Advances in the clinical development of heat shock protein 90 (Hsp90) inhibitors in cancers

Hsp90 is an ATP dependent molecular chaperone protein which integrates multiple oncogenic pathways. As such, Hsp90 inhibition is a promising anti-cancer strategy. Several inhibitors that act on Hsp90 by binding to its N-terminal ATP pocket have entered clinical evaluation. Robust pre-clinical data suggested anti-tumor activity in multiple cancer types. Clinically, encouraging results have been demonstrated in melanoma, acute myeloid leukemia, castrate refractory prostate cancer, non-small cell lung carcinoma and multiple myeloma. In breast cancer, proof-of-concept was demonstrated by first generation Hsp90 inhibitors in combination with trastuzumab mainly in human epidermal growth factor receptor 2 (HER2)+metastatic breast cancer. There are a multitude of second generation Hsp90 inhibitors currently under investigation. To date, however, there is no FDA approved Hsp90 inhibitor nor standardized assay to ascertain Hsp90 inhibition. This review summarizes the current status of both first and second generation Hsp90 inhibitors based on their chemical classification and stage of clinical development. It also discusses the pharmacodynamic assays currently implemented in clinic as well as other novel strategies aimed at enhancing the effectiveness of Hsp90 inhibitors. Ultimately, these efforts will aid in maximizing the full potential of this class of agents. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

热激蛋白90抑制剂

分子伴侣热激蛋白90(heat-shock protein 90,Hsp90)在生物体内具有重要的生理功能,它在许多肿瘤细胞中表达增加。临床研究发现Hsp90抑制剂单一用药或者联合用药都具有较好的抗肿瘤效果,因此目前Hsp90被认为是癌症治疗一个非常有潜力的靶标。本文总结了Hsp90的结构功能、Hsp90抑制剂的作用机理以及Hsp90抑制剂的临床应用前景,希望为设计和开发新的Hsp90抑制剂提供一定的参考。     

An investigation into the potential use of serum Hsp70 as a novel tumour biomarker for Hsp90 inhibitors

Hsp90 inhibitors are under investigation in multiple human clinical trials for the treatment of cancers, including myeloma, breast cancer, prostate, lung, melanoma, gastrointestinal stromal tumour and acute myeloid leukaemia. The pharmacodynamic activity of Hsp90 inhibitors in the clinic is currently assessed by Hsp70 induction in peripheral blood mononuclear cells using Western blot analysis, a method that is laborious, semiquantitative and difficult to implement in the clinic. Since Hsp70 was reported to be secreted by tumour cells and elevated in sera of cancer patients, serum Hsp70 has been evaluated as a potentially more robust, easily and reproducibly measured biomarker of Hsp90 inhibition as an alternative to cytosolic Hsp70. A highly sensitive and specific electrochemiluminescent ELISA was developed to measure serum Hsp70 and employed to evaluate Hsp70 levels in both ex vivo and xenograft samples. In ex vivo studies, maximal secretion of Hsp70 by tumour cells was observed between 48 and 72?h after exposure to Hsp90 inhibitors. In in vivo studies a 3–4-fold increase in serum Hsp70 was observed following treatment with BIIB021 in tumour-bearing mice. Strikingly, secreted Hsp70 was detectable in mice transplanted with human tumours but not in naive mice indicating a direct origination from the transplanted tumours. Analysis of clinical samples revealed low baseline levels (2–15?ng ml^?1) of Hsp70 in the serum of cancer patients and normal donors. Together these findings in laboratory studies and archived cancer patient sera suggest that serum Hsp70 could be a novel biomarker to assess reliably the pharmacological effects of Hsp90 inhibitors in clinical trials, especially under conditions where collection of tumour biopsies is not feasible.     

