A protein–protein interaction inhibitor arrests the cell cycle in Aspergillus fumigatus

Invasive infections caused by Aspergillus fumigatus have high mortality rates, even when treated with the first-line agent voriconazole. The global emergence of azole resistance further increases treatment failure, underscoring the urgent need for antifungals with novel mechanisms of action. The fungal cell cycle is essential for viability and represents an attractive but underexplored target. In A. fumigatus, progression from G2 to M phase requires interaction between the phosphatase NimT and cyclin-dependent kinase NimX. Here, we characterize the role of this interaction and its inhibition by 2-fluoro-4-hydroxybenzonitrile (compound 1), a small molecule that targets the human Cdc25B–Cdk2 interface. Using mutagenesis, we show that NimT residues Arg438 and Arg442 are critical for NimT–NimX binding and confirm they are essential for viability. A co-immunoprecipitation assay demonstrates that compound 1 disrupts the interaction, while live-cell imaging shows that this inhibition arrests cell cycle progression. Our findings provide mechanistic insight into fungal mitosis and highlight cell cycle regulators as promising antifungal drug targets.