HSP90 Shapes the Consequences of Human Genetic Variation.

HSP90 acts as a protein-folding buffer that shapes the manifestations of genetic variation in model organisms. Whether HSP90 influences the consequences of mutations in humans, potentially modifying the clinical course of genetic diseases, remains unknown. By mining data for >1,500 disease-causing mutants, we found a strong correlation between reduced phenotypic severity and a dominant (HSP90 ≥ HSP70) increase in mutant engagement by HSP90. Examining the cancer predisposition syndrome Fanconi anemia in depth revealed that mutant FANCA proteins engaged predominantly by HSP70 had severely compromised function. In contrast, the function of less severe mutants was preserved by a dominant increase in HSP90 binding. Reducing HSP90's buffering capacity with inhibitors or febrile temperatures destabilized HSP90-buffered mutants, exacerbating FA-related chemosensitivities. Strikingly, a compensatory FANCA somatic mutation from an "experiment of nature" in monozygotic twins both prevented anemia and reduced HSP90 binding. These findings provide one plausible mechanism for the variable expressivity and environmental sensitivity of genetic diseases.

Widespread macromolecular interaction perturbations in human genetic disorders.

How disease-associated mutations impair protein activities in the context of biological networks remains mostly undetermined. Although a few renowned alleles are well characterized, functional information is missing for over 100,000 disease-associated variants. Here we functionally profile several thousand missense mutations across a spectrum of Mendelian disorders using various interaction assays. The majority of disease-associated alleles exhibit wild-type chaperone binding profiles, suggesting they preserve protein folding or stability. While common variants from healthy individuals rarely affect interactions, two-thirds of disease-associated alleles perturb protein-protein interactions, with half corresponding to "edgetic" alleles affecting only a subset of interactions while leaving most other interactions unperturbed. With transcription factors, many alleles that leave protein-protein interactions intact affect DNA binding. Different mutations in the same gene leading to different interaction profiles often result in distinct disease phenotypes. Thus disease-associated alleles that perturb distinct protein activities rather than grossly affecting folding and stability are relatively widespread.

The reprogramming of tumor stroma by HSF1 is a potent enabler of malignancy.

Stromal cells within the tumor microenvironment are essential for tumor progression and metastasis. Surprisingly little is known about the factors that drive the transcriptional reprogramming of stromal cells within tumors. We report that the transcriptional regulator heat shock factor 1 (HSF1) is frequently activated in cancer-associated fibroblasts (CAFs), where it is a potent enabler of malignancy. HSF1 drives a transcriptional program in CAFs that complements, yet is completely different from, the program it drives in adjacent cancer cells. This CAF program is uniquely structured to support malignancy in a non-cell-autonomous way. Two central stromal signaling molecules-TGF-ß and SDF1-play a critical role. In early-stage breast and lung cancer, high stromal HSF1 activation is strongly associated with poor patient outcome. Thus, tumors co-opt the ancient survival functions of HSF1 to orchestrate malignancy in both cell-autonomous and non-cell-autonomous ways, with far-reaching therapeutic implications.

HSF1 drives a transcriptional program distinct from heat shock to support highly malignant human cancers.

Heat-Shock Factor 1 (HSF1), master regulator of the heat-shock response, facilitates malignant transformation, cancer cell survival, and proliferation in model systems. The common assumption is that these effects are mediated through regulation of heat-shock protein (HSP) expression. However, the transcriptional network that HSF1 coordinates directly in malignancy and its relationship to the heat-shock response have never been defined. By comparing cells with high and low malignant potential alongside their nontransformed counterparts, we identify an HSF1-regulated transcriptional program specific to highly malignant cells and distinct from heat shock. Cancer-specific genes in this program support oncogenic processes: cell-cycle regulation, signaling, metabolism, adhesion and translation. HSP genes are integral to this program, however, many are uniquely regulated in malignancy. This HSF1 cancer program is active in breast, colon and lung tumors isolated directly from human patients and is strongly associated with metastasis and death. Thus, HSF1 rewires the transcriptome in tumorigenesis, with prognostic and therapeutic implications.

