Targeting Fungal Ras

We reported eight high-resolution crystal structures of CnFTase that define the enzymatic reaction cycle, the basis of ligand selection, and structurally divergent regions of the active site. Our crystal structures of clinically important anticancer FTIs in complex with CnFTase suggest FTIs developed for treatment of cancer are reasonable first generation agents to explore as antifungals. Additionally, our study suggests structure-guided strategies for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions.

Thus, we hypothesize that structure-guided strategies can aid development of agents that specifically target fungal prenyltransferases, which could have broad-spectrum antifungal effects. Our approach takes advantage of the work done by pharmaceutical companies to develop anti-cancer FTIs and GGTIs with good pharmacokinetic properties. We propose structural and functional studies that support Program goals of developing inhibitors to treat invasive fungal infections.

Specific Aims

1. To redirect FTIs developed for cancer chemotherapeutics to antifungal agents using structure-guided approaches. Optimized FTIs have promise as specific broad-spectrum antifungals because FTase is required for fungal viability and pathogenesis. We have determined crystal structures for FTase from C. albicans, C. neoformans, and A. fumigatus and complexes with substrates and FTIs. Our work found structural features conserved across these divergent fungal species that differ from human FTase. We also identified compounds that inhibit C. neoformans FTase in vitro and have antifungal activity in vivo. We propose to extend our studies to other fungal pathogens to identify compounds with improved potency and specificity.
2. To investigate GGTase as an antifungal drug target, and develop potent GGTIs using structure-guided approaches. Our preliminary data supports the role of GGTase in fungal virulence. We published the structure of C. albicans GGTase that reveals divergent structural features from the mammalian enzyme. We propose a structure-guided approach to the development of potent highly specific GGTIs and to extend these studies to the other fungal pathogens.
3. To investigate palmitoylation of Ras-family proteins in fungal pathogenesis, and evaluate its potential for therapeutic targeting. Our preliminary studies show that protein palmitoylation is required for growth and pathogenesis of C. neoformans and A. fumigatus. The exceptional sequence divergence between fungal and mammalian enzymes increases the likelihood of specific fungal palmitoyltransferase inhibition. Based on preliminary genetic experiments identifying the fungal palmitoyltransferases most important for pathogenesis, we will characterize one of these enzymes biochemically, screen for small molecule inhibitors, and initiate structural studies.