Kinetic Analyses of the Siderophore Biosynthesis Inhibitor Salicyl-AMS and Analogues as MbtA Inhibitors and Antimycobacterial Agents
| Autor: | Tezcan Guney, Glennon V. Bythrow, Derek S. Tan, Xuequan Lu, Cheng Ji, Gabrielle Germain, Poornima Mohandas, Sivagami Sundaram Chavadi, Keith Levendosky, Luis E. N. Quadri, Lisa C. Standke |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Siderophore Adenosine 030106 microbiology Mycobacterium smegmatis Antitubercular Agents Siderophores Microbial Sensitivity Tests Biochemistry Article Mycobacterium tuberculosis Ligases 03 medical and health sciences chemistry.chemical_compound Structure-Activity Relationship Biosynthesis Escherichia coli Enzyme kinetics Adenylylation chemistry.chemical_classification biology Molecular Structure Chemistry biology.organism_classification Kinetics 030104 developmental biology Enzyme Drug Design Lead compound Salicylic acid Bacillus subtilis Protein Binding |
| Popis: | 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(i) of salicyl-AMS for inhibition of the salicylic acid adenylation enzyme MbtA from Mycobacterium tuberculosis (MbtA(tb)), 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 [Formula: see text] and analysis of the mechanism of inhibition. For these studies, we optimized production and purification of recombinant MbtA(tb), for which K(m) and k(cat) values were determined, and used the enzyme in conjunction with an MbtA(tb)-optimized, continuous, spectrophotometric assay for MbtA activity and inhibition. The cell-based studies provided an assessment of the antimycobacterial activity and post-antibiotic effect of the nine MbtA(tb) inhibitors. The antimycobacterial properties were evaluated using a strain of non-pathogenic, fast-growing Mycobacterium smegmatis that was genetically engineered for MbtA(tb)-dependent susceptibility to 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(tb) by salicyl-AMS and eight analogues, afford new SAR insights for these inhibitors, and highlight several suitable candidates for future preclinical evaluation. |
| Databáze: | OpenAIRE |
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