Enniatin A Analogues as Novel Hsp90 Inhibitors that Modulate Triple-Negative Breast Cancer.

The 90 kilo-Dalton heat shock protein (Hsp90) is a molecular chaperone that facilitates the maturation of nascent polypeptides into their biologically active conformation. Because many of the >400 known client protein substrates are implicated in the development/progression of cancer, it is hypothesized that Hsp90 inhibition will simultaneously shut down numerous oncogenic pathways. Unfortunately, most of the small molecule Hsp90 inhibitors that have undergone clinical evaluation thus far have failed due to various toxicities. Therefore, the disruption of Hsp90 protein-protein interactions with cochaperones and/or client substrates has been proposed as an alternative way to achieve Hsp90 inhibition without such adverse events. The hexadepsipeptide Enniatin A (EnnA) has recently been reported to be one such inhibitor that also manifests immunogenic activity. Herein, we report preliminary structure-activity relationship (SAR) studies to determine the structural features that confer this unprecedented activity for an Hsp90 inhibitor. Our studies find that EnnA's branching moieties are necessary for its activity, but some structural modifications are tolerated.

Enniatin A inhibits the chaperone Hsp90 and unleashes the immune system against triple-negative breast cancer.

Low response rates and immune-related adverse events limit the remarkable impact of cancer immunotherapy. To improve clinical outcomes, preclinical studies have shown that combining immunotherapies with N-terminal Hsp90 inhibitors resulted in improved efficacy, even though induction of an extensive heat shock response (HSR) and less than optimal dosing of these inhibitors limited their clinical efficacy as monotherapies. We discovered that the natural product Enniatin A (EnnA) targets Hsp90 and destabilizes its client oncoproteins without inducing an HSR. EnnA triggers immunogenic cell death in triple-negative breast cancer (TNBC) syngeneic mouse models and exhibits superior antitumor activity compared to Hsp90 N-terminal inhibitors. EnnA reprograms the tumor microenvironment (TME) to promote CD8+ T cell-dependent antitumor immunity by reducing PD-L1 levels and activating the chemokine receptor CX3CR1 pathway. These findings provide strong evidence for transforming the immunosuppressive TME into a more tumor-hostile milieu by engaging Hsp90 with therapeutic agents involving novel mechanisms of action.

Elucidation of novel TRAP1-Selective inhibitors that regulate mitochondrial processes.

Hsp90 isoform-selective inhibitors represent a new paradigm for novel anti-cancer drugs as each of the four isoforms have specific cellular localization, function, and client proteins. The mitochondrial isoform, TRAP1, is the least understood member of the Hsp90 family due to the lack of small molecule tools to study its biological function. Herein, we report novel TRAP1-selective inhibitors used to interrogate TRAP1's biological function along with co-crystal structures of such compounds bound to the N-terminus of TRAP1. Solution of the co-crystal structure allowed for a structure-based approach that resulted in compound 36, which is a 40 nM inhibitor with >250-fold TRAP1 selectivity over Grp94, the isoform with the highest structural similarity to TRAP1 within the N-terminal ATP binding site. Lead compounds 35 and 36 were found to selectively induce TRAP1 client protein degradation without inducing the heat shock response or disrupting Hsp90-cytosolic clients. They were also shown to inhibit OXPHOS, alter cellular metabolism towards glycolysis, disrupt TRAP1 tetramer stability, and disrupt the mitochondrial membrane potential.

A proteome-wide atlas of lysine-reactive chemistry.

Recent advances in chemical proteomics have begun to characterize the reactivity and ligandability of lysines on a global scale. Yet, only a limited diversity of aminophilic electrophiles have been evaluated for interactions with the lysine proteome. Here, we report an in-depth profiling of >30 uncharted aminophilic chemotypes that greatly expands the content of ligandable lysines in human proteins. Aminophilic electrophiles showed disparate proteomic reactivities that range from selective interactions with a handful of lysines to, for a set of dicarboxaldehyde fragments, remarkably broad engagement of the covalent small-molecule-lysine interactions captured by the entire library. We used these latter 'scout' electrophiles to efficiently map ligandable lysines in primary human immune cells under stimulatory conditions. Finally, we show that aminophilic compounds perturb diverse biochemical functions through site-selective modification of lysines in proteins, including protein-RNA interactions implicated in innate immune responses. These findings support the broad potential of covalent chemistry for targeting functional lysines in the human proteome.

Functionalized Scout Fragments for Site-Specific Covalent Ligand Discovery and Optimization.

