STRUCTURAL INVESTIGATION OF GRR1 AS A KEY COMPONENT IN ANTIFUNGAL TARGET PROTEIN DEGRADATION

Invasive fungal infections represent a global health crisis, necessitating novel therapeutic strategies to overcome the limitations of current antifungals and growing resistance. Targeted Protein Degradation technology, particularly through proteolysis targeting chimera (PROTACs), offers a revolutionary approach. This study addresses this challenge by computationally exploring the fungal specific F-box protein Grr1 (Saccharomyces cerevisiae), as a novel, recruitable E3 ubiquitin ligase for the development of antifungal PROTACs. To this aim, we sought the structural characterization of Grr1-subtrate complex, followed by the identification of binding sites and the proposition of peptide and organic ligands. Initially, we modelled the Grr1 - Gic2 complex and characterized its binding interface. Based on the structural information we designed a peptide ligand targeting Grr1 and investigated its binding affinity and stability. Subsequently, the same binding site was used as the target for a large-scale virtual screening of organic compounds and fragments to identify potential E3 ligase binders. For this purpose, a diverse chemical library was computationally prepared and subjected to molecular docking. Following, hits were prioritized through a multiparameter analysis that integrated docking affinity with physicochemical properties. Additionally, similarity-based clustering algorithm was applied to ensure the selection of structurally diverse chemical scaffolds. The five top-ranking candidates were subjected to all-atom molecular dynamics simulations, followed by clustering analysis, to evaluate the stability of each complex and to identify their predominant conformational states. Thus, for each representative state, the binding free energy was calculated using the MM/PBSA method. Our analysis successfully identified several distinct chemical scaffolds predicted to act as stable Grr1 binders, suggesting they represent viable starting points for the design of experimental E3 ligase binders. This computational investigation provides the first mechanistic evidence supporting the hypothesis that Grr1 constitutes a druggable component of the fungal ubiquitin–proteasome system. The identified ligands constitute a critical first step toward the rational design of Grr1-recruiting binders for PROTAC development. Future efforts will focus on experimentally characterizing their binding and, if validated, on conjugating them to ligands targeting essential proteins in pathogens such as Candida albicans and Fusarium oxysporum. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 306190/2025-7).