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.

There is an urgent need for new strategies to treat invasive fungal infections, which are a leading cause of human mortality. We establish two activities of the natural product beauvericin, which potentiates the activity of the most widely deployed class of antifungal against the leading human fungal pathogens, blocks the emergence of drug resistance, and renders resistant pathogens responsive to treatment in mammalian infection models. Harnessing genome sequencing of beauvericin-resistant mutants, affinity purification of a biotinylated beauvericin analog, and biochemical and genetic assays reveals that beauvericin blocks multidrug efflux and inhibits the global regulator TORC1 kinase, thereby activating protein kinase CK2 and inhibiting the molecular chaperone Hsp90. Substitutions in the multidrug transporter Pdr5 that enable beauvericin efflux impair antifungal efflux, thereby impeding resistance to the drug combination. Thus, dual targeting of multidrug efflux and TOR signaling provides a powerful, broadly effective therapeutic strategy for fungal infectious disease that evades resistance.

Fungal pathogens cause life-threatening infections in immunocompetent and immunocompromised individuals. Millions of people die each year due to fungal infections, comparable to the mortality attributable to tuberculosis or malaria. The three most prevalent fungal pathogens are Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. Fungi are eukaryotes like their human host, making it challenging to identify fungal-specific therapeutics. There is a limited repertoire of antifungals in clinical use, and drug resistance and host toxicity compromise the clinical utility. The three classes of antifungals for treatment of invasive infections are the polyenes, azoles and echinocandins. Understanding mechanisms of resistance to these antifungals has been accelerated by global and targeted approaches, which have revealed that antifungal drug resistance is a complex phenomenon involving multiple mechanisms. Development of novel strategies to block the emergence of drug resistance and render resistant pathogens responsive to antifungals will be critical to treating life-threatening fungal infections.

Invasive fungal infections are a leading cause of human mortality. Effective treatment is hindered by the rapid emergence of resistance to the limited number of antifungal drugs, demanding new strategies to treat life-threatening fungal infections. Here, we explore a powerful strategy to enhance antifungal efficacy against leading human fungal pathogens by using the natural product beauvericin. We found that beauvericin potentiates the activity of azole antifungals against azole-resistant Candida isolates via inhibition of multidrug efflux and that beauvericin itself is effluxed via Yor1. As observed in Saccharomyces cerevisiae, we determined that beauvericin inhibits TOR signaling in Candida albicans. To further characterize beauvericin activity in C. albicans, we leveraged genome sequencing of beauvericin-resistant mutants. Resistance was conferred by mutations in transcription factor genes TAC1, a key regulator of multidrug efflux, and ZCF29, which was uncharacterized. Transcriptional profiling and chromatin immunoprecipitation coupled to microarray analyses revealed that Zcf29 binds to and regulates the expression of multidrug transporter genes. Beyond drug resistance, we also discovered that beauvericin blocks the C. albicans morphogenetic transition from yeast to filamentous growth in response to diverse cues. We found that beauvericin represses the expression of many filament-specific genes, including the transcription factor BRG1. Thus, we illuminate novel circuitry regulating multidrug efflux and establish that simultaneously targeting drug resistance and morphogenesis provides a promising strategy to combat life-threatening fungal infections.

The molecular chaperone Hsp90, essential in all eukaryotes, plays a multifaceted role in promoting survival, virulence, and drug resistance across diverse pathogenic fungal species. The chaperone is also critically important, however, to the pathogen's human host, preventing the use of known clinical Hsp90 inhibitors in antifungal applications due to concomitant host toxicity issues. With the goal of developing Hsp90 inhibitors with acceptable therapeutic indices for the treatment of invasive fungal infections, we initiated a program to design and synthesize potent inhibitors with selective activity against fungal Hsp90 isoforms over their human counterparts. Building on our previously reported derivatization of resorcylate natural products to produce fungal-selective compounds, we have developed a series of synthetic aminopyrazole-substituted resorcylate amides with broad, potent, and fungal-selective Hsp90 inhibitory activity. Herein we describe the synthesis of this series, as well as biochemical structure−activity relationships driving selectivity for the Hsp90 isoforms expressed by Cryptococcus neoformans and Candida albicans, two pathogenic fungi of major clinical importance.