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

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 K of salicyl-AMS for inhibition of the salicylic acid adenylation enzyme MbtA from Mycobacterium tuberculosis (MbtA), 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 ( K, k, k, and t) and analysis of the mechanism of inhibition. For these studies, we optimized production and purification of recombinant MbtA, for which K and k values were determined, and used the enzyme in conjunction with an MbtA-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 MbtA inhibitors. The antimycobacterial properties were evaluated using a strain of nonpathogenic, fast-growing Mycobacterium smegmatis that was genetically engineered for MbtA-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 MbtA by salicyl-AMS and eight analogues, afford new SAR insights for these inhibitors, and highlight several suitable candidates for future preclinical evaluation.