The novel antifungal agent NPD2560 perturbs the Rho1-centered signaling network to induce a cell wall integrity...

Fungal pathogens pose an increasing threat to human health. The limited diversity of antifungal drug classes and the rapid emergence of resistance highlight the urgent need for the identification and development of agents with novel mechanisms of action. Here, we identified NPD2560, a coumarin-derived compound, as an antifungal agent that exhibits fungicidal activity against Candida krusei, a clinically challenging non-albicans Candida species often associated with treatment failure. Chemical-genomic profiling in Saccharomyces cerevisiae identified RHO1 as a major genetic determinant of NPD2560 sensitivity, suggesting perturbation of the Rho1-Fks1 signaling network that regulates fungal cell wall biogenesis and integrity. Mutational analyses revealed that fks1Δ and rho1-3 mutants were hypersensitive to NPD2560, indicating that intact function of this pathway is critical for cellular tolerance to the compound. Biochemical analysis further demonstrated that NPD2560 was associated with a reduction in β-(1,6)-glucan levels, while β-(1,3)-glucan levels were largely unaffected under the conditions tested, leading to activation of the cell wall integrity (CWI) pathway and compensatory chitin deposition. Consistent with these findings, kre6Δ and KRE6(F552I) mutants defective in β-(1,6)-glucan synthase activity showed no cross-resistance, indicating a mechanism distinct from the β-(1,6)-glucan synthase inhibitor jervine, but functionally linked to the Rho1-Fks1 branch. Transcriptomic analysis revealed a robust remodeling response with induction of Rlm1-dependent cell wall genes such as CRH1, KDX1, CWP1, and PIR3. Taken together, these results show that NPD2560 perturbs the Rho1-centered cell wall regulatory network, triggering compensatory cell wall stress responses, and provide a mechanistically distinct chemical probe for dissecting fungal cell wall integrity pathways.IMPORTANCEThe fungal cell wall is an essential structure that determines cell shape, viability, and pathogenicity, representing an ideal therapeutic focus for antifungal intervention. However, most existing antifungal drugs act on ergosterol or β-(1,3)-glucan synthesis, and resistance among non-albicans Candida species remains a serious issue. In this study, we characterized NPD2560, a coumarin-derived compound that functionally perturbs the Rho1-Fks1 signaling pathway involved in cell wall construction. Unlike previously reported coumarin derivatives, which mainly act through oxidative or mitochondrial stress, NPD2560 induces cell wall stress and activates the CWI pathway. These findings provide new mechanistic insights into fungal cell wall control and position NPD2560 as a mechanistic probe and potential lead compound for exploring the cell wall regulatory network.