Francis Burrows

A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors.

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of oncogenic signalling proteins, including HER-2/ErbB2, Akt, Raf-1, Bcr-Abl and mutated p53. Hsp90 inhibitors bind to Hsp90, and induce the proteasomal degradation of Hsp90 client proteins. Although Hsp90 is highly expressed in most cells, Hsp90 inhibitors selectively kill cancer cells compared to normal cells, and the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) is currently in phase I clinical trials. However, the molecular basis of the tumour selectivity of Hsp90 inhibitors is unknown. Here we report that Hsp90 derived from tumour cells has a 100-fold higher binding affinity for 17-AAG than does Hsp90 from normal cells. Tumour Hsp90 is present entirely in multi-chaperone complexes with high ATPase activity, whereas Hsp90 from normal tissues is in a latent, uncomplexed state. In vitro reconstitution of chaperone complexes with Hsp90 resulted in increased binding affinity to 17-AAG, and increased ATPase activity. These results suggest that tumour cells contain Hsp90 complexes in an activated, high-affinity conformation that facilitates malignant progression, and that may represent a unique target for cancer therapeutics.

The high-affinity HSP90-CHIP complex recognizes and selectively degrades phosphorylated tau client proteins

A primary pathologic component of Alzheimers disease (AD) is the formation of neurofibrillary tangles composed of hyperphosphorylated tau (p-tau). Expediting the removal of these p-tau species may be a relevant therapeutic strategy. Here we report that inhibition of Hsp90 led to decreases in p-tau levels independent of heat shock factor 1 (HSF1) activation. A critical mediator of this mechanism was carboxy terminus of Hsp70-interacting protein (CHIP), a tau ubiquitin ligase. Cochaperones were also involved in Hsp90-mediated removal of p-tau, while those of the mature Hsp90 refolding complex prevented this effect. This is the first demonstration to our knowledge that blockade of the refolding pathway promotes p-tau turnover through degradation. We also show that peripheral administration of a novel Hsp90 inhibitor promoted selective decreases in p-tau species in a mouse model of tauopathy, further suggesting a central role for the Hsp90 complex in the pathogenesis of tauopathies. When taken in the context of known high-affinity Hsp90 complexes in affected regions of the AD brain, these data implicate a central role for Hsp90 in the development of AD and other tauopathies and may provide a rationale for the development of novel Hsp90-based therapeutic strategies.

Drugging the cancer chaperone HSP90: combinatorial therapeutic exploitation of oncogene addiction and tumor stress.

Abstract:  The molecular chaperone HSP90 has emerged as an exciting target for cancer treatment. We review the potential advantages of HSP90 inhibitors, particularly the simultaneous combinatorial depletion of multiple oncogenic “client” proteins, leading to blockade of many cancer‐causing pathways and the antagonism of all of the hallmark pathological traits of malignancy. Cancer selectivity is achieved by exploiting cancer “dependencies,” including oncogene addiction and the stressed state of malignant cells. The multiple downstream effects of HSP90 inhibitors should make the development of resistance more difficult than with agents having more restricted effects. We review the various classes of HSP90 inhibitor that have been developed, including the natural products geldanamycin and radicicol and also the purine scaffold and pyrazole/isoxazole class of synthetic small molecule inhibitors. A first‐in‐class HSP90 drug, the geldanamycin analog 17‐AAG, has provided proof of concept for HSP90 inhibition in patients at well tolerated doses and therapeutic activity has been seen. Other inhibitors show promise in preclinical and clinical development. Opportunities and challenges for HSP90 inhibitors are discussed, including use in combination with other agents. Most of the current HSP90 inhibitors act by blocking the essential nucleotide binding and ATPase activity required for chaperone function. Potential new approaches are discussed, for example, interference with cochaperone binding and function in the superchaperone complex. Biomarkers for use with HSP90 inhibitors are described. We stress how basic and translational research has been mutually beneficial and indicate future directions to enhance our understanding of molecular chaperones and their exploitation in cancer and other diseases.

