Inhibitors of fungal GWT1 and MCD4 discovered through Candida albicans fitness test
54th Interscience Conference on Antimicrobial Agents and Chemotherapy Washington, DC | September 08, 2014
Summary: Candida albicans persists as the principal clinically-relevant fungal pathogen in the U.S. Options for treatment are restricted to only three basic classes of agents: amphotericin B, azoles, and echinocandins. Researchers have combined a C. albicans genome-wide reverse genetics small molecule screening strategy (CaFT) and classical drug resistance mapping in S. cerevisiae to identify and mechanistically characterize multiple antifungal inhibitors specifically targeting Gwt1p-dependent acylation or Mcd4p-specific EtNP transferase activity in GPI precursor biosynthesis. They further demonstrated that GPI inhibitors unmask β-glucan and stimulate TNFα in macrophages and that the Mcd4p inhibitor provides a beneficial therapeutic effect in a systemic infection model of candidiasis. GPI biosynthesis should be considered an important target pathway for developing new antifungals.
- To identify novel, target specific inhibitors with antifungal drug-like properties and therapeutic potential, they developed the C. albicans Fitness Test (CaFT) assay as a genomics-based platform.
- Based on the principle of chemically-induced haploinsufficiency, the deletion of one allele of a target gene renders the heterozygote deletion strain hypersensitive to bioactive inhibitors specific to the depleted target.
- To genetically corroborate CaFT profiles which predict novel mechanisms, Saccharomyces cerevisiae drug resistant mutants were isolated and analyzed using Sanger and whole genome sequencing.
- CaFT screening a focused library of synthetic compounds with intrinsic whole cell activity against C. albicans led to the identification of two novel compounds, L365 and L884, each displaying highly selective GWT1-specific CaFT profiles.
- In a CaFT natural product screen, they identified L743, a terpenoid lactone ring-based natural product previously demonstrated to inhibit Mcd4p ethanolamine phosphotransferase activity.
- Both Gwt1p and Mcd4p are essential proteins involved in glycosylphosphatidylinositol (GPI) precursor biosynthesis.
- GPIs are endoplasmic reticulum (ER)-derived post-translational modifications that serve as cell surface anchors for >100 cell wall proteins.