Mechanistic insights into Gwt1-substrate interactions and antifungal drug discovery via molecular dynamics and virtual screening.

Gwt1, an essential acyltransferase in the glycosylphosphatidylinositol (GPI) biosynthesis pathway, is a promising target for the development of high-selectivity antifungal agents. In this study, we combined molecular dynamics (MD) simulations and free energy calculations to characterize the binding mechanism of Gwt1 with its native substrate, palmitoyl-CoA. Our simulations identified key hydrogen-bonding and ionic interactions critical for substrate recognition, particularly involving residues Lys123, Arg181, and Asn432. Potential of mean force (PMF) calculations revealed multiple conformational states of palmitoyl-CoA, including an I-shaped conformation that sterically occludes the GlcN-PI binding site, thereby hindering the acyl transfer step. Leveraging these structural insights, we performed virtual screening targeting the hydrophobic pocket formed by Tyr129, Tyr400, Phe404, and Tyr408, which identified two approved drugs, tivozanib and rosiglitazone, as potential Gwt1 inhibitors. Experimental validation confirmed their antifungal activities against pathogenic fungi, including Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus. This work provides dynamic mechanistic insights into Gwt1 function and offers a rational strategy for repurposing existing drugs as antifungals targeting the GPI pathway.