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.

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Candida auris is an emerging fungal pathogen that exhibits resistance to multiple drugs, including the most commonly prescribed antifungal, fluconazole. Here, we use a combinatorial screening approach to identify a bis-benzodioxolylindolinone (azoffluxin) that synergizes with fluconazole against C. auris. Azoffluxin enhances fluconazole activity through the inhibition of efflux pump Cdr1, thus increasing intracellular fluconazole levels. This activity is conserved across most C. auris clades, with the exception of clade III. Azoffluxin also inhibits efflux in highly azole-resistant strains of Candida albicans, another human fungal pathogen, increasing their susceptibility to fluconazole. Furthermore, azoffluxin enhances fluconazole activity in mice infected with C. auris, reducing fungal burden. Our findings suggest that pharmacologically targeting Cdr1 in combination with azoles may be an effective strategy to control infection caused by azole-resistant isolates of C. auris.

In an effort to expand the stereochemical and structural complexity of chemical libraries used in drug discovery, the Center for Chemical Methodology and Library Development at Boston University has established an infrastructure to translate methodologies accessing diverse chemotypes into arrayed libraries for biological evaluation. In a collaborative effort, the NIH Chemical Genomics Center determined IC 50 's for Plasmodium falciparum viability for each of 2,070 members of the CMLD-BU compound collection using quantitative high-throughput screening across five parasite lines of distinct geographic origin. Three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified, and the nascent structure activity relationships (SAR) from this experiment used to initiate optimization of these chemotypes for further development.T he synthesis of compound libraries embodying structural diversity and complexity is one potential avenue to enabling the discovery of potent and selective bioactive agents (1-3). Synthetic methodology aimed at library design and distribution is the focus of the Center for Chemical Methodology and Library Development at Boston University (CMLD-BU). Currently the CMLD-BU maintains a collection of approximately 2,500 compounds derived from reaction methodologies developed in the Center and associated groups. The CMLD-BU has developed a workflow that leverages focused methodology development (4) and identification of new scaffolds and chemical reactions utilizing multidimensional reaction screening (5) for the synthesis of libraries.In a collaboration between the CMLD-BU and the NIH Chemical Genomics Center (NCGC), an effort to discover unique chemotypes which inhibit the viability of P. falciparum, the causative agent of malaria, is underway. Malaria not only inflicts tremendous suffering and morbidity on the human population, but has dramatically influenced the course of civilization with profound sociological and economic implications (6). In response, there have been significant screening efforts for new antimalarial agents (7,8). Effective treatments continue to be needed in part due to acquired resistance of P. falciparum. Identification of molecular targets affecting the viability of malaria is necessary for efficient drug development as is the discovery of chemotypes active across a variety of geographic isolates. With an aim to improve the process of identifying new compounds and targets, we have used quantitative high-throughput screening (qHTS) to achieve pharmacological fingerprints of chemical libraries (9). This technique tests each compound as a seven point titration covering four orders of magnitude in concentration thus allowing sufficient resolving power by virtue of IC 50 determinations to reveal subtle differences in compound activities between parasite lines. A compound with a differential chemical phenotype (DCP) can enable mapping of target genes using genetic recombination wherein the DCP emerges between two or more...

What We Already Know about This Topic What This Article Tells Us That Is New Background Many general anesthetics were discovered empirically, but primary screens to find new sedative-hypnotics in drug libraries have not used animals, limiting the types of drugs discovered. The authors hypothesized that a sedative-hypnotic screening approach using zebrafish larvae responses to sensory stimuli would perform comparably to standard assays, and efficiently identify new active compounds. Methods The authors developed a binary outcome photomotor response assay for zebrafish larvae using a computerized system that tracked individual motions of up to 96 animals simultaneously. The assay was validated against tadpole loss of righting reflexes, using sedative-hypnotics of widely varying potencies that affect various molecular targets. A total of 374 representative compounds from a larger library were screened in zebrafish larvae for hypnotic activity at 10 µM. Molecular mechanisms of hits were explored in anesthetic-sensitive ion channels using electrophysiology, or in zebrafish using a specific reversal agent. Results Zebrafish larvae assays required far less drug, time, and effort than tadpoles. In validation experiments, zebrafish and tadpole screening for hypnotic activity agreed 100% (n = 11; P = 0.002), and potencies were very similar (Pearson correlation, r > 0.999). Two reversible and potent sedative-hypnotics were discovered in the library subset. CMLD003237 (EC50, ~11 µM) weakly modulated γ-aminobutyric acid type A receptors and inhibited neuronal nicotinic receptors. CMLD006025 (EC50, ~13 µM) inhibited both N-methyl-d-aspartate and neuronal nicotinic receptors. Conclusions Photomotor response assays in zebrafish larvae are a mechanism-independent platform for high-throughput screening to identify novel sedative-hypnotics. The variety of chemotypes producing hypnosis is likely much larger than currently known.