were synthesized, characterized and evaluated for their efflux pump inhibitory activity against ciprofloxacin resistant Staphylococcus aureus. The amides were screened against NorA overexpressing S. aureus SA-1199B and wild type S. aureus SA-1199 using ethidium bromide as NorA efflux pump substrate. EPI C6 was found to be most potent and reduced the MIC of ciprofloxacin by 16 fold followed by C18 which showed 4 fold reduction of MIC. Ethidium bromide efflux inhibition and accumulation assay proved these compounds as NorA inhibitors.

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.