Covalent ligands are a versatile class of chemical probes and drugs that can target noncanonical sites on proteins and display differentiated pharmacodynamic properties. Chemical proteomic methods have been introduced that leverage electrophilic fragments to globally profile the covalent ligandability of nucleophilic residues, such as cysteine and lysine, in native biological systems. Further optimization of these initial ligandability events without resorting to the time-consuming process of individualized protein purification and functional assay development, however, presents a persistent technical challenge. Here, we show that broadly reactive electrophilic fragments, or "scouts", can be converted into site-specific target engagement probes for screening small molecules against a wide array of proteins in convenient gel- and ELISA-based assay formats. We use these assays to expediently optimize a weak potency fragment hit into a sub-µM inhibitor that selectively engages an active-site cysteine in the retinaldehyde reductase AKR1B10. Our findings provide a road map to optimize covalent fragments into more advanced chemical probes without requiring protein purification or structural analysis.

DCAF11 Supports Targeted Protein Degradation by Electrophilic Proteolysis-Targeting Chimeras.

Ligand-induced protein degradation has emerged as a compelling approach to promote the targeted elimination of proteins from cells by directing these proteins to the ubiquitin-proteasome machinery. So far, only a limited number of E3 ligases have been found to support ligand-induced protein degradation, reflecting a dearth of E3-binding compounds for proteolysis-targeting chimera (PROTAC) design. Here, we describe a functional screening strategy performed with a focused library of candidate electrophilic PROTACs to discover bifunctional compounds that degrade proteins in human cells by covalently engaging E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs act through modifying specific cysteines in DCAF11, a poorly characterized E3 ligase substrate adaptor. We further show that DCAF11-directed electrophilic PROTACs can degrade multiple endogenous proteins, including FBKP12 and the androgen receptor, in human prostate cancer cells. Our findings designate DCAF11 as an E3 ligase capable of supporting ligand-induced protein degradation via electrophilic PROTACs.

Chemical proteomic identification of functional cysteines with atypical electrophile reactivities.

Cysteine-directed covalent ligands have emerged as a versatile category of chemical probes and drugs that leverage thiol nucleophilicity to form permanent adducts with proteins of interest. Understanding the scope of cysteines that can be targeted by covalent ligands, as well as the types of electrophiles that engage these residues, represent important challenges for fully realizing the potential of cysteine-directed chemical probe discovery. Although chemical proteomic strategies have begun to address these important questions, only a limited number of electrophilic chemotypes have been explored to date. Here, we describe a diverse set of candidate electrophiles appended to a common core 6-methoxy-1,2,3,4-tetrahydroquinoline fragment and evaluate their global cysteine reactivity profiles in human cancer cell proteomes. This work uncovered atypical reactivity patterns for a discrete set of cysteines, including residues involved in enzymatic catalysis and located in proximity to protein-protein interactions. These findings thus point to potentially preferred electrophilic groups for site-selectively targeting functional cysteines in the human proteome.

An Activity-Guided Map of Electrophile-Cysteine Interactions in Primary Human T Cells.

Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3,000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving the direct functional perturbation and/or degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells and point to electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

The Proteome-Wide Potential for Reversible Covalency at Cysteine.

Reversible covalency, achieved with, for instance, highly electron-deficient olefins, offers a compelling strategy to design chemical probes and drugs that benefit from the sustained target engagement afforded by irreversible compounds, while avoiding permanent protein modification. Reversible covalency has mainly been evaluated for cysteine residues in individual kinases and the broader potential for this strategy to engage cysteines across the proteome remains unexplored. Herein, we describe a mass-spectrometry-based platform that integrates gel filtration with activity-based protein profiling to assess cysteine residues across the human proteome for both irreversible and reversible interactions with small-molecule electrophiles. Using this method, we identify numerous cysteine residues from diverse protein classes that are reversibly engaged by cyanoacrylamide fragment electrophiles, revealing the broad potential for reversible covalency as a strategy for chemical-probe discovery.

Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16.

Ligand-dependent protein degradation has emerged as a compelling strategy to pharmacologically control the protein content of cells. So far, however, only a limited number of E3 ligases have been found to support this process. Here, we use a chemical proteomic strategy that leverages broadly reactive, cysteine-directed electrophilic fragments coupled to selective ligands for intracellular proteins (for example, SLF for FKBP12, JQ1 for BRD4) to screen for heterobifunctional degrader compounds (or proteolysis targeting chimeras, PROTACs) that operate by covalent adduction of E3 ligases. This approach identified DCAF16-a poorly characterized substrate recognition component of CUL4-DDB1 E3 ubiquitin ligases-as a target of electrophilic PROTACs that promote the nuclear-restricted degradation of proteins. We find that only a modest fraction (~10-40%) of DCAF16 needs to be modified to support protein degradation, pointing to the potential for electrophilic PROTACs to induce neosubstrate degradation without substantially perturbing the function of the participating E3 ligase.

