Allosteric inhibition of tRNA synthetase Gln4 by N-pyrimidinyl-ß-thiophenylacrylamides exerts highly selective antifungal activity.

Candida species are among the most prevalent causes of systemic fungal infections, which account for ∼1.5 million annual fatalities. Here, we build on a compound screen that identified the molecule N-pyrimidinyl-ß-thiophenylacrylamide (NP-BTA), which strongly inhibits Candida albicans growth. NP-BTA was hypothesized to target C. albicans glutaminyl-tRNA synthetase, Gln4. Here, we confirmed through in vitro amino-acylation assays NP-BTA is a potent inhibitor of Gln4, and we defined how NP-BTA arrests Gln4's transferase activity using co-crystallography. This analysis also uncovered Met496 as a critical residue for the compound's species-selective target engagement and potency. Structure-activity relationship (SAR) studies demonstrated the NP-BTA scaffold is subject to oxidative and non-oxidative metabolism, making it unsuitable for systemic administration. In a mouse dermatomycosis model, however, topical application of the compound provided significant therapeutic benefit. This work expands the repertoire of antifungal protein synthesis target mechanisms and provides a path to develop Gln4 inhibitors.

A chemical screen identifies structurally diverse metal chelators with activity against the fungal pathogen Candida albicans.

Candida albicans, one of the most prevalent human fungal pathogens, causes diverse diseases extending from superficial infections to deadly systemic mycoses. Currently, only three major classes of antifungal drugs are available to treat systemic infections: azoles, polyenes, and echinocandins. Alarmingly, the efficacy of these antifungals against C. albicans is hindered both by basal tolerance toward the drugs and the development of resistance mechanisms such as alterations of the drug's target, modulation of stress responses, and overexpression of efflux pumps. Thus, the need to identify novel antifungal strategies is dire. To address this challenge, we screened 3,049 structurally-diverse compounds from the Boston University Center for Molecular Discovery (BU-CMD) chemical library against a C. albicans clinical isolate and identified 17 molecules that inhibited C. albicans growth by >80% relative to controls. Among the most potent compounds were CMLD013360, CMLD012661, and CMLD012693, molecules representing two distinct chemical scaffolds, including 3-hydroxyquinolinones and a xanthone natural product. Based on structural insights, CMLD013360, CMLD012661, and CMLD012693 were hypothesized to exert antifungal activity through metal chelation. Follow-up investigations revealed all three compounds exerted antifungal activity against non-albicans Candida, including Candida auris and Candida glabrata, with the xanthone natural product CMLD013360 also displaying activity against the pathogenic mould Aspergillus fumigatus. Media supplementation with metallonutrients, namely ferric or ferrous iron, rescued C. albicans growth, confirming these compounds act as metal chelators. Thus, this work identifies and characterizes two chemical scaffolds that chelate iron to inhibit the growth of the clinically relevant fungal pathogen C. albicansIMPORTANCEThe worldwide incidence of invasive fungal infections is increasing at an alarming rate. Systemic candidiasis caused by the opportunistic pathogen Candida albicans is the most common cause of life-threatening fungal infection. However, due to the limited number of antifungal drug classes available and the rise of antifungal resistance, an urgent need exists for the identification of novel treatments. By screening a compound collection from the Boston University Center for Molecular Discovery (BU-CMD), we identified three compounds representing two distinct chemical scaffolds that displayed activity against C. albicans. Follow-up analyses confirmed these molecules were also active against other pathogenic fungal species including Candida auris and Aspergillus fumigatus. Finally, we determined that these compounds inhibit the growth of C. albicans in culture through iron chelation. Overall, this observation describes two novel chemical scaffolds with antifungal activity against diverse fungal pathogens.

Respiration supports intraphagosomal filamentation and escape of Candida albicans from macrophages.

IMPORTANCE: Candida albicans is a leading human fungal pathogen that often causes life-threatening infections in immunocompromised individuals. The ability of C. albicans to transition between yeast and filamentous forms is key to its virulence, and this occurs in response to many host-relevant cues, including engulfment by host macrophages. While previous efforts identified C. albicans genes required for filamentation in other conditions, the genes important for this morphological transition upon internalization by macrophages remained largely enigmatic. Here, we employed a functional genomic approach to identify genes that enable C. albicans filamentation within macrophages and uncovered a role for the mitochondrial ribosome, respiration, and the SNF1 AMP-activated kinase complex. Additionally, we showed that glucose uptake and glycolysis by macrophages support C. albicans filamentation. This work provides insights into the metabolic dueling that occurs during the interaction of C. albicans with macrophages and identifies vulnerabilities in C. albicans that could serve as promising therapeutic targets.

