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Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer’s disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems., Here, the authors show structural, biochemical, and functional insights into the discovery of epichaperome‐ directed chemical probes for use in central nervous system diseases. Probes emerging from this work have translated to human clinical studies in Alzheimer’s disease and cancer.

Heat shock protein 70 (Hsp70) is a family of proteins with key roles in regulating malignancy. Cancer cells rely on Hsp70 to inhibit apoptosis, regulate senescence and autophagy, and maintain the stability of numerous onco-proteins. Despite these important biological functions in cancer, robust chemical tools that enable the analysis of the Hsp70-regulated proteome in a tumor-by-tumor manner are yet unavailable. Here we take advantage of a recently reported Hsp70 ligand to design and develop an affinity purification chemical toolset for potential use in the investigation of the endogenous Hsp70-interacting proteome in cancer. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins.

The discovery and development of heat shock protein 70 (Hsp70) inhibitors is currently a hot topic in cancer. In the preceding paper in this issue ( 10.1021/jm401551n ), we have described structure-activity relationship studies in the first Hsp70 inhibitor class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the protein. These ligands contained an acrylamide to take advantage of an active cysteine embedded in the allosteric pocket and acted as covalent protein modifiers upon binding. Here, we perform chemical modifications around the irreversible inhibitor scaffold to demonstrate that covalent modification is not a requirement for activity within this class of compounds. The study identifies derivative 27c, which mimics the biological effects of the irreversible inhibitors at comparable concentrations. Collectively, the back-to-back manuscripts describe the first pharmacophores that favorably and selectively interact with a never explored pocket in Hsp70 and provide a novel blueprint for a cancer-oriented development of Hsp70-directed ligands.

Heat shock protein 70 (Hsp70) is an important emerging cancer target whose inhibition may affect multiple cancer-associated signaling pathways and, moreover, result in significant cancer cell apoptosis. Despite considerable interest from both academia and pharmaceutical companies in the discovery and development of druglike Hsp70 inhibitors, little success has been reported so far. Here we describe structure-activity relationship studies in the first rationally designed Hsp70 inhibitor class that binds to a novel allosteric pocket located in the N-terminal domain of the protein. These 2,5'-thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides take advantage of an active cysteine embedded in the allosteric pocket to act as covalent protein modifiers upon binding. The study identifies derivatives 17a and 20a, which selectively bind to Hsp70 in cancer cells. Addition of high nanomolar to low micromolar concentrations of these inhibitors to cancer cells leads to a reduction in the steady-state levels of Hsp70-sheltered oncoproteins, an effect associated with inhibition of cancer cell growth and apoptosis. In summary, the described scaffolds represent a viable starting point for the development of druglike Hsp70 inhibitors as novel anticancer therapeutics.

SummaryHsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the cytosolic but not the organellar human Hsp70s and has biological activity partly by interfering with the formation of active oncogenic Hsp70/Hsp90/client protein complexes. YK5 is a small molecule inhibitor rationally designed to interact with an allosteric pocket of Hsp70 and represents a previously unknown chemical tool to investigate cellular mechanisms associated with Hsp70.

Grp94 is involved in the regulation of a restricted number of proteins and represents a potential target in a host of diseases, including cancer, septic shock, autoimmune diseases, chronic inflammatory conditions, diabetes, coronary thrombosis, and stroke. We have recently identified a novel allosteric pocket located in the Grp94 N-terminal binding site that can be used to design ligands with a 2-log selectivity over the other Hsp90 paralogs. Here we perform extensive SAR investigations in this ligand series and rationalize the affinity and paralog selectivity of choice derivatives by molecular modeling. We then use this to design 18c, a derivative with good potency for Grp94 (IC50 = 0.22 μM) and selectivity over other paralogs (>100- and 33-fold for Hsp90α/β and Trap-1, respectively). The paralog selectivity and target-mediated activity of 18c was confirmed in cells through several functional readouts. Compound 18c was also inert when tested against a large panel of kinases. We show that 18c has biological activity in several cellular models of inflammation and cancer and also present here for the first time the in vivo profile of a Grp94 inhibitor.

Normal cellular function is maintained by coordinated proteome machinery that performs a vast array of activities. Helping the proteome in such roles is the chaperome, a network of molecular chaperones and folding enzymes. The stressed cell contains, at any time, a complex mixture of chaperome complexes; a majority performs ‘housekeeping functions' similarly to non-stressed, normal cells, but a finely-tuned fraction buffers the proteome altered by chronic stress. The stress chaperome is epigenetically distinct from its normal, housekeeping counterpart, providing a basis for its selective targeting by small molecules. We discuss here the development of chaperome inhibitors, and how agents targeting chaperome members in stressed cells are in fact being directed towards chaperome complexes, and their effect is therefore determined by their ability to sample and engage such complexes. A new approach is needed to target and implement chaperome modulators in the investigation of diseases, and we propose that the classical thinking in drug discovery needs adjustment when developing chaperome-targeting drugs.

