Priya Wazir

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.

Escherichia coli N-Acetylglucosamine-1-Phosphate-Uridyltransferase/Glucosamine-1-Phosphate-Acetyltransferase (GlmU) Inhibitory Activity of Terreic Acid Isolated from Aspergillus terreus

Secondary metabolite of Aspergillus terreus, terreic acid, is a reported potent antibacterial that was identified more than 60 years ago, but its cellular target(s) are still unknown. Here we screen its activity against the acetyltransferase domain of a bifunctional enzyme, Escherichia coli N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). An absorbance-based assay was used to screen terreic acid against the acetyltransferase activity of E. coli GlmU. Terreic acid was found to inhibit the acetyltransferase domain of E. coli GlmU with an IC50 of 44.24 ± 1.85 µM. Mode of inhibition studies revealed that terreic acid was competitive with AcCoA and uncompetitive with GlcN-1-P. It also exhibited concentration-dependent killing of E. coli ATCC 25922 up to 4× minimum inhibitory concentration and inhibited the growth of biofilms generated by E. coli. Characterization of resistant mutants established mutation in the acetyltransferase domain of GlmU. Terreic acid was also found to be metabolically stable in the in vitro incubations with rat liver microsome in the presence of a NADPH regenerating system. The studies reported here suggest that terreic acid is a potent antimicrobial agent and support that E. coli GlmU acetyltransferase is a molecular target of terreic acid, resulting in its antibacterial activity.

Development and validation of a highly sensitive LC-ESI-MS/MS method for estimation of IIIM-MCD-211, a novel nitrofuranyl methyl piperazine derivative with potential activity against tuberculosis: Application to drug development

In the present study, a simple, sensitive, specific and rapid liquid chromatography (LC) tandem mass spectrometry (MS/MS) method was developed and validated according to the Food and Drug Administration (FDA) guidelines for estimation of IIIM-MCD-211 (a potent oral candidate with promising action against tuberculosis) in mice plasma using carbamazepine as internal standard (IS). Bioanalytical method consisted of one step protein precipitation for sample preparation followed by quantitation in LC-MS/MS using positive electrospray ionization technique (ESI) operating in multiple reaction monitoring (MRM) mode. Elution was achieved in gradient mode on High Resolution Chromolith RP-18e column with mobile phase comprised of acetonitrile and 0.1% (v/v) formic acid in water at the flow rate of 0.4mL/min. Precursor to product ion transitions (m/z 344.5/218.4 and m/z 237.3/194.2) were used to measure analyte and IS, respectively. All validation parameters were well within the limit of acceptance criteria. The method was successfully applied to assess the pharmacokinetics of the candidate in mice following oral (10mg/kg) and intravenous (IV; 2.5mg/kg) administration. It was also effectively used to quantitate metabolic stability of the compound in mouse liver microsomes (MLM) and human liver microsomes (HLM) followed by its in-vitro-in-vivo extrapolation.

Synthesis and Biological Evaluation of Polar Functionalities Containing Nitrodihydroimidazooxazoles as Anti-TB Agents

Novel polar functionalities containing 6-nitro-2,3-dihydroimidazooxazole (NHIO) analogues were synthesized to produce a compound with enhanced solubility. Polar functionalities including sulfonyl, uridyl, and thiouridyl-bearing NHIO analogues were synthesized and evaluated against Mycobacterium tuberculosis (MTB) H37Rv. The aqueous solubility of compounds with MIC values ≤0.5 μg/mL were tested, and six compounds showed enhanced aqueous solubility. The best six compounds were further tested against resistant (Rif(R) and MDR) and dormant strains of MTB and tested for cytotoxicity in HepG2 cell line. Based on its overall in vitro characteristics and solubility profile, compound 6d was further shown to possess high microsomal stability, solubility under all tested biological conditions (PBS, SGF and SIF), and favorable oral in vivo pharmacokinetics and in vivo efficacy.

Pharmacokinetics, pharmacodynamics and safety profiling of IS01957, a preclinical candidate possessing dual activity against inflammation and nociception

ABSTRACT In spite of unprecedented advances in modern systems of medicine, there is necessity for exploration of traditional plant based secondary metabolites or their semisynthetic derivatives which may results in better therapeutic activity, low toxicity and favourable pharmacokinetics. In this context, computational model based predictions aid medicinal chemists in rational development of new chemical entity having unfavourable pharmacokinetic properties which is a major hurdle for its further development as a drug molecule. Para‐coumaric acid (p‐CA) and its derivatives found to be have promising antiinflammatory and analgesic activity. IS01957, a p‐CA derivative has been identified as dual acting molecule against inflammation and nociception. Therefore, objective of the present study was to investigate pharmacokinetics, efficacy and safety profile based on in‐silico, in‐vitro and in‐vivo model to assess drug likeliness. In the present study, it has excellent pharmacological action in different animal models for inflammation and nociception. Virtual pharmacokinetics related properties of IS01957 have resemblance between envision and experimentation with a few deviations. It has also acceptable safety pharmacological profile in various animal models for central nervous system (CNS), gastro intestinal tract (GIT)/digestive system and cardiovascular system (CVS). Finally, further development of IS01957 is required based on its attractive preclinical profiles. HighlightsIS01957 have excellent drug likeliness as a preclinical candidate.It has dual activity against inflammation and nociception.It has resemblance in pharmacokinetics between envision and experimental outcomes.It has acceptable safety profile for CNS, CVS and GIT under experimental conditions.