DYRK-family kinases regulate Candida albicans morphogenesis and virulence through the Ras1/PKA pathway.

IMPORTANCE: Candida albicans is an opportunistic human fungal pathogen that frequently causes life-threatening infections in immunocompromised individuals. To cause disease, the fungus employs several virulence traits, including its ability to transition between yeast and filamentous states. Previous work identified a role for the kinase Yak1 in regulating C. albicans filamentation. Here, we demonstrate that Yak1 regulates morphogenesis through the canonical cAMP/PKA pathway and that this regulation is environmentally contingent, as host-relevant concentrations of CO2 bypass the requirement of Yak1 for C. albicans morphogenesis. We show a related kinase, Pom1, is important for filamentation in the absence of Yak1 under these host-relevant conditions, as deletion of both genes blocked filamentous growth under all conditions tested. Finally, we demonstrate that Yak1 is required for filamentation in a mouse model of C. albicans dermatitis using genetic and pharmacological approaches. Overall, our results expand our understanding of how Yak1 regulates an important virulence trait in C. albicans.

Potent Antifungal Activity of Penta-O-galloyl-ß-d-Glucose against Drug-Resistant Candida albicans, Candida auris, and Other Non-albicans Candida Species.

Among fungal pathogens, infections by drug-resistant Candida species continue to pose a major challenge to healthcare. This study aimed to evaluate the activity of the bioactive natural product, penta-O-galloyl-ß-d-glucose (PGG) against multidrug-resistant (MDR) Candida albicans, MDR Candida auris, and other MDR non-albicans Candida species. Here, we show that PGG has a minimum inhibitory concentration (MIC) of 0.25-8 µg mL-1 (0.265-8.5 µM) against three clinical strains of C. auris and a MIC of 0.25-4 µg mL-1 (0.265-4.25 µM) against a panel of other MDR Candida species. Our cytotoxicity studies found that PGG was well tolerated by human kidney, liver, and epithelial cells with an IC50 > 256 µg mL-1 (>272 µM). We also show that PGG is a high-capacity iron chelator and that deletion of key iron homeostasis genes in C. albicans rendered strains hypersensitive to PGG. In conclusion, PGG displayed potent anti-Candida activity with minimal cytotoxicity for human cells. We also found that the antifungal activity of PGG is mediated through an iron-chelating mechanism, suggesting that the compound could prove useful as a topical treatment for superficial Candida infections.

Multifactor transcriptional control of alternative oxidase induction integrates diverse environmental inputs to enable fungal virulence.

Metabolic flexibility enables fungi to invade challenging host environments. In Candida albicans, a common cause of life-threatening infections in humans, an important contributor to flexibility is alternative oxidase (Aox) activity. Dramatic induction of this activity occurs under respiratory-stress conditions, which impair the classical electron transport chain (ETC). Here, we show that deletion of the inducible AOX2 gene cripples C. albicans virulence in mice by increasing immune recognition. To investigate further, we examined transcriptional regulation of AOX2 in molecular detail under host-relevant, ETC-inhibitory conditions. We found that multiple transcription factors, including Rtg1/Rtg3, Cwt1/Zcf11, and Zcf2, bind and regulate the AOX2 promoter, conferring thousand-fold levels of inducibility to AOX2 in response to distinct environmental stressors. Further dissection of this complex promoter revealed how integration of stimuli ranging from reactive species of oxygen, nitrogen, and sulfur to reduced copper availability is achieved at the transcriptional level to regulate AOX2 induction and enable pathogenesis.

Identification of triazenyl indoles as inhibitors of fungal fatty acid biosynthesis with broad-spectrum activity.

