title: Dequalinium, a new inhibitor of Mycobacterium tuberculosis mycothiol ligase identified by high-throughput screening.
abstract: Mycothiol ligase (MshC) is a key enzyme in the biosynthesis of mycothiol, a small molecular weight thiol that is unique to actinomycetes and whose primary role is to maintain intracellular redox balance and remove toxins. MshC catalyzes the adenosine triphosphate (ATP)-dependent condensation of cysteine and glucosamine-inositol (GI) to produce cysteine-glucosamine-inositol (CGI). MshC is essential to Mycobacterium tuberculosis and therefore represents an attractive target for chemotherapeutic intervention. A screening protocol was developed to identify MshC inhibitors based on quantification of residual ATP using a coupled luminescent assay. The protocol was used to screen a library of 3100 compounds in a 384-well plate format (Z'>or=0.65). Fifteen hits (0.48%) were identified from the screen, and 2 hits were confirmed in a secondary assay that measures production of CGI. The structures of both hits contain N-substituted quinolinium moieties, and the more potent of the 2-namely, dequalinium chloride-inhibits MshC with an IC50 value of 24+/-1 microM. Further studies showed dequalinium to be an ATP-competitive inhibitor of MshC, to bind MshC with a KD of 0.22 microM, and to inhibit the growth of M. tuberculosis under aerobic and anaerobic conditions with minimum inhibitory and anaerobic bactericidal concentrations of 1.2 and 0.3 microg/mL, respectively. The screening protocol described is robust and has enabled the identification of new MshC inhibitors.
authors.affiliation: Laboratory of Bioorganic Chemistry, Biotechnology Unit, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20817, USA.
authors.forename: Maria-Teresa
authors.initials: MT
authors.lastname: Gutierrez-Lugo
authors.forename: Heather
authors.initials: H
authors.lastname: Baker
authors.forename: Joseph
authors.initials: J
authors.lastname: Shiloach
authors.forename: Helena
authors.initials: H
authors.lastname: Boshoff
authors.forename: Carole A
authors.initials: CA
authors.lastname: Bewley
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors; D002352:Carrier Proteins / Q000037:antagonists & inhibitors; D003545:Cysteine / Q000378:metabolism*; D003868:Dequalinium / Q000032:analysis* / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis / Q000494:pharmacology*; D006020:Glycopeptides / Q000378:metabolism*; D007294:Inositol / Q000378:metabolism*; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors*; D008163:Luminescent Measurements; D058619:Maltose-Binding Proteins; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology*
chemical_list: D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D006020:Glycopeptides; D058619:Maltose-Binding Proteins; C089265:mycothiol; D007294:Inositol; D003868:Dequalinium; D008025:Ligases; D003545:Cysteine