Therapeutic and diagnostic implications of Hsp90 activation

Abstract The molecular chaperone heat-shock protein 90 (Hsp90) is involved in the stabilization and conformational maturation of many signaling proteins that are deregulated in cancers. Hsp90 inhibition results in the proteasomal degradation of these client proteins and leads to potent antitumor activity. The Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) is presently in clinical trials. Recent work has identified the role of Hsp90 in multiple signal transduction pathways and revealed that the molecular mechanism of tumor selectivity by Hsp90 inhibitors is the result of an activated, high-affinity conformation of Hsp90 in tumors. This review discusses these recent advances in the understanding of tumor Hsp90 for the treatment and diagnosis of cancer. In addition, the role of Hsp90 in non-oncological diseases will also be discussed.

HSP induction mediates selective clearance of tau phosphorylated at proline-directed Ser/Thr sites but not KXGS (MARK) sites.

Neurofibrillary tangles (NFTs) are a characteristic neuropathological feature of Alzheimers disease (AD), and molecular chaperones appear to be involved in the removal of disease‐associated hyperphosphorylated tau, a primary component of NFTs. Here, novel HSP90 inhibitors were used to examine the impact of chaperone elevation on clearance of different tau species in transfected cells using a unique quantitative assay. The HSP90 inhibitors reduced levels of tau phosphorylated at proline‐directed Ser/Thr sites (pS202/T205, pS396/S404) and conformationally altered (MC‐1) tau species, an epitope that is immeasurable by standard Western blot techniques. The selective clearance of these phospho‐tau species and MC‐1 tau was mediated via the proteasome, while lysosomal‐mediated tau degradation seems to lack specificity for certain tau species, suggesting a more general role in total tau removal. Interestingly, tau phosphorylated at S262/S356 within the tau microtubule binding domain was minimally affected by chaperone induction. Overall, our data show that chaperone induction results in the selective clearance of specific phospho‐tau and conformationally altered tau species mediated by the proteasome; however, the apparent stability of pS262/S356 tau may also explain why MARK is able to regulate normal tau function yet still be linked to the initiation of pathogenic tau hyperphosphorylation in AD.

BIIB021, an orally available, fully synthetic small-molecule inhibitor of the heat shock protein Hsp90

Inhibition of heat shock protein 90 (Hsp90) results in the degradation of oncoproteins that drive malignant progression, inducing cell death, making Hsp90 a target of substantial interest for cancer therapy. BIIB021 is a novel, fully synthetic inhibitor of Hsp90 that binds competitively with geldanamycin in the ATP-binding pocket of Hsp90. In tumor cells, BIIB021 induced the degradation of Hsp90 client proteins including HER-2, AKT, and Raf-1 and up-regulated expression of the heat shock proteins Hsp70 and Hsp27. BIIB021 treatment resulted in growth inhibition and cell death in cell lines from a variety of tumor types at nanomolar concentrations. Oral administration of BIIB021 led to the degradation of Hsp90 client proteins measured in tumor tissue and resulted in the inhibition of tumor growth in several human tumor xenograft models. Studies to investigate the antitumor effects of BIIB021 showed activity on both daily and intermittent dosing schedules, providing dose schedule flexibility for clinical studies. Assays measuring the HER-2 protein in tumor tissue and the HER-2 extracellular domain in plasma were used to show interdiction of the Hsp90 pathway and utility as potential biomarkers in clinical trials for BIIB021. Together, these data show that BIIB021 is a promising new oral inhibitor of Hsp90 with antitumor activity in preclinical models.[Mol Cancer Ther 2009;8(4):921–9]

Hsp90 Activation and Cell Cycle Regulation

The widely-expressed molecular chaperone heat shock protein 90 (Hsp90) regulates several important cellular processes via its’ repertoire of ‘client’ proteins. Signal transduction pathways controlled by Hsp90 contribute to all major components of the malignant phenotype, so Hsp90 inhibitors are under investigation as anticancer agents. Since Hsp90 is also expressed at high levels in many normal tissues, it was unclear why Hsp90 inhibitors such as 17-allylamino-geldanamycin (17-AAG) have selective antitumor activity in animals and are well tolerated clinically. Recent findings indicate that Hsp90 is largely latent in unstressed normal cells, but tumor Hsp90 becomes completely utilized during malignant progression, resulting in an activation-dependent conformational shift that radically increases 17-AAG binding affinity in cancer cells. In this article, the implications of this discovery are discussed, with particular reference to cell cycle regulation in normal and malignant cells, and the consequences of inducing cell cycle arrest with Hsp90 inhibitors.