Author Correction: Mitochondrial metabolism promotes adaptation to proteotoxic stress.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mitochondrial metabolism promotes adaptation to proteotoxic stress.

The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but are key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome inhibitor sensitivity. When cells were forced to use oxidative phosphorylation rather than glycolysis, they became proteasome-inhibitor resistant. This mitochondrial state, however, creates a unique vulnerability: sensitivity to the small molecule compound elesclomol. Genome-wide CRISPR-Cas9 screening showed that a single gene, encoding the mitochondrial reductase FDX1, could rescue elesclomol-induced cell death. Enzymatic function and nuclear-magnetic-resonance-based analyses further showed that FDX1 is the direct target of elesclomol, which promotes a unique form of copper-dependent cell death. These studies explain a fundamental mechanism by which cells adapt to proteotoxic stress and suggest strategies to mitigate proteasome inhibitor resistance.

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.

Enhanced Efflux Pump Expression in Candida Mutants Results in Decreased Manogepix Susceptibility.

Manogepix is a broad-spectrum antifungal agent that inhibits glycosylphosphatidylinositol (GPI) anchor biosynthesis. Using whole-genome sequencing, we characterized two efflux-mediated mechanisms in the fungal pathogens Candida albicans and Candida parapsilosis that resulted in decreased manogepix susceptibility. In C. albicans, a gain-of-function mutation in the transcription factor gene ZCF29 activated expression of ATP-binding cassette transporter genes CDR11 and SNQ2 In C. parapsilosis, a mitochondrial deletion activated expression of the major facilitator superfamily transporter gene MDR1.

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.

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.

FKBP12 contributes to α-synuclein toxicity by regulating the calcineurin-dependent phosphoproteome.

Calcineurin is an essential Ca2+-dependent phosphatase. Increased calcineurin activity is associated with α-synuclein (α-syn) toxicity, a protein implicated in Parkinson's Disease (PD) and other neurodegenerative diseases. Calcineurin can be inhibited with Tacrolimus through the recruitment and inhibition of the 12-kDa cis-trans proline isomerase FK506-binding protein (FKBP12). Whether calcineurin/FKBP12 represents a native physiologically relevant assembly that occurs in the absence of pharmacological perturbation has remained elusive. We leveraged α-syn as a model to interrogate whether FKBP12 plays a role in regulating calcineurin activity in the absence of Tacrolimus. We show that FKBP12 profoundly affects the calcineurin-dependent phosphoproteome, promoting the dephosphorylation of a subset of proteins that contributes to α-syn toxicity. Using a rat model of PD, partial elimination of the functional interaction between FKBP12 and calcineurin, with low doses of the Food and Drug Administration (FDA)-approved compound Tacrolimus, blocks calcineurin's activity toward those proteins and protects against the toxic hallmarks of α-syn pathology. Thus, FKBP12 can endogenously regulate calcineurin activity with therapeutic implications for the treatment of PD.

Suppression of 19S proteasome subunits marks emergence of an altered cell state in diverse cancers.

The use of proteasome inhibitors to target cancer's dependence on altered protein homeostasis has been greatly limited by intrinsic and acquired resistance. Analyzing data from thousands of cancer lines and tumors, we find that those with suppressed expression of one or more 19S proteasome subunits show intrinsic proteasome inhibitor resistance. Moreover, such proteasome subunit suppression is associated with poor outcome in myeloma patients, where proteasome inhibitors are a mainstay of treatment. Beyond conferring resistance to proteasome inhibitors, proteasome subunit suppression also serves as a sentinel of a more global remodeling of the transcriptome. This remodeling produces a distinct gene signature and new vulnerabilities to the proapoptotic drug, ABT-263. This frequent, naturally arising imbalance in 19S regulatory complex composition is achieved through a variety of mechanisms, including DNA methylation, and marks the emergence of a heritably altered and therapeutically relevant state in diverse cancers.

Withaferin A and Withanolide D Analogues with Dual Heat-Shock-Inducing and Cytotoxic Activities: Semisynthesis and Biological Evaluation.

