Chemical Genomics-Based Antifungal Drug Discovery: Targeting Glycosylphosphatidylinositol (GPI) Precursor Biosynthesis

Abstract

Steadily increasing antifungal drug resistance and persistent high rates of fungal-associated mortality highlight the dire need for the development of novel antifungals. Characterization of inhibitors of one enzyme in the GPI anchor pathway, Gwt1, has generated interest in the exploration of targets in this pathway for further study. Utilizing a chemical genomics-based screening platform referred to as the Candida albicans fitness test (CaFT), we have identified novel inhibitors of Gwt1 and a second enzyme in the glycosylphosphatidylinositol (GPI) cell wall anchor pathway, Mcd4. We further validate these targets using the model fungal organism Saccharomyces cerevisiae and demonstrate the utility of using the facile toolbox that has been compiled in this species to further explore target specific biology. Using these compounds as probes, we demonstrate that inhibition of Mcd4 as well as Gwt1 blocks the growth of a broad spectrum of fungal pathogens and exposes key elicitors of pathogen recognition. Interestingly, a strong chemical synergy is also observed by combining Gwt1 and Mcd4 inhibitors, mirroring the demonstrated synthetic lethality of combining conditional mutants of GWT1 and MCD4. We further demonstrate that the Mcd4 inhibitor M720 is efficacious in a murine infection model of systemic candidiasis. Our results establish Mcd4 as a promising antifungal target and confirm the GPI cell wall anchor synthesis pathway as a promising antifungal target area by demonstrating that effects of inhibiting it are more general than previously recognized.

A complex game of hide and seek: the search for new antifungals

“…and A. fumigatus, which were comparable to those of CFG and AmB. 190 Overall, molecules that can inhibit GPI biosynthesis have great potential as novel drug classes that may act as either as monotherapy or may be used in conjunction with another fungicidal agent.…”

“…10). 190 However, both G884 and G365 were not active against C. krusei and C. glabrata . G884 was also inactive against A. fumigatus.…”

“…G884 was also inactive against A. fumigatus. 190 Another enzyme in the GPI biosynthesis that has been studied as a potential drug target is Mcd4, an ethanolamine phosphotransferase responsible for transferring phosphoethanolamine to the first mannose molecule during GPI biosynthesis. 191, 192 The terpenoid lactone compounds M743 ( 47a , Fig.…”

Generating anchors only to lose them: The unusual story of glycosylphosphatidylinositol anchor biosynthesis and remodeling in yeast and fungi

“…Mcd4 is also identified as a potential target of anti‐fungals. M720, a derivative of YW3548/M743 (a naturally occurring terpenoid lactone), is a yeast‐specific inhibitor which shows antifungal effects in a murine model without toxicity to host . Due to a synthetic lethal interaction between GWT1 and MCD4 , inhibitors of both enzymes can target a broad spectrum of fungal infections .…”

“…M720, a derivative of YW3548/M743 (a naturally occurring terpenoid lactone), is a yeast‐specific inhibitor which shows antifungal effects in a murine model without toxicity to host . Due to a synthetic lethal interaction between GWT1 and MCD4 , inhibitors of both enzymes can target a broad spectrum of fungal infections . Not only do these inhibitors induce UPR in fungal cells but they also cause exposure of the β‐glucan layer of fungal cells due to absence of GPI‐APs at the cell surface, leading to better clearance by the host immune response .…”