Revelation and cloning of valinomycin synthetase genes in Streptomyces lavendulae ACR-DA1 and their expression analysis under different fermentation and elicitation conditions

Streptomyces species are amongst the most exploited microorganisms due to their ability to produce a plethora of secondary metabolites with bioactive potential, including several well known drugs. They are endowed with immense unexplored potential and substantial efforts are required for their isolation as well as characterization for their bioactive potential. Unexplored niches and extreme environments are host to diverse microbial species. In this study, we report Streptomyces lavendulae ACR-DA1, isolated from extreme cold deserts of the North Western Himalayas, which produces a macrolactone antibiotic, valinomycin. Valinomycin is a K+ ionophoric non-ribosomal cyclodepsipeptide with a broad range of bioactivities including antibacterial, antifungal, antiviral and cytotoxic/anticancer activities. Production of valinomycin by the strain S. lavendulae ACR-DA1 was studied under different fermentation conditions like fermentation medium, temperature and addition of biosynthetic precursors. Synthetic medium at 10°C in the presence of precursors i.e. valine and pyruvate showed enhanced valinomycin production. In order to assess the impact of various elicitors, expression of the two genes viz. vlm1 and vlm2 that encode components of heterodimeric valinomycin synthetase, was analyzed using RT-PCR and correlated with quantity of valinomycin using LC-MS/MS. Annelid, bacterial and yeast elicitors increased valinomycin production whereas addition of fungal and plant elicitors down regulated the biosynthetic genes and reduced valinomycin production. This study is also the first report of valinomycin biosynthesis by Streptomyces lavendulae.

Determination of ZSTK474, a novel Pan PI3K inhibitor in mouse plasma by LC–MS/MS and its application to Pharmacokinetics.

Graphical abstract Figure. No caption available. HighlightsSimple, robust, rapid, reproducible method development of ZSTK474 for routine analysis.Full validated LCMS/MS Method.Successfully applied to pharmacokinetics study.Mice pharmacokinetics is first time reported. ABSTRACT ZSTK474, a promising novel anticancer molecule derived from s‐triazine, found to have antitumor activities against different cancer cell lines. However, neither LCMS method nor pharmacokinetics of ZSTK474 has been reported till now. A sensitive, simple, short and specific liquid chromatography tandem mass spectrometry (LCMS/MS) method was developed for the quantification of ZSTK474 in mouse plasma accordance with the US Food and Drug Administration guidelines. Extraction of drug molecule was carried out using protein precipitation. Chromatographic analyte separation was achieved on Atlantis dC18 (4.6 × 50 mm, 3 &mgr;m). Composition of isocratic mobile phase consists of 90% acetonitrile and 0.2% formic acid, at 0.7 mL/min flow rate, having short 2.5 min run time. Method development was validated and found to be linear over a dynamic range between 1.9–1000 ng/mL; having a correlation coefficient (r 2) ≥ 0.9978. The analyte was found to be stable under short and long term storage conditions. LCMS/MS method developed was validated and found to be selective, reproducible, precise and accurate to quantify ZSTK474 in plasma samples, and first time successfully applied to pharmacokinetic studies. Pharmacokinetic data showed fast absorption attaining Cmax at 0.25 h and half life (t1/2) 5.18 h after oral administration of ZSTK474 at 20 mg/kg in mouse.

Re-Validation of New Develop Highly Sensitive, Simple LCMS/MS Method for the Estimation of Rohitukine and its Application in ADME/Pre-Clinical Pharmacokinetics

The purpose of the research was to develop a simple, rapid, accurate, reproducible and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for determination of Rohitukine, a chromone alkaloid in plasma. The chromatographic separation was achieved with high resolution RP18e Chromolith column (100 × 4.6 mm, 2 μm) employing a isocratic composition of organic solvent acetonitrile with 0.1% (v/v) formic acid (80:20, %v/v) at a flow rate of 0.5 mL/min. Triple quadrupole mass spectrometry with positive electrospray ionization (ESI) technique operating in multiple reaction monitoring used to estimate MS/MS ion transitions like 306.05>245.10 and 306.05>231.05 for Rohitukine and 330.30>97.0 for IS. Simple single step protein precipitate method was used for sample preparation. The method was validated for specificity, linearity, accuracy, precision, recovery, matrix effect and stability as per FDA guidelines. Linearity of the analyte was acquired throughout the concentration range from 0.1 ng/mL to 1000 ng/mL in mice plasma. Pharmacokinetic study was performed on female BALB/c mice through oral (20 mg/kg) and intravenous (2 mg/kg) route where the oral bioavailability of Rohitukine obtained was 84%. The bioanalytical method was successfully used for determination of plasma protein binding study, permeability and microsomal stability in mouse, rat and human liver microsomes.