Rising drug resistance among pathogenic fungi, paired with a limited antifungal arsenal, poses an increasing threat to human health. To identify antifungal compounds, we screened the RIKEN natural product depository against representative isolates of four major human fungal pathogens. This screen identified NPD6433, a triazenyl indole with broad-spectrum activity against all screening strains, as well as the filamentous mold Aspergillus fumigatus. Mechanistic studies indicated that NPD6433 targets the enoyl reductase domain of fatty acid synthase 1 (Fas1), covalently inhibiting its flavin mononucleotide-dependent NADPH-oxidation activity and arresting essential fatty acid biosynthesis. Robust Fas1 inhibition kills Candida albicans, while sublethal inhibition impairs diverse virulence traits. At well-tolerated exposures, NPD6433 extended the lifespan of nematodes infected with azole-resistant C. albicans. Overall, identification of NPD6433 provides a tool with which to explore lipid homeostasis as a therapeutic target in pathogenic fungi and reveals a mechanism by which Fas1 function can be inhibited.

The Trisubstituted Isoxazole MMV688766 Exerts Broad-Spectrum Activity against Drug-Resistant Fungal Pathogens through Inhibition of Lipid Homeostasis.

Candida species are among the most prevalent causes of systemic fungal infection, posing a growing threat to public health. While Candida albicans is the most common etiological agent of systemic candidiasis, the frequency of infections caused by non-albicans Candida species is rising. Among these is Candida auris, which has emerged as a particular concern. Since its initial discovery in 2009, it has been identified worldwide and exhibits resistance to all three principal antifungal classes. Here, we endeavored to identify compounds with novel bioactivity against C. auris from the Medicines for Malaria Venture's Pathogen Box library. Of the five hits identified, the trisubstituted isoxazole MMV688766 emerged as the only compound displaying potent fungicidal activity against C. auris, as well as other evolutionarily divergent fungal pathogens. Chemogenomic profiling, as well as subsequent metabolomic and phenotypic analyses, revealed that MMV688766 disrupts cellular lipid homeostasis, driving a decrease in levels of early sphingolipid intermediates and fatty acids and a concomitant increase in lysophospholipids. Experimental evolution to further probe MMV688766's mode of action in the model fungus Saccharomyces cerevisiae revealed that loss of function of the transcriptional regulator HAL9 confers resistance to MMV688766, in part through the upregulation of the lipid-binding chaperone HSP12, a response that appears to assist in tolerating MMV688766-induced stress. The novel mode of action we have uncovered for MMV688766 against drug-resistant fungal pathogens highlights the broad utility of targeting lipid homeostasis to disrupt fungal growth and how screening structurally-diverse chemical libraries can provide new insights into resistance-conferring stress responses of fungi. IMPORTANCE As widespread antimicrobial resistance threatens to propel the world into a postantibiotic era, there is a pressing need to identify mechanistically distinct antimicrobial agents. This is of particular concern when considering the limited arsenal of drugs available to treat fungal infections, coupled with the emergence of highly drug-resistant fungal pathogens, including Candida auris. In this work, we demonstrate that existing libraries of drug-like chemical matter can be rich resources for antifungal molecular scaffolds. We discovered that the small molecule MMV688766, from the Pathogen Box library, displays previously undescribed broad-spectrum fungicidal activity through perturbation of lipid homeostasis. Characterization of the mode of action of MMV688766 provided new insight into the protective mechanisms fungi use to cope with the disruption of lipid homeostasis. Our findings highlight that elucidating the genetic circuitry required to survive in the presence of cellular stress offers powerful insights into the biological pathways that govern this important phenotype.

Calcineurin Inhibitors Synergize with Manogepix to Kill Diverse Human Fungal Pathogens.

Invasive fungal infections have mortality rates of 30-90%, depending on patient co-morbidities and the causative pathogen. The frequent emergence of drug resistance reduces the efficacy of currently approved treatment options, highlighting an urgent need for antifungals with new modes of action. Addressing this need, fosmanogepix (N-phosphonooxymethylene prodrug of manogepix; MGX) is the first in a new class of gepix drugs, and acts as a broad-spectrum, orally bioavailable inhibitor of the essential fungal glycosylphosphatidylinositol (GPI) acyltransferase Gwt1. MGX inhibits the growth of diverse fungal pathogens and causes accumulation of immature GPI-anchored proteins in the fungal endoplasmic reticulum. Relevant to the ongoing clinical development of fosmanogepix, we report a synergistic, fungicidal interaction between MGX and inhibitors of the protein phosphatase calcineurin against important human fungal pathogens. To investigate this synergy further, we evaluated a library of 124 conditional expression mutants covering 95% of the genes encoding proteins involved in GPI-anchor biosynthesis or proteins predicted to be GPI-anchored. Strong negative chemical-genetic interactions between the calcineurin inhibitor FK506 and eleven GPI-anchor biosynthesis genes were identified, indicating that calcineurin signalling is required for fungal tolerance to not only MGX, but to inhibition of the GPI-anchor biosynthesis pathway more broadly. Depletion of these GPI-anchor biosynthesis genes, like MGX treatment, also exposed fungal cell wall (1→3)-ß-D-glucans. Taken together, these findings suggest the increased risk of invasive fungal infections associated with use of calcineurin inhibitors as immunosuppressants may be mitigated by their synergistic fungicidal interaction with (fos)manogepix and its ability to enhance exposure of immunostimulatory glucans.

