Much Ado About Antibacterials: Dynamics and Drug Design
The Graduate School, Stony Brook University: Stony Brook, NY.
The post genomic era has led to a glut of new putative targets. However, there is a serious dearth of novel chemotherapeutics. It has become clear in recent years that apart from optimizing the thermodynamics of a drug-target interaction, a cell-wide understanding of a drug's mode of action is indispensable to prevent the attrition commonly observed in the drug discovery pipeline. The type II fatty acid biosynthesis (FASII) pathway is a validated target for antibacterial drug design. In this study we focus on two targets of the bacterial FASII; a trans-2-enoyl-ACP reductase and a &beta-ketoacyl-ACP synthase, both essential for bacterial survival. InhA is the enoyl-ACP reductase in Mycobacterium tuberculosis. SAR analysis based on a triclosan lead has resulted in a range of inhibitors with slow dissociation rates and hence long residence times on the target, which we believe to be important for in vivo efficacy. A structural explanation of the slow-off kinetics is crucial for designing inhibitors with longer residence times. We have used NMR to explore the relationship between protein dynamics and enzyme inhibition. KasA, the &beta-keto-acyl-ACP synthase in MTB, is targeted by various natural products including thiolactomycin (TLM) though with very modest activity in vitro. We have used inter-ligand NOEs to understand the relative orientation of TLM and a pantetheine fragment bound to KasA. Based on our data we have synthesized molecules with not only improved binding affinities but also longer residence times on KasA. Lastly, we have addressed questions of target quantitation and turnover in bacteria and the effect of drugs on bacterial homeostasis. We have used a mass spectrometry based approach to quantify cellular target concentrations and their rates of synthesis and degradation. Our data has yielded insights into the mechanism of the post-antibiotic effect and the influence that drugs have on target levels in the cell. These data have helped us envision a utopian scenario where a drug with a long residence time on a target that has a slow turnover, would show potent in vivo efficacy.