Although the Hsp90 chaperone family, comprised in humans of four paralogs, Hsp90α, Hsp90β, Grp94 and Trap-1, has important roles in malignancy, the contribution of each paralog to the cancer phenotype is poorly understood. This is in large part because reagents to study paralog-specific functions in cancer cells have been unavailable. Here we combine compound library screening with structural and computational analyses to identify purine-based chemical tools that are specific for Hsp90 paralogs. We show that Grp94 selectivity is due to the insertion of these compounds into a new allosteric pocket. We use these tools to demonstrate that cancer cells use individual Hsp90 paralogs to regulate a client protein in a tumor-specific manner and in response to proteome alterations. Finally, we provide new mechanistic evidence explaining why selective Grp94 inhibition is particularly efficacious in certain breast cancers.

We here describe the first reported comprehensive analysis of Hsp90 paralogue affinity and selectivity in the clinical Hsp90 inhibitor chemotypes. This has been possible through the development of a versatile experimental assay based on a new FP-probe (16a) that we both describe here. The assay can test rapidly and accurately the binding affinity of all major Hsp90 chemotypes and has a testing range that spans low nanomolar to millimolar binding affinities. We couple this assay with a computational analysis that allows for rationalization of paralogue selectivity and defines not only the major binding modes that relay pan-paralogue binding or, conversely, paralogue selectivity, but also identifies molecular characteristics that impart such features. The methods developed here provide a blueprint for parsing out the contribution of the four Hsp90 paralogues to the perceived biological activity with the current Hsp90 chemotypes and set the ground for the development of paralogue selective inhibitors.

A number of compounds from different chemical classes are known to bind competitively to the ATP-pocket of Hsp90 and inhibit its chaperone function. The natural product geldanamycin was the first reported inhibitor of Hsp90 and since then synthetic inhibitors from purine, isoxazole and indazol-4-one chemical classes have been discovered and are currently or soon to be in clinical trials for the treatment of cancer. In spite of a similar binding mode to Hsp90, distinct biological profiles were demonstrated amongst these molecules, both in vitro and in vivo. To better understand the molecular basis for these dissimilarities, we report here the synthesis of chemical tools for three Hsp90 inhibitor classes. These agents will be useful for probing tumor-by-tumor the Hsp90 complexes isolated by specific inhibitors. Such information will lead to better understanding of tumor specific molecular markers to aid in their clinical development. It will also help to elucidate the molecular basis for the biological differences observed among Hsp90 inhibitors.

The chemotherapy of lymphatic filariasis relies upon drugs such as diethylcarbamazine and ivermectin that largely target the microfilarial stages of the parasite, necessitating continued treatment over the long reproductive life span of the adult worm. The identification of compounds that target adult worms has been a long-term goal of WHO. Here we describe a fluorescence polarization assay for the identification of compounds that target Hsp90 in adult filarial worms. The assay was originally developed to identify inhibitors of Hsp90 in tumor cells, and relies upon the ability of small molecules to inhibit the binding of fluorescently labelled geldanamycin to Hsp90. We demonstrate that the assay works well with soluble extracts of Brugia, while extracts of the free-living nematode C. elegans fail to bind the probe, in agreement with data from other experiments. The assay was validated using known inhibitors of Hsp90 that compete with geldanamycin for binding to Hsp90, including members of the synthetic purine-scaffold series of compounds. The efficacy of some of these compounds against adult worms was confirmed in vitro. Moreover, the assay is sufficiently sensitive to differentiate between binding of purine-scaffold compounds to human and Brugia Hsp90. The assay is suitable for high-throughput screening and provides the first example of a format with the potential to identify novel inhibitors of Hsp90 in filarial worms and in other parasitic species where Hsp90 may be a target., Author Summary Helminth diseases of humans remain a major problem in many parts of the tropics. Treatment of these parasitic infections is restricted to a limited number of drugs and few new compounds are in development. One of the major obstacles to the development of new therapeutics is the lack of high-throughput screens that can be adapted to parasitic species for the identification of small molecule inhibitors. Here we present a simple, inexpensive assay for the identification of inhibitors of Hsp90 in filarial worms. The assay, first described for the identification of Hsp90 inhibitors in tumor cells, does not require recombinant protein but relies upon the ability of a fluorescently labelled drug to bind to Hsp90 in the context of a soluble fraction of worm homogenate. We validated the assay using known inhibitors of Hsp90, including derivatives of the synthetic purine-scaffold series of Hsp90 inhibitors and were able to show a differential sensitivity to these compounds between human and Brugia Hsp90.

