Hsp90 Inhibitory Activity Research Articles

A Library of Noviosylated Coumarin Analogues

The DNA gyrase inhibitor, novobiocin, was recently shown to inhibit Hsp90 via a previously unrecognized C-terminal ATP-binding site. Previous structure-activity relationship studies identified key moieties that appear important for Hsp90 inhibitory activity. In an effort to provide a more efficacious lead compound, a parallel library of noviosylated coumarin analogues was prepared.

Total Synthesis of Herbimycin A

[structure: see text] Hsp90 has recently emerged as a promising biological target for treatment of cancer. Herbimycin A and other members of the benzoquinoid ansamycin class of natural products are known to inhibit Hsp90 activity. The total synthesis of herbimycin A was achieved from the commercially available Roche ester 1 by using allylmetals to control the stereogenic centers at C6, C7, C10, C11, and C12 and a ring-closing metathesis to control the (Z)-double bond of the (E,Z)-dienic moiety.

Novobiocin: Redesigning a DNA Gyrase Inhibitor for Selective Inhibition of Hsp90

Novobiocin is a member of the coumermycin family of antibiotics and is a well-established inhibitor of DNA gyrase. Recent studies have shown that novobiocin binds to a previously unrecognized ATP-binding site at the C-terminus of Hsp90 and induces degradation of Hsp90-dependent client proteins at approximately 700 microM. In an effort to develop more efficacious inhibitors of the C-terminal binding site, a library of novobiocin analogues was prepared and initial structure-activity relationships revealed. These data suggested that the 4-hydroxy moiety of the coumarin ring and the 3'-carbamate of the noviose appendage were detrimental to Hsp90 inhibitory activity. In an effort to confirm these findings, 4-deshydroxy novobiocin (DHN1) and 3'-descarbamoyl-4-deshydroxynovobiocin (DHN2) were prepared and evaluated against Hsp90. Both compounds were significantly more potent than the natural product, and DHN2 proved to be more active than DHN1. In an effort to determine whether these moieties are important for DNA gyrase inhibition, these compounds were tested for their ability to inhibit DNA gyrase and found to exhibit significant reduction in gyrase activity. Thus, we have established the first set of compounds that clearly differentiate between the C-terminus of Hsp90 and DNA gyrase, converted a well-established gyrase inhibitor into a selective Hsp90 inhibitor, and confirmed essential structure-activity relationships for the coumermycin family of antibiotics.

Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators

Although androgen receptor (AR)-mediated signaling is central to prostate cancer, the ability to modulate AR signaling states is limited. Here we establish a chemical genomic approach for discovery and target prediction of modulators of cancer phenotypes, as exemplified by AR signaling. We first identify AR activation inhibitors, including a group of structurally related compounds comprising celastrol, gedunin, and derivatives. To develop an in silico approach for target pathway identification, we apply a gene expression-based analysis that classifies HSP90 inhibitors as having similar activity to celastrol and gedunin. Validating this prediction, we demonstrate that celastrol and gedunin inhibit HSP90 activity and HSP90 clients, including AR. Broadly, this work identifies new modes of HSP90 modulation through a gene expression-based strategy.

Structural and Quantum Chemical Studies of 8-Aryl-sulfanyl Adenine Class Hsp90 Inhibitors

Hsp90 chaperones play a critical role in modulating the activity of many cell signaling proteins and are an attractive target for anti-cancer therapeutics. We report here the structures of the water soluble 8-aryl-sulfanyl adenine class Hsp90 inhibitors, 1 (PU-H71) and 2 (PU-H64), in complex with the N-terminal domain of human Hsp90alpha. The conformation of 1 when bound to Hsp90 differs from previously reported 8-aryl adenine Hsp90 inhibitors including 3 (PU24FCl). While the binding mode for 3 places the 2'-halide of the 8-aryl group on top of the adenine ring, for 1 and 2, we show that the 2'-halide is rotated approximately 180 degrees away. This difference explains the opposing trends in Hsp90 inhibitory activity for the 2'-halo derivatives of the 3',4',5'-trimethoxy series where Cl > Br > I compared to the 4',5'-methylenedioxy series where I > Br > Cl. We also present quantum chemical calculations of 2 and its analogues that illuminate their basis for Hsp90 inhibition. The calculated conformation of 2 agreed well with the crystallographically observed conformations of 1 and 2. The predictive nature of the calculations has allowed the exploration of additional derivatives based on the 8-aryl adenine scaffold.