Withanolides constitute a valuable class of bioactive natural products because some members of the class are known to exhibit cytotoxic activity and also induce a cytoprotective heat-shock response. In order to understand the relationship between their structures and these dual bioactivities of the withanolide scaffold, we obtained 25 analogues of withaferin A (WA) and withanolide D (WD) including 17 new compounds by semisynthesis involving chemical and microbial transformations. Hitherto unknown 16ß-hydroxy analogues of WA and WD were prepared by their reaction with triphenylphosphine/iodine, providing unexpected 5ß-hydroxy-6α-iodo analogues (iodohydrins) followed by microbial biotransformation with Cunninghamella echinulata and base-catalyzed cyclization of the resulting 16ß-hydroxy iodohydrins. Evaluation of these 25 withanolide analogues for their cytotoxicity and heat-shock-inducing activity (HSA) confirmed the known structure-activity relationships for WA-type withanolides and revealed that WD analogues were less active in both assays compared to their corresponding WA analogues. The 5ß,6ß-epoxide moiety of withanolides contributed to their cytotoxicity but not HSA. Introduction of a 16ß-OAc group to 4,27-di- O-acetyl-WA enhanced cytotoxicity and decreased HSA, whereas introduction of the same group to 4- O-acetyl-WD decreased both activities.

Nontoxic antimicrobials that evade drug resistance.

Drugs that act more promiscuously provide fewer routes for the emergence of resistant mutants. This benefit, however, often comes at the cost of serious off-target and dose-limiting toxicities. The classic example is the antifungal amphotericin B (AmB), which has evaded resistance for more than half a century. We report markedly less toxic amphotericins that nevertheless evade resistance. They are scalably accessed in just three steps from the natural product, and they bind their target (the fungal sterol ergosterol) with far greater selectivity than AmB. Hence, they are less toxic and far more effective in a mouse model of systemic candidiasis. To our surprise, exhaustive efforts to select for mutants resistant to these more selective compounds revealed that they are just as impervious to resistance as AmB. Thus, highly selective cytocidal action and the evasion of resistance are not mutually exclusive, suggesting practical routes to the discovery of less toxic, resistance-evasive therapies.

HSP90 empowers evolution of resistance to hormonal therapy in human breast cancer models.

The efficacy of hormonal therapies for advanced estrogen receptor-positive breast cancers is limited by the nearly inevitable development of acquired resistance. Efforts to block the emergence of resistance have met with limited success, largely because the mechanisms underlying it are so varied and complex. Here, we investigate a new strategy aimed at the very processes by which cancers evolve resistance. From yeast to vertebrates, heat shock protein 90 (HSP90) plays a unique role among molecular chaperones by promoting the evolution of heritable new traits. It does so by regulating the folding of a diverse portfolio of metastable client proteins, many of which mediate adaptive responses that allow organisms to adapt and thrive in the face of diverse challenges, including those posed by drugs. Guided by our previous work in pathogenic fungi, in which very modest HSP90 inhibition impairs resistance to mechanistically diverse antifungals, we examined the effect of similarly modest HSP90 inhibition on the emergence of resistance to antiestrogens in breast cancer models. Even though this degree of inhibition fell below the threshold for proteotoxic activation of the heat-shock response and had no overt anticancer activity on its own, it dramatically impaired the emergence of resistance to hormone antagonists both in cell culture and in mice. Our findings strongly support the clinical testing of combined hormone antagonist-low-level HSP90 inhibitor regimens in the treatment of metastatic estrogen receptor-positive breast cancer. At a broader level, they also provide promising proof of principle for a generalizable strategy to combat the pervasive problem of rapidly emerging resistance to molecularly targeted therapeutics.

Calcineurin determines toxic versus beneficial responses to α-synuclein.