Low-dose Hsp90 inhibitors tumor-selectively sensitize bladder cancer cells to chemoradiotherapy

Although radical cystectomy with urinary diversion is the standard treatment for muscle-invasive bladder cancer (MIBC), loss of native bladder frequently impairs patient's quality of life (QOL). Bladder-sparing approach incorporating chemoradiotherapy (CRT) improves QOL while not compromising survival outcomes in MIBC patients. In this approach, complete response to induction CRT is a prerequisite for bladder preservation and favorable oncological outcomes. We investigated a strategy to potentiate CRT response of bladder cancer cells by using Hsp90 inhibitors in preclinical models. Hsp90 inhibitors at low concentrations, which did not exert cytocidal effects but inactivated key anti-apoptotic proteins including erbB2, Akt, and NF-κB, efficiently sensitized bladder cancer cells (T24, 5637 and UM-UC-3 cells) to in vitro CRT by enhancing apoptosis. Importantly, the sensitizing effects were not observed in primarily cultured normal human urothelial cells. We also showed that CRT induces accumulation of nuclear phospho-Akt, which antagonizes apoptosis, and that Hsp90 inhibitors block the cellular process. Hsp90 inhibition sensitized bladder cancer cells to in vitro CRT more effectively than sole or combined inhibition of erbB2 and Akt. In mice UM-UC-3 tumor xenografts model, Hsp90 inhibitors successfully potentiated anti-tumor activity of CRT. These results encourage clinical trials of Hsp90 inhibitors to overcome CRT resistance in patients with MIBC.     

2,4-dihydroxy benzaldehyde derived Schiff bases as small molecule Hsp90 inhibitors: Rational identification of a new anticancer lead

Hsp90 is a molecular chaperone that heals diverse array of biomolecules ranging from multiple oncogenic proteins to the ones responsible for development of resistance to chemotherapeutic agents. Moreover they are over-expressed in cancer cells as a complex with co-chaperones and under-expressed in normal cells as a single free entity. Hence inhibitors of Hsp90 will be more effective and selective in destroying cancer cells with minimum chances of acquiring resistance to them. In continuation of our goal to rationally develop effective small molecule azomethines against Hsp90, we designed few more compounds belonging to the class of 2,4-dihydroxy benzaldehyde derived imines (1–13) with our validated docking protocol. The molecules exhibiting good docking score were synthesized and their structures were confirmed by IR, ¹H NMR and mass spectral analysis. Subsequently, they were evaluated for their potential to suppress Hsp90 ATPase activity by Malachite green assay. The antiproliferative effect of the molecules were examined on PC3 prostate cancer cell lines by adopting 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay methodology. Finally, schiff base 13 emerged as the lead molecule for future design and development of Hsp90 inhibitors as anticancer agents.     

Hsp90 inhibitors as novel cancer chemotherapeutic agents

Heat shock protein 90 (Hsp90) is a molecular chaperone whose association is required for the stability and function of multiple mutated, chimeric and over-expressed signaling proteins that promote the growth and/or survival of cancer cells. Hsp90 client proteins include mutated p53, Bcr-Abl, Raf-1, Akt, ErbB2 and hypoxia-inducible factor 1α (HIF-1α). Hsp90 inhibitors, by interacting specifically with a single molecular target, cause the destabilization and eventual degradation of Hsp90 client proteins, and they have shown promising antitumor activity in preclinical model systems. One Hsp90 inhibitor, 17-allylaminogeldanamycin (17AAG), is currently in phase I clinical trial. Because of the chemoprotective activity of several proteins that are Hsp90 clients, the combination of an Hsp90 inhibitor with a standard chemotherapeutic agent could dramatically increase the in vivo efficacy of the therapeutic agent.     

Heat shock protein 90: The cancer chaperone

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of a number of conditionally activated and/or expressed signalling proteins, as well as multiple mutated, chimeric, and/or over-expressed signalling proteins, that promote cancer cell growth and/or survival. Hsp90 inhibitors are unique in that, although they are directed towards a specific molecular target, they simultaneously inhibit multiple cellular signalling pathways. By inhibiting nodal points in multiple overlapping survival pathways utilized by cancer cells, combination of an Hsp90 inhibitor with standard chemotherapeutic agents may dramatically increase the in vivo efficacy of the standard agent. Hsp90 inhibitors may circumvent the characteristic genetic plasticity that has allowed cancer cells to eventually evade the toxic effects of most molecularly targeted agents. The mechanism-based use of Hsp90 inhibitors, both alone and in combination with other drugs, should be effective toward multiple forms of cancer. Further, because Hsp90 inhibitors also induce Hsf-1-dependent expression of Hsp70, and because certain mutated Hsp90 client proteins are neurotoxic, these drugs display ameliorative properties in several neurodegenerative disease models, suggesting a novel role for Hsp90 inhibitors in treating multiple pathologies involving neurodegeneration.     