Geldanamycin, Radicicol, and Chimeric Inhibitors of the Hsp90 Nterminal ATP Binding Site

Natural products have continued to drive the development of new chemotherapeutics and elucidation of new biological targets for the treatment of disease. Since Whitesell and Neckers' original discovery that geldanamycin does not directly inhibit v-Src, but instead manifests its biological activity through inhibition of the Hsp90 molecular chaperone, additional natural products and natural product derivatives have been identified and developed to inhibit the Hsp90 protein folding machinery. 17-AAG, a geldanamycin analogue, is currently in clinical trials for the treatment of several types of cancer. Recent work has produced improved radicicol analogues that show promising Hsp90 inhibitory activity in vitro. In addition, chimeric molecules of these two natural products are active in vitro and represent a novel class of Hsp90 inhibitors for cancer treatment. In addition to their chemotherapeutic uses, natural product inhibitors and their derivatives have been utilized to probe the biological mechanisms by which Hsp90 inhibition regulates tumor cell growth. As a consequence of these studies, the molecular chaperones have emerged as an exciting new class of therapeutic targets. This review will highlight the utility of the natural products, geldanamycin and radicicol, as well as improved analogues and the activities exhibited by these compounds against various cancer cell lines.

ZAP-70 Expressed in CLL Is an Hsp90 Client Protein and Its Level of Expression Can Be Downregulated by Specific Hsp90 Inhibitors.

Hsp90 is a ubiquitous molecular chaperone protein that is involved in the folding, activation and assembly of many client proteins, including mediators of signal transduction, cell-cycle control and transcriptional regulation. In order to exert its function on client proteins, Hsp90 requires the formation of an active protein complex composed of cochaperone molecules and an active ATP binding site. Proteins identified as Hsp90 client proteins include transmembrane tyrosine kinases [HER-2/neu, epidermal growth factor receptor (EGFR), MET and insulin-like growth factor-1 receptor (IGF-1R)], metastable signaling proteins (Akt, Raf-1 and IKK), mutated signaling proteins (p53, Kit, Flt3 and v-src), chimeric signaling proteins (NPM-ALK, Bcr-Abl), steroid receptors (androgen, estrogen and progesterone receptors), cell-cycle regulators (cdk4, cdk6) and apoptosis related proteins. It has been postulated that malignant progression and cancer prognosis may be associated with the presence of activated Hsp90 which exists in heightened complexes with cochaperone proteins.The zeta-associated protein of 70 kD (ZAP-70), is a protein kinase found in a subset of patients with chronic lymphocytic leukemia (CLL) and its expression correlates with disease progression, poor clinical outcome, decreased overall survival and early requirement for treatment. ZAP-70 promotes phosphorylation of downstream signaling molecules after engagement of the B cell receptor (BCR). Since ZAP-70 is a marker of disease progression in CLL we hypothesized that its abnormal over-expression and function in CLL B cells may depend on activated Hsp90. To test this, we performed immunoprecipitation, western blot and flow cytometry analyses on CLL samples from patients that differ in their level of ZAP-70 expression (ZAP-70 positive and negative based on a cutoff expression of 20% by flow cytometry). We observed that in ZAP-70 positive CLL samples (n=4) the majority of Hsp90 present in the cytoplasm was in a complexed-activated form, whereas in ZAP-70 negative samples (n=4) most Hsp90 was found to be non-complexed. In addition, we found that ZAP-70 co-immunoprecipitated with Hsp90, suggesting that it is an Hsp90 client protein. In addition, the level of ZAP-70 expression was decreased 30–40% in cells treated for 24 hours with Hsp90 specific inhibitors at nanomolar concentrations.Together, our data show that CLL B cells that express ZAP-70 have larger amounts of activated Hsp90 than those with negligible expression of this tyrosine kinase protein. In addition, we found that ZAP-70 is an Hsp90 client protein and its level of expression could be downregulated by using specific Hsp90 inhibitors. These findings suggest an important role for Hsp90 as a prognostic marker in CLL and provide the rationale to investigate, in clinical trials, the activity of Hsp90 inhibitors and their effect on ZAP-70 expression.

Hsp90: an emerging target for breast cancer therapy.

