Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of infectious mortality, with 1.5 million deaths reported in 2024. The rapid emergence of drug-resistant TB strains highlights the need for new therapeutic strategies targeting novel, essential bacterial pathways. Acetohydroxyacid synthase (AHAS), a key enzyme in branched-chain amino acid biosynthesis, is absent in humans and represents an attractive but underexplored drug target. Structural insights into native Mtb AHAS are crucial to enable rational drug design.
We employed an integrated approach combining enzyme inhibition assays, in vivo efficacy studies, and high-resolution single-particle cryo-electron microscopy (cryo-EM) to investigate Mtb AHAS as a drug target. A panel of commercial herbicidal AHAS inhibitors was screened to evaluate both their inhibitory effects on MtbAHAS enzymatic activity and their antimycobacterial potency.
Among tested compounds, florasulam (FS) emerged as a potent MtbAHAS inhibitor (Ki = 0.23 µM), displaying strong antimycobacterial activity (MIC = 0.52 µM). FS effectively inhibited M. tuberculosis growth in vitro, including within infected macrophages, and reduced pulmonary bacterial load by 13-fold in a murine infection model.
Native, non-crosslinked Mtb AHAS was purified and subjected to single-particle cryo-EM, achieving a reconstruction at 3.7 Å resolution. The structure reveals a conserved catalytic architecture and a clearly defined herbicide-binding pocket, offering key insights into ligand–protein interactions.
This study reports the first high-resolution structure of native MtbAHAS, addressing a major gap in TB drug discovery. The in vivo efficacy and favourable safety profile of FS strongly support its repurposing potential as a lead compound. These structural insights provide a foundation for structure-guided design and optimization of AHAS inhibitors as a novel class of anti-TB agents. Our findings demonstrate the feasibility of targeting amino acid biosynthesis in M. tuberculosis using repurposed herbicidal scaffolds.