title: Benzothiazole Derivative as a Novel Mycobacterium tuberculosis Shikimate Kinase Inhibitor: Identification and Elucidation of Its Allosteric Mode of Inhibition.
abstract: 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.
authors.affiliation: Discovery Informatics Division, CSIR-Indian Institute of Integrative Medicine , Canal Road, Jammu 180001, India.
authors.forename: Rukmankesh
authors.initials: R
authors.lastname: Mehra
authors.affiliation: Clinical Microbiology Division, CSIR-Indian Institute of Integrative Medicine , Canal Road, Jammu 180001, India.|Academy of Scientific and Innovative Research, CSIR-Indian Institute of Integrative Medicine , Canal Road, Jammu 180001, India.
authors.forename: Vikrant Singh
authors.initials: VS
authors.lastname: Rajput
authors.affiliation: Discovery Informatics Division, CSIR-Indian Institute of Integrative Medicine , Canal Road, Jammu 180001, India.
authors.forename: Monika
authors.initials: M
authors.lastname: Gupta
authors.affiliation: Clinical Microbiology Division, CSIR-Indian Institute of Integrative Medicine , Canal Road, Jammu 180001, India.
authors.forename: Reena
authors.initials: R
authors.lastname: Chib
authors.affiliation: Discovery Informatics Division, CSIR-Indian Institute of Integrative Medicine , Canal Road, Jammu 180001, India.
authors.forename: Amit
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Instrumentation Division, CSIR-Indian Institute of Integrative Medicine , Canal Road, Jammu 180001, India.
authors.forename: Priya
authors.initials: P
authors.lastname: Wazir
authors.affiliation: Clinical Microbiology Division, CSIR-Indian Institute of Integrative Medicine , Canal Road, Jammu 180001, India.|Academy of Scientific and Innovative Research, CSIR-Indian Institute of Integrative Medicine , Canal Road, Jammu 180001, India.
authors.forename: Inshad Ali
authors.initials: IA
authors.lastname: Khan
authors.affiliation: Discovery Informatics Division, CSIR-Indian Institute of Integrative Medicine , Canal Road, Jammu 180001, India.|Academy of Scientific and Innovative Research, CSIR-Indian Institute of Integrative Medicine , Canal Road, Jammu 180001, India.
authors.forename: Amit
authors.initials: A
authors.lastname: Nargotra
mesh_terms: D000495:Allosteric Site / Q000187:drug effects; D000995:Antitubercular Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D052160:Benzothiazoles / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D020134:Catalytic Domain; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: D000995:Antitubercular Agents; D052160:Benzothiazoles; D004791:Enzyme Inhibitors; D017853:Phosphotransferases (Alcohol Group Acceptor); C022642:shikimate kinase; C005465:benzothiazole