Vital residues-orientated rational design of butenolide inhibitors targeting Of ChtI

An effective approach for discovering small molecular inhibitors is the residues-oriented strategy based on enzyme analysis. In this study, we employed a rational approach to design and synthesize a library of butenolide analogues (Ia-f and IIa-f) targeting Trp107, utilizing reported piperonyl butenolide as lead compound. Notably, the most compounds IIa-f (R2 = NO2) exhibited slightly higher inhibitory potency against Of ChtI compared to compounds Ia-f (R2 = Br). Molecular mechanism studies unveiled a crucial hydrogen bond interaction between the NO2 group and Trp107, explaining the enhanced binding affinities. Compounds IIe and IIf, both bearing NO2 on the benzene ring at the R2 position, displayed the highest inhibitory activity, with Ki values of 0.87 and 0.68 μM, respectively. Our findings highlight the potential of designing inhibitors with high enzymatic activity by structurally optimizing compounds based on the distinct interaction modes with crucial residues in the binding cavity of Of ChtI.