Inhibition of heat shock protein (Hsp) 27 potentiates the suppressive effect of Hsp90 inhibitors in targeting breast cancer stem-like cells

Heat shock protein (Hsp) 90 is an ATP-dependent chaperone and its expression has been reported to be associated with poor prognosis of breast cancer. Cancer stem cells (CSCs) are particular subtypes of cells in cancer which have been demonstrated to be important to tumor initiation, drug resistance and metastasis. In breast cancer, breast CSCs (BCSCs) are identified as CD24-CD44 + cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH+). Although the clinical trials of Hsp90 inhibitors in breast cancer therapy are ongoing, the BCSC targeting effect of them remains unclear. In the present study, we discovered that the expression of Hsp90α was increased in ALDH + human breast cancer cells. Geldanamycin (GA), a Hsp90 inhibitor, could suppress ALDH + breast cancer cells in a dose dependent manner. We are interesting in the insufficiently inhibitory effect of low dose GA treatment. It was correlated with the upregulation of Hsp27 and Hsp70. By co-treatment with HSP inhibitors, quercetin or KNK437 potentiated BCSCs, which determined with ALDH+ population or mammosphere cells, toward GA inhibition, as well as anti-proliferation and anti-migration effects of GA. With siRNA mediated gene silencing, we found that knockdown of Hsp27 could mimic the effect of HSP inhibitors to potentiate the BCSC targeting effect of GA. In conclusion, combination of HSP inhibitors with Hsp90 inhibitors could serve as a potential solution to prevent the drug resistance and avoid the toxicity of high dose of Hsp90 inhibitors in clinical application. Furthermore, Hsp27 may play a role in chemoresistant character of BCSCs.     

Modified biphenyl Hsp90 C-terminal inhibitors for the treatment of cancer

Heat Shock Protein 90 (Hsp90) is a molecular chaperone under clinical investigation for the treatment of neurodegenerative diseases and cancer. Neuroprotective Hsp90 C-terminal inhibitors (novologues) contain a biaryl ring system, and include KU-596, which was modified and investigated for potential anti-cancer activity. Incorporation of a benzamide group onto the biaryl novologues in lieu of the acetamide yielded compounds that manifest anti-cancer activity. Further exploration of the central phenyl ring led to compounds with enhanced anti-proliferative activity. The design, synthesis, and evaluation of these new analogs against breast and prostate cancer cell lines is reported herein, where it was found that 8b and 10 manifest potent anti-proliferative activity and a robust degradation of Hsp90 client-dependent proteins.     

Mitochondrial-targeted Hsp90 C-terminal inhibitors manifest anti-proliferative activity

The development of C-terminal heat shock protein 90 kDa (Hsp90) inhibitors has emerged as a potential treatment for cancer. Similarly, small molecules that target the mitochondria have proven to be efficacious towards cancer, as the reprogramming of mitochondrial function is often associated with oncogenic transformation. Herein, we report the development of triphenylphosphonium (TPP)-conjugated Hsp90 C-terminal inhibitors, their anti-proliferative activity, and accumulation in the mitochondria. In general, TPP-conjugated Hsp90 C-terminal inhibitors were found to manifest increased activity against various cancer cell lines when compared to the parent compounds.     

