Antifungal Activity of Gepinacin Scaffold Glycosylphosphatidylinositol Anchor Biosynthesis Inhibitors with Improved Metabolic Stability.

The glycosylphosphatidylinositol anchor biosynthesis inhibitor gepinacin demonstrates broad-spectrum antifungal activity and negligible mammalian toxicity in culture but is metabolically labile. The stability and bioactivity of 39 analogs were tested in vitro to identify LCUT-8, a stabilized lead with increased potency and promising single-dose pharmacokinetics. Unfortunately, no antifungal activity was seen at the maximum dosing achievable in a neutropenic rabbit model. Nevertheless, structure-activity relationships identified here suggest strategies to further improve compound potency, solubility, and stability.

Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus.

New strategies are needed to counter the escalating threat posed by drug-resistant fungi. The molecular chaperone Hsp90 affords a promising target because it supports survival, virulence and drug-resistance across diverse pathogens. Inhibitors of human Hsp90 under development as anticancer therapeutics, however, exert host toxicities that preclude their use as antifungals. Seeking a route to species-selectivity, we investigate the nucleotide-binding domain (NBD) of Hsp90 from the most common human fungal pathogen, Candida albicans. Here we report structures for this NBD alone, in complex with ADP or in complex with known Hsp90 inhibitors. Encouraged by the conformational flexibility revealed by these structures, we synthesize an inhibitor with >25-fold binding-selectivity for fungal Hsp90 NBD. Comparing co-crystals occupied by this probe vs. anticancer Hsp90 inhibitors revealed major, previously unreported conformational rearrangements. These insights and our probe's species-selectivity in culture support the feasibility of targeting Hsp90 as a promising antifungal strategy.

Inhibiting mitochondrial phosphate transport as an unexploited antifungal strategy.

The development of effective antifungal therapeutics remains a formidable challenge because of the close evolutionary relationship between humans and fungi. Mitochondrial function may present an exploitable vulnerability because of its differential utilization in fungi and its pivotal roles in fungal morphogenesis, virulence, and drug resistance already demonstrated by others. We now report mechanistic characterization of ML316, a thiohydantoin that kills drug-resistant Candida species at nanomolar concentrations through fungal-selective inhibition of the mitochondrial phosphate carrier Mir1. Using genetic, biochemical, and metabolomic approaches, we established ML316 as the first Mir1 inhibitor. Inhibition of Mir1 by ML316 in respiring yeast diminished mitochondrial oxygen consumption, resulting in an unusual metabolic catastrophe marked by citrate accumulation and death. In a mouse model of azole-resistant oropharyngeal candidiasis, ML316 reduced fungal burden and enhanced azole activity. Targeting Mir1 could provide a new, much-needed therapeutic strategy to address the rapidly rising burden of drug-resistant fungal infection.

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

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.

Inhibiting GPI anchor biosynthesis in fungi stresses the endoplasmic reticulum and enhances immunogenicity.

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 ß-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.

A rhizosphere fungus enhances Arabidopsis thermotolerance through production of an HSP90 inhibitor.

The molecular chaperone HEAT SHOCK PROTEIN90 (HSP90) is essential for the maturation of key regulatory proteins in eukaryotes and for the response to temperature stress. Earlier, we have reported that fungi living in association with plants of the Sonoran desert produce small molecule inhibitors of mammalian HSP90. Here, we address whether elaboration of the HSP90 inhibitor monocillin I (MON) by the rhizosphere fungus Paraphaeosphaeria quadriseptata affects plant HSP90 and plant environmental responsiveness. We demonstrate that MON binds Arabidopsis (Arabidopsis thaliana) HSP90 and can inhibit the function of HSP90 in lysates of wheat (Triticum aestivum) germ. MON treatment of Arabidopsis seedlings induced HSP101 and HSP70, conserved components of the stress response. Application of MON, or growth in the presence of MON, allowed Arabidopsis wild type but not AtHSP101 knockout mutant seedlings to survive otherwise lethal temperature stress. Finally, cocultivation of P. quadriseptata with Arabidopsis enhanced plant heat stress tolerance. These data demonstrate that HSP90-inhibitory compounds produced by fungi can influence plant growth and responses to the environment.

HspBP1, an Hsp70 cochaperone, has two structural domains and is capable of altering the conformation of the Hsp70 ATPase domain.

We present here the first structural information for HspBP1, an Hsp70 cochaperone. Using circular dichroism, HspBP1 was determined to be 35% helical. Although HspBP1 is encoded by seven exons, limited proteolysis shows that it has only two structural domains. Domain I, amino acids 1-83, is largely unstructured. Domain II, amino acids 84-359, is predicted to be 43% helical using circular dichroism. Using limited proteolysis we have also shown that HspBP1 association changes the conformation of the ATPase domain of Hsp70. Only domain II of HspBP1 is required to bring about this conformational change. Truncation mutants of HspBP1 were tested for their ability to inhibit the renaturation of luciferase and bind to Hsp70 in reticulocyte lysate. A carboxyl terminal truncation mutant that was slightly longer than domain I was inactive in these assays, but domain II was sufficient to perform both functions. Domain II was less active than full-length HspBP1 in these assays, and addition of amino acids from domain I improved both functions. These studies show that HspBP1 domain II can bind Hsp70, change the conformation of the ATPase domain, and inhibit Hsp70-associated protein folding.