Targeting fungal membrane homeostasis with imidazopyrazoindoles impairs azole resistance and biofilm formation.

Fungal infections cause more than 1.5 million deaths annually. With an increase in immune-deficient susceptible populations and the emergence of antifungal drug resistance, there is an urgent need for novel strategies to combat these life-threatening infections. Here, we use a combinatorial screening approach to identify an imidazopyrazoindole, NPD827, that synergizes with fluconazole against azole-sensitive and -resistant isolates of Candida albicans. NPD827 interacts with sterols, resulting in profound effects on fungal membrane homeostasis and induction of membrane-associated stress responses. The compound impairs virulence in a Caenorhabditis elegans model of candidiasis, blocks C. albicans filamentation in vitro, and prevents biofilm formation in a rat model of catheter infection by C. albicans. Collectively, this work identifies an imidazopyrazoindole scaffold with a non-protein-targeted mode of action that re-sensitizes the leading human fungal pathogen, C. albicans, to azole antifungals.

Inhibiting C-4 Methyl Sterol Oxidase with Novel Diazaborines to Target Fungal Plant Pathogens.

With resistance to current agricultural fungicides rising, a great need has emerged for new antifungals with unexploited targets. In response, we report a novel series of diazaborines with potent activity against representative fungal plant pathogens. To identify their mode of action, we selected for resistant isolates using the model fungus Saccharomyces cerevisiae. Whole-genome sequencing of independent diazaborine-resistant lineages identified a recurring mutation in ERG25, which encodes a C-4 methyl sterol oxidase required for ergosterol biosynthesis in fungi. Haploinsufficiency and allele-swap experiments provided additional genetic evidence for Erg25 as the most biologically relevant target of our diazaborines. Confirming Erg25 as putative target, sterol profiling of compound-treated yeast revealed marked accumulation of the Erg25 substrate, 4,4-dimethylzymosterol and depletion of both its immediate product, zymosterol, as well as ergosterol. Encouraged by these mechanistic insights, the potential utility of targeting Erg25 with a diazaborine was demonstrated in soybean-rust and grape-rot models of fungal plant disease.

HSF2 cooperates with HSF1 to drive a transcriptional program critical for the malignant state.

Heat shock factor 1 (HSF1) is well known for its role in the heat shock response (HSR), where it drives a transcriptional program comprising heat shock protein (HSP) genes, and in tumorigenesis, where it drives a program comprising HSPs and many noncanonical target genes that support malignancy. Here, we find that HSF2, an HSF1 paralog with no substantial role in the HSR, physically and functionally interacts with HSF1 across diverse types of cancer. HSF1 and HSF2 have notably similar chromatin occupancy and regulate a common set of genes that include both HSPs and noncanonical transcriptional targets with roles critical in supporting malignancy. Loss of either HSF1 or HSF2 results in a dysregulated response to nutrient stresses in vitro and reduced tumor progression in cancer cell line xenografts. Together, these findings establish HSF2 as a critical cofactor of HSF1 in driving a cancer cell transcriptional program to support the anabolic malignant state.

Adaptive laboratory evolution in S. cerevisiae highlights role of transcription factors in fungal xenobiotic resistance.

In vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in Saccharomyces cerevisiae. Sequence analysis identified many genes contributing to the resistance phenotype as well as numerous amino acids in potential targets that may play a role in compound binding. Our work shows that compound-target pairs can be conserved across multiple species. The set of 25 most frequently mutated genes was enriched for transcription factors, and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function SNVs found in a 170 amino acid domain in the two Zn2C6 transcription factors YRR1 and YRM1 (p < 1 × 10-100). This remarkable enrichment for transcription factors as drug resistance genes highlights their important role in the evolution of antifungal xenobiotic resistance and underscores the challenge to develop antifungal treatments that maintain potency.

