Distinct binding modes shape exploitable asymmetries between echinocandin and triterpenoid resistance landscapes in Fks1

Echinocandins are the first-line treatment for invasive fungal infections and work by inhibiting the fungal {beta}-1,3-glucan synthase Fks1. Recently, a first-in-class triterpenoid Fks1 inhibitor, ibrexafungerp, was approved for clinical use. The extent to which the difference between these drug classes alters the landscape of resistance mutations in Fks1 is unknown. We used deep mutational scanning (DMS) to quantify the effects of all single-amino-acid variants spanning 159 residues across the three canonical Fks1 resistance hotspots and flanking regions in Saccharomyces cerevisiae. Drug selections revealed widespread position-specific cross-resistance and differential resistance, including an ibrexafungerp-specific resistance cluster downstream of hotspot 2 that defines a novel triterpenoid-specific resistance hotspot. We defined this as hotspot 4 and redefined the boundaries of the remaining three canonical echinocandin hotspots, based on the mutational landscape of our DMS analyses. Susceptibility testing of individual FKS1 variants of S. cerevisiae and Candidozyma auris strains confirmed conserved class-specific and cross-resistance patterns. Structure-guided ligand placement, molecular dynamics-based simulated annealing, and DMS-informed pose selection linked drug-specific phenotypes to distinct interaction networks, providing a molecular rationale for the observed differential resistance patterns. These data provide a framework for predicting antifungal resistance, informing treatment strategies and future drug design.