Inhibitors of heat shock protein 90 activity: A novel class of tumor radiosensitizers

The 90-kDa heat shock protein (Hsp90) is one of the major chaperones in eukaryotes and catalyzes the maturation and activation of its client proteins. Oncogene products, hormones or growth factor receptors, and key components of signaling pathways are responsible for the malignant growth of tumors or their resistance to chemotherapy and radiotherapy; some of these molecules were identified among the client proteins of Hsp90. Upon the inhibition of Hsp90 chaperone function, such client proteins are inactivated and rapidly degraded, which leads to simultaneous blocking of multiple pathways that are essential for malignant cell proliferation and survival; therefore, pharmacological inhibitors of the Hsp90 chaperone activity could be potentially used in anticancer therapy. Several Hsp90 inhibitors are currently being tested in preclinical or phase I–III clinical trials, either as single agents or in combination with other anticancer drugs or irradiation treatment. In this review, we summarize the data regarding the characterization of Hsp90 inhibitors as efficient radiosensitizers of tumor cells. We also discuss molecular mechanisms and the selectivity of radiosensitization induced by Hsp90 inhibition, as well as a possibility of their application to improve the outcome of radiotherapy.     

Induction of Premature Senescence by Hsp90 Inhibition in Small Cell Lung Cancer

Background

The molecular chaperone Hsp90 is a promising new target in cancer therapy and selective Hsp90 inhibitors are currently in clinical trials. Previously these inhibitors have been reported to induce either cell cycle arrest or cell death in cancer cells. Whether the cell cycle arrest is reversible or irreversible has not generally been assessed. Here we have examined in detail the cell cycle arrest and cell death responses of human small cell lung cancer cell lines to Hsp90 inhibition.

Methodology/Principal Findings

In MTT assays, small cell lung cancer cells showed a biphasic response to the Hsp90 inhibitors geldanamycin and radicicol, with low concentrations causing proliferation arrest and high concentrations causing cell death. Assessment of Hsp90 intracellular activity using loss of client protein expression showed that geldanamycin concentrations that inhibited Hsp90 correlated closely with those causing proliferation arrest but not cell death. The proliferation arrest induced by low concentrations of geldanamycin was not reversed for a period of over thirty days following drug removal and showed features of senescence. Rare populations of variant small cell lung cancer cells could be isolated that had additional genetic alterations and no longer underwent irreversible proliferation arrest in response to Hsp90 inhibitors.

Conclusions/Significance

We conclude that: (1) Hsp90 inhibition primarily induces premature senescence, rather than cell death, in small cell lung cancer cells; (2) small cell lung cancer cells can bypass this senescence through further genetic alterations; (3) Hsp90 inhibitor-induced cell death in small cell lung cancer cells is due to inhibition of a target other than cytosolic Hsp90. These results have implications with regard to how these inhibitors will behave in clinical trials and for the design of future inhibitors in this class.     

5′‐N‐ethylcarboxamidoadenosine is not a paralog‐specific Hsp90 inhibitor

The molecular chaperone Hsp90 facilitates the folding and modulates activation of diverse substrate proteins. Unlike other heat shock proteins such as Hsp60 and Hsp70, Hsp90 plays critical regulatory roles by maintaining active states of kinases, many of which are overactive in cancer cells. Four Hsp90 paralogs are expressed in eukaryotic cells: Hsp90α/β (in the cytosol), Grp94 (in the endoplasmic reticulum), Trap1 (in mitochondria). Although numerous Hsp90 inhibitors are being tested in cancer clinical trials, little is known about why different Hsp90 inhibitors show specificity among Hsp90 paralogs. The paralog specificity of Hsp90 inhibitors is likely fundamental to inhibitor efficacy and side effects. In hopes of gaining insight into this issue we examined NECA (5′‐N‐ethylcarboxamidoadenosine), which has been claimed to be an example of a highly specific ligand that binds to one paralog, Grp94, but not cytosolic Hsp90. To our surprise we find that NECA inhibits many different Hsp90 proteins (Grp94, Hsp90α, Trap1, yeast Hsp82, bacterial HtpG). NMR experiments demonstrate that NECA can bind to the N‐terminal domains of Grp94 and Hsp82. We use ATPase competition experiments to quantify the inhibitory power of NECA for different Hsp90 proteins. This scale: Hsp82 > Hsp90α > HtpG ≈ Grp94 > Trap1, ranks Grp94 as less sensitive to NECA inhibition. Because NECA is primarily used as an adenosine receptor agonist, our results also suggest that cell biological experiments utilizing NECA may have confounding effects from cytosolic Hsp90 inhibition.     