Rieske head domain dynamics and indazole-derivative inhibition of Candida albicans complex III.

Electron transfer between respiratory complexes drives transmembrane proton translocation, which powers ATP synthesis and membrane transport. The homodimeric respiratory complex III (CIII2) oxidizes ubiquinol to ubiquinone, transferring electrons to cytochrome c and translocating protons through a mechanism known as the Q cycle. The Q cycle involves ubiquinol oxidation and ubiquinone reduction at two different sites within each CIII monomer, as well as movement of the head domain of the Rieske subunit. We determined structures of Candida albicans CIII2 by cryoelectron microscopy (cryo-EM), revealing endogenous ubiquinone and visualizing the continuum of Rieske head domain conformations. Analysis of these conformations does not indicate cooperativity in the Rieske head domain position or ligand binding in the two CIIIs of the CIII2 dimer. Cryo-EM with the indazole derivative Inz-5, which inhibits fungal CIII2 and is fungicidal when administered with fungistatic azole drugs, showed that Inz-5 inhibition alters the equilibrium of Rieske head domain positions.

Genetic analysis of Hsp90 function in Cryptococcus neoformans highlights key roles in stress tolerance and virulence.

The opportunistic human fungal pathogen Cryptococcus neoformans has tremendous impact on global health, causing 181,000 deaths annually. Current treatment options are limited, and the frequent development of drug resistance exacerbates the challenge of managing invasive cryptococcal infections. In diverse fungal pathogens, the essential molecular chaperone Hsp90 governs fungal survival, drug resistance, and virulence. Therefore, targeting this chaperone has emerged as a promising approach to combat fungal infections. However, the role of Hsp90 in supporting C. neoformans pathogenesis remains largely elusive due to a lack of genetic characterization. To help dissect the functions of Hsp90 in C. neoformans, we generated a conditional expression strain in which HSP90 is under control of the copper-repressible promoter CTR4-2. Addition of copper to culture medium depleted Hsp90 transcript and protein levels in this strain, resulting in compromised fungal growth at host temperature; increased sensitivity to stressors, including the azole class of antifungals; altered C. neoformans morphology; and impaired melanin production. Finally, leveraging the fact that copper concentrations vary widely in different mouse tissues, we demonstrated attenuated virulence for the CTR4-2p-HSP90 mutant specifically in an inhalation model of Cryptococcus infection. During invasion and establishment of infection in this mouse model, the pathogen is exposed to the relatively high copper concentrations found in the lung as compared to blood. Overall, this work generates a tractable genetic system to study the role of Hsp90 in supporting the pathogenicity of C. neoformans and provides proof-of-principle that targeting Hsp90 holds great promise as a strategy to control cryptococcal infection.

A small molecule produced by Lactobacillus species blocks Candida albicans filamentation by inhibiting a DYRK1-family kinase.

The fungus Candida albicans is an opportunistic pathogen that can exploit imbalances in microbiome composition to invade its human host, causing pathologies ranging from vaginal candidiasis to fungal sepsis. Bacteria of the genus Lactobacillus are colonizers of human mucosa and can produce compounds with bioactivity against C. albicans. Here, we show that some Lactobacillus species produce a small molecule under laboratory conditions that blocks the C. albicans yeast-to-filament transition, an important virulence trait. It remains unexplored whether the compound is produced in the context of the human host. Bioassay-guided fractionation of Lactobacillus-conditioned medium linked this activity to 1-acetyl-ß-carboline (1-ABC). We use genetic approaches to show that filamentation inhibition by 1-ABC requires Yak1, a DYRK1-family kinase. Additional biochemical characterization of structurally related 1-ethoxycarbonyl-ß-carboline confirms that it inhibits Yak1 and blocks C. albicans biofilm formation. Thus, our findings reveal Lactobacillus-produced 1-ABC can prevent the yeast-to-filament transition in C. albicans through inhibition of Yak1.

The macrophage-derived protein PTMA induces filamentation of the human fungal pathogen Candida albicans.

