Synergistic Lethality of a Binary Inhibitor of Mycobacterium tuberculosis KasA

Abstract

We report GSK3011724A (DG167) as a binary inhibitor of β-ketoacyl-ACP synthase (KasA) in <i>Mycobacterium tuberculosis</i> Genetic and biochemical studies established KasA as the primary target. The X-ray crystal structure of the KasA-DG167 complex refined to 2.0-Å resolution revealed two interacting DG167 molecules occupying nonidentical sites in the substrate-binding channel of KasA. The binding affinities of KasA to DG167 and its analog, 5g, which binds only once in the substrate-binding channel, were determined, along with the KasA-5g X-ray crystal structure. DG167 strongly augmented the <i>in vitro</i> activity of isoniazid (INH), leading to synergistic lethality, and also synergized in an acute mouse model of <i>M. tuberculosis</i> infection. Synergistic lethality correlated with a unique transcriptional signature, including upregulation of oxidoreductases and downregulation of molecular chaperones. The lead structure-activity relationships (SAR), pharmacokinetic profile, and detailed interactions with the KasA protein that we describe may be applied to evolve a next-generation therapeutic strategy for tuberculosis (TB).<b>IMPORTANCE</b> Cell wall biosynthesis inhibitors have proven highly effective for treating tuberculosis (TB). We discovered and validated members of the indazole sulfonamide class of small molecules as inhibitors of <i>Mycobacterium tuberculosis</i> KasA-a key component for biosynthesis of the mycolic acid layer of the bacterium's cell wall and the same pathway as that inhibited by the first-line antitubercular drug isoniazid (INH). One lead compound, DG167, demonstrated synergistic lethality in combination with INH and a transcriptional pattern consistent with bactericidality and loss of persisters. Our results also detail a novel dual-binding mechanism for this compound as well as substantial structure-activity relationships (SAR) that may help in lead optimization activities. Together, these results suggest that KasA inhibition, specifically, that shown by the DG167 series, may be developed into a potent therapy that can synergize with existing antituberculars.