Natural iminosugar (+)-lentiginosine inhibits ATPase and chaperone activity of hsp90

Heat shock protein 90 (Hsp90) is a significant target in the development of rational cancer therapy due to its role at the crossroads of multiple signaling pathways associated with cell proliferation and cell viability. The relevance of Hsp90 as a therapeutic target for numerous diseases states has prompted the identification and optimization of novel Hsp90 inhibitors as an emerging therapeutic strategy. We performed a screening aimed to identify novel Hsp90 inhibitors among several natural compounds and we focused on the iminosugar (+)-lentiginosine, a natural amyloglucosidases inhibitor, for its peculiar bioactivity profile. Characterization of Hsp90 inhibition was performed using a panel of chemical and biological approaches, including limited proteolysis, biochemical and cellular assays. Our result suggested that the middle domain of Hsp90, as opposed to its ATP-binding pocket, is a promising binding site for new classes of Hsp90 inhibitors with multi-target anti-cancer potential.     

Co-Crystalization and In Vitro Biological Characterization of 5-Aryl-4-(5-Substituted-2-4-Dihydroxyphenyl)-1,2,3-Thiadiazole Hsp90 Inhibitors

A potential therapeutic strategy for targeting cancer that has gained much interest is the inhibition of the ATP binding and ATPase activity of the molecular chaperone Hsp90. We have determined the structure of the human Hsp90α N-terminal domain in complex with a series of 5-aryl-4-(5-substituted-2-4-dihydroxyphenyl)-1,2,3-thiadiazoles. The structures provide the molecular details for the activity of these inhibitors. One of these inhibitors, ICPD 34, causes a structural change that affects a mobile loop, which adopts a conformation similar to that seen in complexes with ADP, rather than the conformation generally seen with the pyrazole/isoxazole-resorcinol class of inhibitors. Competitive binding to the Hsp90 N-terminal domain was observed in a biochemical assay, and these compounds showed antiproliferative activity and induced apoptosis in the HCT116 human colon cancer cell line. These inhibitors also caused induction of the heat shock response with the upregulation of Hsp72 and Hsp27 protein expression and the depletion of Hsp90 clients, CRAF, ERBB2 and CDK4, thus confirming that antiproliferative activity was through the inhibition of Hsp90. The presence of increased levels of the cleavage product of PARP indicated apoptosis in response to Hsp90 inhibitors. This work provides a framework for the further optimization of thiadiazole inhibitors of Hsp90. Importantly, we demonstrate that the thiadiazole inhibitors display a more limited core set of interactions relative to the clinical trial candidate NVP-AUY922, and consequently may be less susceptible to resistance derived through mutations in Hsp90.     

Hsp90 phosphorylation,Wee1 and the cell cycle

Heat Shock Protein 90 (Hsp90) is an essential molecular chaperone in eukaryotic cells, and it maintains the functional conformation of a subset of proteins that are typically key components of multiple regulatory and signaling networks mediating cancer cell proliferation, survival, and metastasis. It is possible to selectively inhibit Hsp90 using natural products such as geldanamycin (GA) or radicicol (RD), which have served as prototypes for development of synthetic Hsp90 inhibitors. These compounds bind within the ADP/ATP-binding site of the Hsp90 N-terminal domain to inhibit its ATPase activity. As numerous N-terminal domain inhibitors are currently undergoing extensive clinical evaluation, it is important to understand the factors that may modulate in vivo susceptibility to these drugs. We recently reported that Wee1Swe1-mediated, cell cycle-dependent, tyrosine phosphorylation of Hsp90 affects GA binding and impacts cancer cell sensitivity to Hsp90 inhibition. This phosphorylation also affects Hsp90 ATPase activity and its ability to chaperone a selected group of clients, comprised primarily of protein kinases. Wee1 regulates the G2/M transition. Here we present additional data demonstrating that tyrosine phosphorylation of Hsp90 by Wee1Swe1 is important for Wee1Swe1 association with Hsp90 and for Wee1Swe1 stability. Yeast expressing non-phosphorylatable yHsp90-Y24F, like swe1? yeast, undergo premature nuclear division that is insensitive to G2/M checkpoint arrest. These findings demonstrate the importance of Hsp90 phosphorylation for proper cell cycle regulation.