Identification of KasA as the cellular target of an anti-tubercular scaffold

Abstract

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis β-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related β-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis.

Fatty Acid Biosynthesis: Chain‐Length Regulation and Control

“…Similar uncoupling was observed in baker's yeast type I FAS, when the active‐site cysteine residue was blocked . Another intensively studied KS enzyme is M. tuberculosis KasA, because it is a relevant drug target . KasA has been analyzed in terms of structure and function, and recently also by computational means .…”

Development of a whole‐cell high‐throughput phenotypic screen to identify inhibitors of mycobacterial amino acid biosynthesis

“…Target identification commonly involves the WGS of spontaneous‐resistant mutants against compounds that have shown anti‐tubercular activity from a whole throughput screening campaign. There are a number of reported successes when using this approach . However, to avoid the discovery and progression of compounds that inhibit inappropriate targets, we have developed a new strategy to identify inhibitors of a specific set of biochemical pathways: amino acid biosynthesis.…”

“…Subsequent to the discovery of an inhibitor of an amino acid biosynthetic pathway, the precise mode of action can then be determined. This can be achieved through a number of techniques, including WGS of resistant isolates, and over‐expression studies of the genes involved in the particular biosynthetic pathway . Although it is known that the control compounds MSM and SMM target the essential IlvB1 enzyme, WGS of resistant isolates was performed to identify resistance‐conferring mutations within the gene.…”

“…There are a number of reported successes when using this approach. 3,[19][20][21] However, to avoid the discovery and progression of compounds that inhibit inappropriate targets, we have developed a new strategy to identify inhibitors of a specific set of biochemical pathways: amino acid biosynthesis. We have designed a high-throughput phenotypic screen, which can be used to find inhibitors of any amino acid biosynthetic pathway, or tailored to that of a defined amino acid.…”

Colworth prize lecture 2016: exploiting new biological targets from a whole-cell phenotypic screening campaign for TB drug discovery

“…A wealth of basic biochemistry and biology has gone into understanding the mechanism of this pro-drug and has primed the field for study of this pathway. A new family of indazole sulfonamides were found to possess anti-tubercular activity against KasA [27]. KasA is a condensing enzyme in the FAS-II fatty acid synthesis pathway and is essential for viability in mycobacteria [28].…”