Calcineurin (CN) is a highly conserved Ca(2+)-calmodulin (CaM)-dependent phosphatase that senses Ca(2+) concentrations and transduces that information into cellular responses. Ca(2+) homeostasis is disrupted by α-synuclein (α-syn), a small lipid binding protein whose misfolding and accumulation is a pathological hallmark of several neurodegenerative diseases. We report that α-syn, from yeast to neurons, leads to sustained highly elevated levels of cytoplasmic Ca(2+), thereby activating a CaM-CN cascade that engages substrates that result in toxicity. Surprisingly, complete inhibition of CN also results in toxicity. Limiting the availability of CaM shifts CN's spectrum of substrates toward protective pathways. Modulating CN or CN's substrates with highly selective genetic and pharmacological tools (FK506) does the same. FK506 crosses the blood brain barrier, is well tolerated in humans, and is active in neurons and glia. Thus, a tunable response to CN, which has been conserved for a billion years, can be targeted to rebalance the phosphatase's activities from toxic toward beneficial substrates. These findings have immediate therapeutic implications for synucleinopathies.

Synthesis of Aza-Rocaglates via ESIPT-Mediated (3+2) Photocycloaddition.

Synthesis of aza-rocaglates, nitrogen-containing analogues of the rocaglate natural products, is reported. The route features ESIPT-mediated (3+2) photocycloaddition of 1-alkyl-2-aryl-3-hydroxyquinolinones with the dipolarophile methyl cinnamate. A continuous photoflow reactor was utilized for photocycloadditions. An array of compounds bearing the hexahydrocyclopenta[b]indole core structure was synthesized and evaluated in translation inhibition assays.

Fitness trade-offs restrict the evolution of resistance to amphotericin B.

The evolution of drug resistance in microbial pathogens provides a paradigm for investigating evolutionary dynamics with important consequences for human health. Candida albicans, the leading fungal pathogen of humans, rapidly evolves resistance to two major antifungal classes, the triazoles and echinocandins. In contrast, resistance to the third major antifungal used in the clinic, amphotericin B (AmB), remains extremely rare despite 50 years of use as monotherapy. We sought to understand this long-standing evolutionary puzzle. We used whole genome sequencing of rare AmB-resistant clinical isolates as well as laboratory-evolved strains to identify and investigate mutations that confer AmB resistance in vitro. Resistance to AmB came at a great cost. Mutations that conferred resistance simultaneously created diverse stresses that required high levels of the molecular chaperone Hsp90 for survival, even in the absence of AmB. This requirement stemmed from severe internal stresses caused by the mutations, which drastically diminished tolerance to external stresses from the host. AmB-resistant mutants were hypersensitive to oxidative stress, febrile temperatures, and killing by neutrophils and also had defects in filamentation and tissue invasion. These strains were avirulent in a mouse infection model. Thus, the costs of evolving resistance to AmB limit the emergence of this phenotype in the clinic. Our work provides a vivid example of the ways in which conflicting selective pressures shape evolutionary trajectories and illustrates another mechanism by which the Hsp90 buffer potentiates the emergence of new phenotypes. Developing antibiotics that deliberately create such evolutionary constraints might offer a strategy for limiting the rapid emergence of drug resistance.

Biomimetic kinetic resolution: highly enantio- and diastereoselective transfer hydrogenation of aglain ketones to access flavagline natural products.

We have previously reported asymmetric syntheses and absolute configuration assignments of the aglains (+)-ponapensin and (+)-elliptifoline and proposed a biosynthetic kinetic resolution process to produce enantiomeric rocaglamides and aglains. Herein, we report a biomimetic approach for the synthesis of enantiomerically enriched aglains and rocaglamides via kinetic resolution of a bridged ketone utilizing enantioselective transfer hydrogenation. The methodology has been employed to synthesize and confirm the absolute stereochemistries of the pyrimidone rocaglamides (+)-aglaiastatin and (-)-aglaroxin C. Additionally, the enantiomers and racemate of each metabolite were assayed for inhibition of the heat-shock response, cytotoxicity, and translation inhibition.

HSP90 and the chaperoning of cancer.

Standing watch over the proteome, molecular chaperones are an ancient and evolutionarily conserved class of proteins that guide the normal folding, intracellular disposition and proteolytic turnover of many of the key regulators of cell growth, differentiation and survival. This essential guardian function is subverted during oncogenesis to allow malignant transformation and to facilitate rapid somatic evolution. Pharmacologically 'bribing' the essential guard duty of the chaperone HSP90 (heat-shock protein of 90 kDa) seems to offer a unique anticancer strategy of considerable promise.