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Dequalinium, a New Inhibitor of Mycobacterium tuberculosis Mycothiol Ligase Identified by High-Throughput Screening

Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland

Heather Baker

Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland

Joseph Shiloach

Biotechnology Unit, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland

Helena Boshoff

Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland

Carole A. Bewley

Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, caroleb{at}mail.nih.gov

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' 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 µM. Further studies showed dequalinium to be an ATP-competitive inhibitor of MshC, to bind MshC with a K_D of 0.22 µM, 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 µg/mL, respectively. The screening protocol described is robust and has enabled the identification of new MshC inhibitors. (Journal of Biomolecular Screening 2009:643-652)

Key Words: competitive inhibitor • luminescent assay • mycothiol biosynthesis • screening • small molecular weight thiol

This version was published on July 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 6, 643-652 (2009)
DOI: 10.1177/1087057109335743


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