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Biochemistry. 2019 Feb 12;58(6):833-847. doi: 10.1021/acs.biochem.8b01153. Epub 2019 Jan 10.

Kinetic Analyses of the Siderophore Biosynthesis Inhibitor Salicyl-AMS and Analogues as MbtA Inhibitors and Antimycobacterial Agents.

Author information

1
Department of Biology, Brooklyn College , City University of New York , 2900 Bedford Avenue , Brooklyn , New York 11210 , United States.
2
Biology Program, Graduate Center , City University of New York , 365 Fifth Avenue , New York , New York 10016 , United States.
3
Chemical Biology Program, Sloan Kettering Institute , Memorial Sloan Kettering Cancer Center , 1275 York Avenue , New York , New York 10065 , United States.
4
Pharmacology Program, Weill Cornell Graduate School of Medical Sciences , Memorial Sloan Kettering Cancer Center , 1275 York Avenue , New York , New York 10065 , United States.
5
Tri-Institutional Research Program , Memorial Sloan Kettering Cancer Center , 1275 York Avenue , New York , New York 10065 , United States.
6
Biochemistry Program, Graduate Center , City University of New York , 365 Fifth Avenue , New York , New York 10016 , United States.

Abstract

There is a paramount need for expanding the drug armamentarium to counter the growing problem of drug-resistant tuberculosis. Salicyl-AMS, an inhibitor of salicylic acid adenylation enzymes, is a first-in-class antibacterial lead compound for the development of tuberculosis drugs targeting the biosynthesis of salicylic-acid-derived siderophores. In this study, we determined the Ki of salicyl-AMS for inhibition of the salicylic acid adenylation enzyme MbtA from Mycobacterium tuberculosis (MbtAtb), designed and synthesized two new salicyl-AMS analogues to probe structure-activity relationships (SAR), and characterized these two analogues alongside salicyl-AMS and six previously reported analogues in biochemical and cell-based studies. The biochemical studies included determination of kinetic parameters ( Kiapp, konapp, koff, and tR) and analysis of the mechanism of inhibition. For these studies, we optimized production and purification of recombinant MbtAtb, for which Km and kcat values were determined, and used the enzyme in conjunction with an MbtAtb-optimized, continuous, spectrophotometric assay for MbtA activity and inhibition. The cell-based studies provided an assessment of the antimycobacterial activity and postantibiotic effect of the nine MbtAtb inhibitors. The antimycobacterial properties were evaluated using a strain of nonpathogenic, fast-growing Mycobacterium smegmatis that was genetically engineered for MbtAtb-dependent susceptibility to MbtA inhibitors. This convenient model system greatly facilitated the cell-based studies by bypassing the methodological complexities associated with the use of pathogenic, slow-growing M. tuberculosis. Collectively, these studies provide new information on the mechanism of inhibition of MbtAtb by salicyl-AMS and eight analogues, afford new SAR insights for these inhibitors, and highlight several suitable candidates for future preclinical evaluation.

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