Evasion of killing by immune cells is crucial for fungal survival in the host. For the human fungal pathogen Candida albicans, internalization by macrophages induces a transition from yeast to filaments that promotes macrophage death and fungal escape. Nutrient deprivation, alkaline pH, and oxidative stress have been implicated as triggers of intraphagosomal filamentation; however, the impact of other host-derived factors remained unknown. Here, we show that lysates prepared from macrophage-like cell lines and primary macrophages robustly induce C. albicans filamentation. Enzymatic treatment of lysate implicates a phosphorylated protein, and bioactivity-guided fractionation coupled to mass spectrometry identifies the immunomodulatory phosphoprotein PTMA as a candidate trigger of C. albicans filamentation. Immunoneutralization of PTMA within lysate abolishes its activity, strongly supporting PTMA as a filament-inducing component of macrophage lysate. Adding to the known repertoire of physical factors, this work implicates a host protein in the induction of C. albicans filamentation within immune cells.

Inhibition of translation initiation factor eIF4a inactivates heat shock factor 1 (HSF1) and exerts anti-leukemia activity in AML.

Eukaryotic initiation factor 4A (eIF4A), the enzymatic core of the eIF4F complex essential for translation initiation, plays a key role in the oncogenic reprogramming of protein synthesis, and thus is a putative therapeutic target in cancer. As important component of its anticancer activity, inhibition of translation initiation can alleviate oncogenic activation of HSF1, a stress-inducible transcription factor that enables cancer cell growth and survival. Here, we show that primary acute myeloid leukemia (AML) cells exhibit the highest transcript levels of eIF4A1 compared to other cancer types. eIF4A inhibition by the potent and specific compound rohinitib (RHT) inactivated HSF1 in these cells, and exerted pronounced in vitro and in vivo anti-leukemia effects against progenitor and leukemia-initiating cells, especially those with FLT3-internal tandem duplication (ITD). In addition to its own anti-leukemic activity, genetic knockdown of HSF1 also sensitized FLT3-mutant AML cells to clinical FLT3 inhibitors, and this synergy was conserved in FLT3 double-mutant cells carrying both ITD and tyrosine kinase domain mutations. Consistently, the combination of RHT and FLT3 inhibitors was highly synergistic in primary FLT3-mutated AML cells. Our results provide a novel therapeutic rationale for co-targeting eIF4A and FLT3 to address the clinical challenge of treating FLT3-mutant AML.

Exploring boron applications in modern agriculture: A structure-activity relationship study of a novel series of multi-substitution benzoxaboroles for identification of potential fungicides.

Several boron-containing small molecules have been approved by the US FDA to treat human diseases. We explored potential applications of boron-containing compounds in modern agriculture by pursuing multiple research and development programs. Here, we report a novel series of multi-substitution benzoxaboroles (1-36), a compound class that we recently reported as targeting geranylgeranyl transferase I (GGTase I) and thereby inhibiting protein prenylation (Kim et al., 2020). These compounds were designed, synthesized, and tested against the agriculturally important fungal pathogens Mycosphaerella fijiensis and Colletotrichum sublineolum in a structure-activity relationship (SAR) study. Compounds 13, 28, 30, 34 and 36 were identified as active leads with excellent antifungal MIC95 values in the range of 1.56-3.13 ppm against M. fijiensis and 0.78-3.13 ppm against C. sublineolum.

Fungal-Selective Resorcylate Aminopyrazole Hsp90 Inhibitors: Optimization of Whole-Cell Anticryptococcal Activity and Insights into the Structural Origins of Cryptococcal Selectivity.

The essential eukaryotic chaperone Hsp90 regulates the form and function of diverse client proteins, many of which govern thermotolerance, virulence, and drug resistance in fungal species. However, use of Hsp90 inhibitors as antifungal therapeutics has been precluded by human host toxicities and suppression of immune responses. We recently described resorcylate aminopyrazoles (RAPs) as the first class of Hsp90 inhibitors capable of discriminating between fungal (Cryptococcus neoformans, Candida albicans) and human isoforms of Hsp90 in biochemical assays. Here, we report an iterative structure-property optimization toward RAPs capable of inhibiting C. neoformans growth in culture. In addition, we report the first X-ray crystal structures of C. neoformans Hsp90 nucleotide binding domain (NBD), as the apoprotein and in complexes with the non-species-selective Hsp90 inhibitor NVP-AUY922 and three RAPs revealing unique ligand-induced conformational rearrangements, which reaffirm the hypothesis that intrinsic differences in protein flexibility can confer selective inhibition of fungal versus human Hsp90 isoforms.