T218 and K376 Control Phenamacril Sensitivity in Fusarium solani Myosin I and Enable Structure-BasedAntifungal Discovery

Phenamacril (PHA) is a Fusarium-specific fungicide targeting myosin I, but its sensitivity differs among Fusarium species. The molecular basis of this differential sensitivity remains unclear. Here, we investigated how site-specific variations in Fusarium solani myosin I (FsMyoI) affect PHA sensitivity and evaluated FsMyoI as a target for inhibitor discovery. Molecular dynamics simulations, site-directed mutagenesis, binding assays, and ATPase activity measurements revealed that FsMyoIK376M and FsMyoIT218S mutations reshaped the dynamics of key functional regions, influencing PHA binding and ATPase inhibition. These findings guided virtual screening and led to three hits with confirmed antifungal activity and target engagement. These results provide structural insight into Fusarium species-specific PHA sensitivity and highlight FsMyoI as a potential target for novel antifungal development.