Structure Based Design of a Grp94-Selective Inhibitor: Exploiting a Key Residue in Grp94 To Optimize Paralog-Selective Binding.

Grp94 and Hsp90, the ER and cytoplasmic hsp90 paralogs, share a conserved ATP-binding pocket that has been targeted for therapeutics. Paralog-selective inhibitors may lead to drugs with fewer side effects. Here, we analyzed 1 (BnIm), a benzyl imidazole resorcinylic inhibitor, for its mode of binding. The structures of 1 bound to Hsp90 and Grp94 reveal large conformational changes in Grp94 but not Hsp90 that expose site 2, a binding pocket adjacent to the central ATP cavity that is ordinarily blocked. The Grp94:1 structure reveals a flipped pose of the resorcinylic scaffold that inserts into the exposed site 2. We exploited this flipped binding pose to develop a Grp94-selective derivative of 1. Our structural analysis shows that the ability of the ligand to insert its benzyl imidazole substituent into site 1, a different side pocket off the ATP binding cavity, is the key to exposing site 2 in Grp94.

Trifunctional High-Throughput Screen Identifies Promising Scaffold To Inhibit Grp94 and Treat Myocilin-Associated Glaucoma.

Gain-of-function mutations within the olfactomedin (OLF) domain of myocilin result in its toxic intracellular accumulation and hasten the onset of open-angle glaucoma. The absence of myocilin does not cause disease; therefore, strategies aimed at eliminating myocilin could lead to a successful glaucoma treatment. The endoplasmic reticulum Hsp90 paralog Grp94 accelerates OLF aggregation. Knockdown or pharmacological inhibition of Grp94 in cells facilitates clearance of mutant myocilin via a non-proteasomal pathway. Here, we expanded our support for targeting Grp94 over cytosolic paralogs Hsp90α and Hsp90ß. We then developed a high-throughput screening assay to identify new chemical matter capable of disrupting the Grp94/OLF interaction. When applied to a blind, focused library of 17 Hsp90 inhibitors, our miniaturized single-read in vitro thioflavin T -based kinetics aggregation assay exclusively identified compounds that target the chaperone N-terminal nucleotide binding site. In follow up studies, one compound (2) decreased the extent of co-aggregation of Grp94 with OLF in a dose-dependent manner in vitro, and enabled clearance of the aggregation-prone full-length myocilin variant I477N in cells without inducing the heat shock response or causing cytotoxicity. Comparison of the co-crystal structure of compound 2 and another non-selective hit in complex with the N-terminal domain of Grp94 reveals a docking mode tailored to Grp94 and explains its selectivity. A new lead compound has been identified, supporting a targeted chemical biology assay approach to develop a protein degradation-based therapy for myocilin-associated glaucoma by selectively inhibiting Grp94.

Isoform-selective Hsp90 inhibition rescues model of hereditary open-angle glaucoma.

The heat shock protein 90 (Hsp90) family of molecular chaperones regulates protein homeostasis, folding, and degradation. The ER-resident Hsp90 isoform, glucose-regulated protein 94 (Grp94), promotes the aggregation of mutant forms of myocilin, a protein associated with primary open-angle glaucoma. While inhibition of Grp94 promotes the degradation of mutant myocilin in vitro, to date no Grp94-selective inhibitors have been investigated in vivo. Here, a Grp94-selective inhibitor facilitated mutant myocilin degradation and rescued phenotypes in a transgenic mouse model of hereditary primary open-angle glaucoma. Ocular toxicities previously associated with pan-Hsp90 inhibitors were not evident with our Grp94-selective inhibitor, 4-Br-BnIm. Our study suggests that selective inhibition of a distinct Hsp90 family member holds translational promise for ocular and other diseases associated with cell stress and protein misfolding.

Resorcinol-Based Grp94-Selective Inhibitors.

Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum resident of the 90 kDa heat shock protein (Hsp90) family and represents a promising therapeutic target for the treatment of several diseases. Grp94 is the most unique member of the 90 kDa heat shock protein family due to a five amino acid insertion into its primary sequence, which creates hydrophobic subpockets exclusive to Grp94 that can be utilized for selective inhibition. The first resorcinol-based Grp94-selective inhibitor to take advantage of the hydrophobic S2 subpocket has been developed and shown to manifest low nanomolar affinity and ∼10-fold selectivity for Grp94. Furthermore, these Grp94-selective inhibitors manifest low micromolar GI50 values against multiple myeloma cells, supporting Grp94 as an emerging target for the treatment of this disease.

Second Generation Grp94-Selective Inhibitors Provide Opportunities for the Inhibition of Metastatic Cancer.

Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum (ER) resident isoform of the 90 kDa heat shock protein (Hsp90) family and its inhibition represents a promising therapeutic target for the treatment of many diseases. Modification of the first generation cis-amide bioisostere imidazole to alter the angle between the resorcinol ring and the benzyl side chain via cis-amide replacements produced compounds with improved Grp94 affinity and selectivity. Structure-activity relationship studies led to the discovery of compound 30, which exhibits 540 nm affinity and 73-fold selectivity towards Grp94. Grp94 is responsible for the maturation and trafficking of proteins associated with cell signaling and motility, including select integrins. The Grp94-selective inhibitor 30 was shown to exhibit potent anti-migratory effects against multiple aggressive and metastatic cancers.

Development of Glucose Regulated Protein 94-Selective Inhibitors Based on the BnIm and Radamide Scaffold.

Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum resident of the heat shock protein 90 kDa (Hsp90) family of molecular chaperones. Grp94 associates with many proteins involved in cell adhesion and signaling, including integrins, Toll-like receptors, immunoglobulins, and mutant myocilin. Grp94 has been implicated as a target for several therapeutic areas including glaucoma, cancer metastasis, and multiple myeloma. While 85% identical to other Hsp90 isoforms, the N-terminal ATP-binding site of Grp94 possesses a unique hydrophobic pocket that was used to design isoform-selective inhibitors. Incorporation of a cis-amide bioisostere into the radamide scaffold led to development of the original Grp94-selective inhibitor, BnIm. Structure-activity relationship studies have now been performed on the aryl side chain of BnIm, which resulted in improved analogues that exhibit better potency and selectivity for Grp94. These analogues also manifest superior antimigratory activity in a metastasis model as well as enhanced mutant myocilin degradation in a glaucoma model compared to BnIm.

Natural Product Inspired N-Terminal Hsp90 Inhibitors: From Bench to Bedside?

The 90 kDa heat shock proteins (Hsp90) are responsible for the conformational maturation of nascent polypeptides and the rematuration of denatured proteins. Proteins dependent upon Hsp90 are associated with all six hallmarks of cancer. Upon Hsp90 inhibition, protein substrates are degraded via the ubiquitin-proteasome pathway. Consequentially, inhibition of Hsp90 offers a therapeutic opportunity for the treatment of cancer. Natural product inhibitors of Hsp90 have been identified in vitro, which have served as leads for the development of more efficacious inhibitors and analogs that have entered clinical trials. This review highlights the development of natural product analogs, as well as the development of clinically important inhibitors that arose from natural products.

Exploiting the interaction between Grp94 and aggregated myocilin to treat glaucoma.

Gain-of-function mutations in the olfactomedin domain of the MYOC gene facilitate the toxic accumulation of amyloid-containing myocilin aggregates, hastening the onset of the prevalent ocular disorder primary open-angle glaucoma. Aggregation of wild-type myocilin has been reported in other glaucoma subtypes, suggesting broader relevance of misfolded myocilin across the disease spectrum, but the absence of myocilin does not cause disease. Thus, strategies aimed at eliminating myocilin could be therapeutically relevant for glaucoma. Here, a novel and selective Grp94 inhibitor reduced the levels of several mutant myocilin proteins as well as wild-type myocilin when forced to misfold in cells. This inhibitor rescued mutant myocilin toxicity in primary human trabecular meshwork cells. Mechanistically, in vitro kinetics studies demonstrate that Grp94 recognizes on-pathway aggregates of the myocilin olfactomedin domain (myoc-OLF), accelerates rates of aggregation and co-precipitates with myoc-OLF. These results indicate that aberrant myocilin quaternary structure drives Grp94 recognition, rather than peptide motifs exposed by unfolded protein. Inhibition of Grp94 ameliorates the effects of Grp94-accelerated myoc-OLF aggregation, and Grp94 remains in solution. In cells, when wild-type myocilin is driven to misfold and aggregate, it becomes a client of Grp94 and sensitive to Grp94 inhibition. Taken together, the interaction of Grp94 with myocilin aggregates can be manipulated by cellular environment and genetics; this process can be exploited with Grp94 inhibitors to promote the clearance of toxic forms of myocilin.

An Efficient Synthesis of 4(5)-Benzyl-L-Histidines Employing Catalytic Transfer Hydrogenolysis at Elevated Temperatures.

An efficient two-step synthesis of 4(5)-benzyl-L-histidine from L-histidine was developed. A Pictet-Spengler reaction between L-histidine and benzaldehyde in the presence of excess strong base yielded 4-phenylspinacine within one hour. Catalytic transfer hydrogenolysis in methanol at reflux using ammonium formate rapidly converted 4-L-phenylspinacine to 4(5)-benzyl-L-histidine within five minutes. No racemization of the final product 4(5)-benzyl-L-histidine was observed using the Marfey reagent. To show the utility of this methodology, a series of fluorinated benzylhistidines is presented.