title: Screening, identification, and characterization of mechanistically diverse inhibitors of the Mycobacterium tuberculosis enzyme, pantothenate kinase (CoaA).
abstract: The authors describe the discovery of anti-mycobacterial compounds through identifying mechanistically diverse inhibitors of the essential Mycobacterium tuberculosis (Mtb) enzyme, pantothenate kinase (CoaA). Target-driven drug discovery technologies often work with purified enzymes, and inhibitors thus discovered may not optimally inhibit the form of the target enzyme predominant in the bacterial cell or may not be available at the desired concentration. Therefore, in addition to addressing entry or efflux issues, inhibitors with diverse mechanisms of inhibition (MoI) could be prioritized before hit-to-lead optimization. The authors describe a high-throughput assay based on protein thermal melting to screen large numbers of compounds for hits with diverse MoI. Following high-throughput screening for Mtb CoaA enzyme inhibitors, a concentration-dependent increase in protein thermal stability was used to identify true binders, and the degree of enhancement or reduction in thermal stability in the presence of substrate was used to classify inhibitors as competitive or non/uncompetitive. The thermal shift-based MoI assay could be adapted to screen hundreds of compounds in a single experiment as compared to traditional biochemical approaches for MoI determination. This MoI was confirmed through mechanistic studies that estimated K(ie) and K(ies) for representative compounds and through nuclear magnetic resonance-based ligand displacement assays.
authors.affiliation: AstraZeneca R & D, Bangalore, India. janani.venkatraman@astrazeneca.com
authors.forename: Janani
authors.initials: J
authors.lastname: Venkatraman
authors.forename: Jyothi
authors.initials: J
authors.lastname: Bhat
authors.forename: Suresh M
authors.initials: SM
authors.lastname: Solapure
authors.forename: Jatheendranath
authors.initials: J
authors.lastname: Sandesh
authors.forename: Debasmita
authors.initials: D
authors.lastname: Sarkar
authors.forename: Sundaram
authors.initials: S
authors.lastname: Aishwarya
authors.forename: Kakoli
authors.initials: K
authors.lastname: Mukherjee
authors.forename: Santanu
authors.initials: S
authors.lastname: Datta
authors.forename: Krishnan
authors.initials: K
authors.lastname: Malolanarasimhan
authors.forename: Balachandra
authors.initials: B
authors.lastname: Bandodkar
authors.forename: Kaveri S
authors.initials: KS
authors.lastname: Das
mesh_terms: D001681:Biological Assay; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors*
chemical_list: D004791:Enzyme Inhibitors; D017853:Phosphotransferases (Alcohol Group Acceptor); C023088:pantothenate kinase