Rationally designed high-affinity 2-amino-6-halopurine heat shock protein 90 inhibitors that exhibit potent antitumor activity

Heat shock protein 90 (Hsp90) is a molecular chaperone protein implicated in stabilizing the conformation and maintaining the function of many cell-signaling proteins. Many oncogenic proteins are more dependent on Hsp90 in maintaining their conformation, stability, and maturation than their normal counterparts. Furthermore, recent data show that Hsp90 exists in an activated form in malignant cells but in a latent inactive form in normal tissues, suggesting that inhibitors selective for the activated form could provide a high therapeutic index. Hence, Hsp90 is emerging as an exciting new target for the treatment of cancer. We now report on a novel series of 2-amino-6-halopurine Hsp90 inhibitors exemplified by 2-amino-6-chloro-9-(4-iodo-3,5-dimethylpyridin-2-ylmethyl)purine (30). These highly potent inhibitors (IC50 of 30 = 0.009 microM in a HER-2 degradation assay) also display excellent antiproliferative activity against various tumor cell lines (IC50 of 30 = 0.03 microM in MCF7 cells). Moreover, this class of inhibitors shows higher affinity for the activated form of Hsp90 compared to our earlier 8-sulfanylpurine Hsp90 inhibitor series. When administered orally to mice, these compounds exhibited potent tumor growth inhibition (>80%) in an N87 xenograft model, similar to that observed with 17-allylamino-17-desmethoxygeldanamycin (17-AAG), which is a compound currently in phase I/II clinical trials.

Development of a high throughput drug screening assay for the detection of changes in tau levels - Proof of concept with HSP90 inhibitors

Therapeutic development for Alzheimers disease has largely focused on the removal of beta amyloid because of its suggested role in the primary agent in initiating the disease process. However, with the recent discovery of mutations that result as pathologic buildup of tau in the absence of amyloid pathology, tau is beginning to be recognized as a potential target for drug discovery. We have developed a high-throughput drug screening method that allows for direct intracellular quantitation of tau protein species, enabling the fast, reliable detection of these changes. We have identified a family of small, blood brain barrier penetrant heat shock protein 90 inhibitors that significantly reduce tau protein levels in vitro. Western blot analysis demonstrated a clear inverse correlation between the tau levels and the increase in HSP27, HSP40 and HSP90. Modifications to this assay will further allow the specific analysis of pathologically relevant species. Using this assay, we have demonstrated that a class of HSP90 inhibitors is able to significantly lower intracellular tau levels most likely through induction of a heat shock response.

Identification of new biomarkers for clinical trials of Hsp90 inhibitors

The selective heat shock protein 90 (HSP90) inhibitor 17-allyamino-17-demethoxygeldanamycin (17-AAG) is currently in phase I/II clinical studies at numerous institutions. Heretofore, the biomarkers to detect 17-AAG bioactivity (Hsp70, Raf-1, and cyclin-dependent kinase 4) had to be analyzed by Western blot of cellular samples, either from tumor biopsies or peripheral blood leukocytes, a method that is both laborious and invasive. We have identified two new biomarkers [insulin-like growth factor binding protein-2 (IGFBP2) and HER-2 extracellular domain] that can be readily detected in patient sera by ELISA. Both secreted proteins are derived from or regulated by Hsp90 client proteins, raising hopes that they might be sensitive serum markers of HSP90 inhibitor activity. Several structurally unrelated HSP90 inhibitors dose-dependently decreased secretion of both IGFBP-2 and HER-2 extracellular domain into culture medium, and both proteins were more sensitive to HSP90 inhibitors than previously identified biomarkers. In sera from BT474 tumor-bearing mice, both IGFBP-2 and HER-2 extracellular domain were down-regulated by 17-AAG in a time-dependent and dose-dependent manner, coincident with the degradation of HER-2 and attenuation of AKT activity in the tumors. Furthermore, IGFBP-2 levels at the end of treatment correlated with residual tumor load, suggesting that IGFBP-2 might serve as an early indicator of therapeutic response. In addition, we also found that both IGFBP-2 and HER-2 extracellular domain levels are elevated in patient sera from several cancer types, suggesting that these novel secreted biomarkers could be valuable pharmacodynamic tools in clinical trials of HSP90 inhibitors. [Mol Cancer Ther 2006;5(5):1256–64]