Background: Heat shock protein 70 family members play an important role in cancer. They are up-regulated in wide variety of tumors and the increased Hsp70 protein expression correlates with metastases, resistance to treatment and poor prognosis. Multiple mechanisms explain cancer cells dependence on Hsp70, such as inhibition of apoptosis by Hsp70, induction of autophagy and control of stability of onco-proteins. These Hsp70 activities are mediated in cancer by its ability to chaperone and interact with a large number of proteins in a cell-specific, context dependent manner. Hypothesis: Reagents that enable the capture of tumor-specific Hsp70 complexes facilitate the identification of context-dependent Hsp70 interactomes. Results: Our laboratory recently reported the identification of a novel allosteric site located in the nucleotide binding domain of Hsp70 (Chem Biol 2013). It has also reported the discovery of ligands that bind to the allosteric pocket of Hsp70, inhibit its function in cancer cells and result in anti-cancer activity (J Med Chem 2013). Structure-activity relationship studies in this ligand series gave insights on the attachment of specific linkers for the design of Hsp70-related chemical tools. Here we present the design of Hsp70-directed reagents and use biochemical and cell-based methods to validate Hsp70-directed affinity purification probes. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins. Significance: The knowledge derived from the use of such reagents will be extremely valuable not only to understand tumor-specific roles of Hsp70 and associated mechanisms but also to develop rational strategies for the clinical implementation of these agents to cancer treatment. They may also provide clues on the altered functional proteome in individual tumors, a quest yet elusive by today's proteomics methods. Citation Format: Anna A. Rodina, Tony Taldone, Yanlong Kang, Pallav Patel, John Koren, Pengrong Yan, Erica DaGama Gomes, Chenghua Yang, Maulik Patel, Liza Shrestha, Stefan Ochiana, Ronnie Maharaj, Alexander Gozman, Marc Cox, Hediye Erdjument-Bromage, Ronald Hendrickson, Leandro Cerchietti, Ari Melnick, Monica Guzman, Gabriela Chiosis. Development of chemical tools to study the endogenous Hsp70 interactome in malignant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1733. doi:10.1158/1538-7445.AM2015-1733

Glucose Regulated Protein 94 (Grp94) is an Hsp90 paralog localized in the lumen of the endoplasmic reticulum (ER) of higher eukaryotes. While the mechanisms associated with Grp94-pathogenic expression are still actively studied, the focus thus far of most cancer related studies has been primarily on the immunogenic activity of Grp94/peptide complexes and the involvement of this protein in the secretion of IGF-I and IGF-II and the regulation of Toll-like receptors and integrins. This status quo has recently changed when work from our lab has shown that in certain breast cancers the maintenance of a high density HER2 species at the plasma membrane and its associated aberrant signaling also requires Grp94, rendering these tumors highly sensitive to Grp94 inhibition. The discovery of selective and potent Grp94 inhibitors has been hindered due to the high similarity of the ATP-binding regulatory pocket of the Hsp90 paralogs. However, recent work from our lab has shown that this apparent roadblock can be overcome by using library screening and structural and computational analysis to discover purine-based molecules that show 100-fold selectivity for the Grp94 isoform. Herein, we detail for the first time the structure activity relationship of the selective purine derived Grp94 molecules. This work provides insights on how to overcome the high structural similarity of Hsp90s in the ligand binding pocket, and also reports selective and therapeutically relevant Grp94 inhibitors based on a purine scaffold. These initial inhibitors have potent activity against cancer cells providing an important platform for the development of Grp94 inhibitors with drug-like features and potential for clinical translation. Citation Format: Stefan O. Ochiana, Tony Taldone, Hardik J. Patel, Pallav Patel, Yan Pengrong, Weilin Sun, Anna Rodina, Smit Shah, Daniel T. Gewirth, Gabriela Chiosis. Development of selective GRP94 inhibitors for the treatment of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2831. doi:10.1158/1538-7445.AM2015-2831

