Adh1-Programmed SNF1 Phosphogradients Decrypt Morphogenesis in Candida albicans: Chemical Interrogation Unveils Hyphal Transition Thresholds

While Candida albicans alcohol dehydrogenase I (Adh1) conventionally functions as an alcohol dehydrogenase, this study builds upon previous work to redefine its novel role in regulating hyphal morphogenesis and elucidates the underlying mechanisms. Adh1 knockout strains exhibited hyperfilamentation, suggesting that Adh1 directly regulates true hyphal development, independent of its canonical metabolic activity. Leveraging this phenotype, a ‘reverse screening strategy’ identified 2-hydroxyanthraquinone (HAQ) through high-throughput screening as a potent inhibitor of biofilm formation and hyphal growth by targeting Adh1. Biochemical and structural analyses confirmed HAQ's direct binding to Adh1's F224/A254/Q257 interface. Mechanistically, affinity purification-mass spectrometry revealed Adh1 modulates the SNF1 signalling axis by accelerating SNF1 dephosphorylation via interactions with Bmh1/Ssb1 regulators, thereby inhibiting hyphal conversion. HAQ disrupted these interactions, reducing SNF1 phosphorylation levels in an Adh1-dependent manner. This work establishes Adh1 as both an endogenous SNF1 pathway suppressor and an exogenous drug target, while demonstrating the efficacy of phenotype-driven discovery pipelines. The findings provide a novel antifungal strategy targeting virulence-regulating metabolic enzymes and validate HAQ as a lead compound for therapeutic development against C. albicans pathogenicity. Graphical Based on the ADH1 knockout strain, HAQ was identified through forward/reverse genetic screening of 114 natural products and analysis of protein ligand and protein–protein interactions. With the help of HAQ, a new mechanism for regulating the phosphorylation level of Adh1 –SNF1 has been revealed, in which HAQ binds to Adh1 to release dephosphorylation factors Bmh1 and Ssb1, thereby reducing the phosphorylation level of SNF1 and inhibiting hyphal growth.