Competitive Affinity-Based Protein Profiling Reveals Potential Antifungal Targets of 1,2,3-Triazole Hydrazide in <i>Fusarium graminearum</i>

In previous research, 1,2,3-triazole hydrazide NAU-6ad exhibited remarkable broad-spectrum antifungal activity. However, the specific targets of NAU-6ad remained unknown. Initially, we excluded three targets─succinate dehydrogenase, laccase, and ergosterol synthase─commonly associated with hydrazide derivatives mentioned in the literature. Subsequently, we developed two types of photoprobes: one incorporating diazirine (<b>DA</b>) and the other phenyl tetrazole (<b>TZ</b>), both featuring terminal alkynes for bioorthogonal reactions. Using these two sets of probes, a total of 52 potential targets were identified through competitive affinity-based proteome profiling. Notably, Ndufs6 and I1RC94 were consistently identified by both sets. The overexpression or knockout of Ndufs6, a subunit of complex I, led to significant changes in sensitivity to NAU-6ad in <i>F. graminearum</i>. Similarly, the knockout of other subunits of complex I, specifically Ndufs2, Ndufv1, and Ndufa9, altered the sensitivity of <i>F. graminearum</i> to NAU-6ad, indicating that NAU-6ad might act upon complex I. Further validation was provided by enzyme activity tests, ATP content assays, pyruvate addition assays, and molecular docking, collectively reinforcing the hypothesis that NAU-6ad might function as a complex I inhibitor.