The Endoplasmic Reticulum (ER) HSP90 paralog, Glucose Regulated Protein 94 (Grp94) is a molecular chaperone often overexpressed in tumors. Clinically, expression of Grp94 correlates with advanced stage and poor survival in a variety of cancers, and is closely linked to cancer growth and metastasis. The majority of the cancer-related studies on Grp94 have focused narrowly on the immunogenic activity of Grp94/peptide complexes and the involvement of this protein in the secretion of IGF-I and IGF-II and the regulation of Toll-like receptors and integrins. Recently however, novel mechanistic understanding emerged regarding the cancer specific roles of Grp94: the important and unexpected tumor specific translocation of Grp94 from ER to plasma membrane to regulate the altered expression and function of plasma membrane associated cancer-proteins and its role in sustaining their transforming ability. In this respect, we found that the high density HER2 formations at the plasma membrane of HER2-overexpressing breast cancer cells necessitate Grp94 (Nat Chem Biol 2013 9(11):677-84). Inhibition of Grp94 in these cells was sufficient to destabilize plasma membrane HER2, inhibit its signaling properties, target HER2 towards a degradative pathway and as a result kill the cancer cell. We here use a chemical biology approach combined with classical methods to produce preliminary evidence for an unanticipated oncogenic role for Grp94 in maintaining high density receptor tyrosine kinases (RTKs) at the plasma membrane, particularly in cancer cells where RTKs are required to channel an amplified signaling. Our data indicate that under conditions in which stress is imposed on the cell by proteome alterations (i.e. RTK overexpression), the chaperoning function of Grp94 is vital for proper functioning of RTKs. In these cells Grp94 translocates to the plasma membrane where it functions to maintain an active conformation of RTKs and to stabilize downstream signaling through the receptor. In contrast, we find no effect of Grp94 inhibition in cells with normal or physiological RTK function/expression. Our findings provide a strong rationale for the use of chemical tools in the investigation of Grp94 associated oncogenic mechanisms and support the development of Grp94 inhibitors as a novel targeted therapy for the treatment of cancers dependent on increased signaling through plasma membrane RTKs, many of which tend to have an aggressive presentation. Citation Format: Pengrong Yan, Hardik Patel, Pallav Patel, Stefan Ochiana, Weilin Sun, Smit Shah, Paola Finotti, Cynthia Leifer, Zihai Li, Daniel Gewirth, Tony Taldone, Gabriela Chiosis. Tumor-specific regulation of receptor tyrosine kinases by Grp94. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4716. doi:10.1158/1538-7445.AM2015-4716

The 70 kDa heat shock protein (Hsp70) plays an important role in cancer and its pharmacological modulation with small molecules may represent a useful therapeutic approach. The discovery of such agents has been hampered by the lack of a full length crystal structure of human Hsp70. We constructed a homology model of the human Hsp70, which we investigated for potential druggable sites using SiteMap tool. An allosteric site in the nucleotide binding domain was identified and used to design potent Hsp70 modulators. Analysis of the Glide docking resulted in an understanding of important interactions between designed ligands, such as YK5, and the Hsp70 protein. When tested in breast cancer cells, YK5 led to depletion of onco-proteins, induced apoptosis and inhibited growth. YK5 is to our knowledge the first rationally designed small molecule inhibitor of Hsp70, and thus, represents a novel chemical tool to investigate its potential in cancer and other diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3895. doi:1538-7445.AM2012-3895

Background. The cancerous phenotype evolves through accumulation of a large repertoire of elements that allow for increased proliferative potential, self-sufficiency in growth signals and resistance to apoptotic and antiproliferative signals. While much is now gained in understanding the many factors that lead to a malignant phenotype, little remains known on how cancer cells are able to accumulate and to survive with such complexity and high load of aberrant processes. Study design. Here we use the novel dual Hsp70 and Hsc70 inhibitor, YK5, and its biotinylated derivative, YK55, to investigate the interactome of cytosolic Hsp70s. Results. We show that heat shock protein 70 family members (Hsp70s) keep active conformations of key oncoproteins and inhibit the function of tumor suppressors. Hsp70s also buffer levels of activated molecules by maintaining them in a conformation that alters their dephosphorylation by phosphatases. Pharmacologic inhibition of Hsp70s by YK5 impairs oncoprotein stability and function, restores tumor suppressor activity, affects major cancer hallmarks and is selectively lethal to cancer cells. We show that enhanced affinity of YK5 for cancer cell-Hsp70s species is an essential mechanism for providing selective tumor lethality. Conclusion. By demonstrating that pharmacologic dual inhibitors of Hsp70 and Hsc70, such as YK5, confer specific and broad tumor lethality, and interact selectively with tumor Hsp70s species, this work proposes Hsp70s inhibitors as potential anti-cancer therapeutics with a large spectrum of activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2579. doi:10.1158/1538-7445.AM2011-2579