Discovery, Synthesis and Evaluation of a Ketol‐Acid Reductoisomerase Inhibitor

Ketol‐acid reductoisomerase (KARI), the second enzyme in the branched‐chain amino acid biosynthesis pathway, is a potential drug target for bacterial infections including Mycobacterium tuberculosis. Here, we have screened the Medicines for Malaria Venture Pathogen Box against purified M. tuberculosis (Mt) KARI and identified two compounds that have Ki values below 200 nm. In Mt cell susceptibility assays one of these compounds exhibited an IC50 value of 0.8 μm. Co‐crystallization of this compound, 3‐((methylsulfonyl)methyl)‐2H‐benzo[b][1,4]oxazin‐2‐one (MMV553002), in complex with Staphylococcus aureus KARI, which has 56 % identity with Mt KARI, NADPH and Mg2+ yielded a structure to 1.72 Å resolution. However, only a hydrolyzed product of the inhibitor (i.e. 3‐(methylsulfonyl)‐2‐oxopropanic acid, missing the 2‐aminophenol attachment) is observed in the active site. Surprisingly, Mt cell susceptibility assays showed that the 2‐aminophenol product is largely responsible for the anti‐TB activity of the parent compound. Thus, 3‐(methylsulfonyl)‐2‐oxopropanic acid was identified as a potent KARI inhibitor that could be further explored as a potential biocidal agent and we have shown 2‐aminophenol, as an anti‐TB drug lead, especially given it has low toxicity against human cells. The study highlights that careful analysis of broad screening assays is required to correctly interpret cell‐based activity data. Potent pathogens: E4P, the hydrolyzed product of compound E4 (MMV553002) from the Pathogen box, is a potent inhibitor of ketol‐acid reductoisomerase (KARI) of Mycobacterium tuberculosis with potential for a novel biocide development.