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Slow Onset Inhibition of KasA by Thiolactomycin: Mechanistic Insights for Lead Optimization

Mycobacterium tuberculosis (MTB) is a global health threat and novel chemotherapeutics are urgently needed for the treatment of multi‐drug resistant and extensively drug resistant strains. In our search for novel TB chemotherapeutics we are focusing on KasA, the β‐keto‐acyl ACP synthase enzyme in the type II fatty acid biosynthesis pathway (FASII), which is a validated target for drug discovery. KasA, which catalyzes a Claisen condensation to initiate each round of fatty acid elongation, is inhibited by the natural product thiolactomycin (TLM). In order to direct the synthesis of more potent TLM analogues, we are using fluorescence spectroscopy and enzyme inhibition studies to analyze the interaction of TLM with KasA. Inhibition studies of ketoacyl synthase enzymes by TLM routinely include a preincubation step prior to substrate addition. We have shown that while TLM binds relatively weakly to the free enzyme, the compound is a slow onset high affinity inhibitor of the KasA acyl‐enzyme intermediate. Currently we are extending these studies to C171Q KasA, a mutant thought to mimic the acyl enzyme intermediate. In parallel experiments, interligand‐NOE NMR studies are being used to optimize the binding of TLM to KasA. These data are being used to guide the synthesis of additional TLM analogs.