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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., Genome Canada (Firm), Genome Quebec

In fungi, the anchoring of proteins to the plasma membrane via their covalent attachment to glycosylphosphatidylinositol (GPI) is essential and thus provides a valuable point of attack for the development of antifungal therapeutics. Unfortunately, studying the underlying biology of GPI-anchor synthesis is difficult, especially in medically relevant fungal pathogens because they are not genetically tractable. Compounding difficulties, many of the genes in this pathway are essential in Saccharomyces cerevisiae. Here, we report the discovery of a new small molecule christened gepinacin (for GPI acylation inhibitor) which selectively inhibits Gwt1, a critical acyltransferase required for the biosynthesis of fungal GPI anchors. After delineating the target specificity of gepinacin using genetic and biochemical techniques, we used it to probe key, therapeutically relevant consequences of disrupting GPI anchor metabolism in fungi. We found that, unlike all three major classes of antifungals in current use, the direct antimicrobial activity of this compound results predominantly from its ability to induce overwhelming stress to the endoplasmic reticulum. Gepinacin did not affect the viability of mammalian cells nor did it inhibit their orthologous acyltransferase. This enabled its use in co-culture experiments to examine Gwt1’s effects on host–pathogen interactions. In isolates of Candida albicans, the most common fungal pathogen in humans, exposure to gepinacin at sublethal concentrations impaired filamentation and unmasked cell wall β-glucan to stimulate a pro-inflammatory cytokine response in macrophages. Gwt1 is a promising antifungal drug target, and gepanacin is a useful probe for studying how disrupting GPI-anchor synthesis impairs viability and alters host–pathogen interactions in genetically intractable fungi., Howard Hughes Medical Institute

The Hsp70 family protein members are involved in many diverse processes that are essential for cell survival. The Hsp70 cochaperones control and specify Hsp70 interactions by modifying Hsp70 activity and linking Hsp70 proteins to specific cellular processes. Understanding the mechanistic details of how cochaperones co-opt Hsp70 for specific functions in a cell may provide insight into the workings of specific cellular functions as well as a broader understanding of how multifunctional proteins are regulated. HspBP1 is an Hsp70 cochaperone that binds to the ATPase domain of Hsp70 and inhibits its ATPase activity. The purpose of these studies was to gain a basic understanding of the Hsp70/HspBP1 interaction. The first goal was to define the structural domains of HspBP1 and explore the secondary structure of those domains. The next goal was to determine if HspBP1's ability to bind to Hsp70 and inhibit its activity segregated to its distinct protein domains. Finally, the effects of HspBP1 association on Hsp70 structure were investigated. It was determined from these studies that although HspBP1 is encoded by seven exons, it has only two structural domains as determined by limited proteolysis. Domain I, amino acids 1-83, is largely unstructured. Domain II, amino acids 84-359 is predicted to be 43% helical using circular dichroism. It was also shown that domain II is sufficient for binding to the Hsp70 ATPase domain and inhibiting luciferase renaturation although domain I was required for full activity. These studies also describe a novel activity for HspBP1, the ability to change the conformation of the ATPase domain. Only domain II of HspBP1 is required to bring about this conformational change. The studies presented here are the first to examine HspBP1's structure and determine how its structural domains interact with Hsp70. Prior to these studies, no structural information had been reported for an HspBPl family member. This information, along with the discovery that H