Rapidly evolving insights into the specific molecular genetic abnormalities that drive the growth and metastasis of breast cancer have led to the development of targeted therapeutics that do not rely on the generalized disruption of DNA metabolism and cell division for activity. Of particular interest are inhibitors of cellular signal transduction pathways involving tyrosine kinases as well as selective modulators of steroid hormone signaling, histone acetylation, angiogenesis and tumor cell apoptosis. Unique within this array of promising new agents, however, are compounds that target heat shock protein 90 (Hsp90). This molecular chaperone associates with a distinct, but surprisingly diverse, set of proteins that are referred to as Hsp90 client proteins. Hsp90 binds to these clients, and plays a key role in regulating their stability and function. Many of the proteins chaperoned by Hsp90 are involved in breast cancer progression and resistance to therapy, including the estrogen receptor, receptor tyrosine kinases of the erbB family, Akt, and mutant p53. Several small molecule inhibitors of Hsp90 have been identified that can deplete cellular levels of multiple oncogenic client proteins simultaneously by enhancing their ubiquitination and proteasome-mediated degradation. The activity of Hsp90 inhibitors has been well validated in preclinical breast cancer models, both in single-agent studies and in combination with conventional chemotherapy. One of these inhibitors, 17-allylamino, 17-demethoxygeldanamycin (17-AAG, NSC 330507) has recently completed phase I testing. The agent was well tolerated at drug exposures that were shown to cause modulation of Hsp90 client protein levels. Given the redundancy and complexity of the molecular abnormalities present in most breast cancers, the ability of Hsp90 inhibitors to alter the activity of multiple oncogenic targets may prove of unique therapeutic benefit.

Effects of the histone deacetylase inhibitor (HDACI) LAQ824 on histone acetylation, Hsp70 and c-Raf in peripheral blood lymphocytes from patients with advanced solid tumours enrolled in a phase I clinical trial

3023 Background: Several HDACIs, including LAQ824, are now undergoing clinical evaluation. Preclinical data indicate that as well as promoting histone acetylation, these agents can also affect the activity of the molecular chaperone Hsp90 resulting in increased expression of Hsp70 and degradation of key client proteins such as c-Raf. Hsp90 represents an important target for novel therapeutics (Fuino et al. Mol. Cancer Ther. 2003). The purpose of this study was to determine whether an HDACI-mediated impact on Hsp90 could be detected clinically. The assessment was performed on peripheral blood lymphocytes (PBL) taken from patients participating in a Phase I trial of LAQ824. Methods: A dose-escalating, open-label, Phase I trial was undertaken, with LAQ824 being administered by i.v. infusion on days 1–3 q21 days. PBL were collected at various timepoints on days 1, 3, 4, 5 and 8, lysed and assayed for acetylated histone H3 and H4, Hsp70 and c-Raf expression using Western blotting and, in some cases, ELISA. Histone extracts and whole cell lysates from two human colorectal tumour cell lines (HCT116 and HT29) either untreated or treated with LAQ824 were analysed as controls. Results: A consistent increase in acetylation of histones H3 and H4 in PBL lysates on days of treatment with LAQ824 was observed from doses of 36mg/m2 (50% MTD) and maintained for at least 24 hours. Additionally, in selected samples from patients treated at 36 mg/m2 and above, an effect on Hsp90 activity was demonstrated with increased expression of Hsp70 and decreased c-Raf levels over a similar time course. These results were consistent with those obtained from LAQ824 treated HCT116 and HT29 cells. Conclusions: These observed events in PBL suggest that as well as increasing histone acetylation, HDAC inhibition by LAQ824 does inhibit Hsp90 activity in patients at doses that are generally well-tolerated. Additional evaluation of these pharmacodynamic markers in tumour biopsies is ongoing. The biological importance of these non-histone mediated effects requires further study. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Novartis

Pharmacological and genetic analysis of 90-kDa heat shock isoprotein-aryl hydrocarbon receptor complexes.

The 90-kDa heat shock protein (Hsp90) is an abundant chaperone that regulates a diverse set of intracellular signaling proteins. Drugs that inhibit Hsp90 activity have been useful in the identification of novel Hsp90-dependent signaling pathways. One class of inhibitory compounds disrupts Hsp90-dependent processes by binding to the N-terminal ATPase/p23-binding domain of Hsp90, whereas a second inhibitor class binds within the C-terminal domain. We used signaling by aryl hydrocarbon receptor (AhR), an Hsp90-dependent transcription factor, as a functional probe to study the effects of Hsp90 inhibitors in yeast strains with deletion mutations of individual Hsp90 and p23 cochaperone genes. The more abundant and constitutively expressed Hsp90 isoform, Hsc82, functioned best in supporting AhR signaling. Deletion of the more inducible isoform, Hsp82, had no effect on signaling. AhR complexes containing Hsc82 were preferentially sensitive to the effects of low concentrations of the N-terminal inhibitors radicicol and herbimycin A. However, both Hsp90 isoforms were equally sensitive to the AhR-specific effects of novobiocin, which binds to the C terminus. Hsp90 inhibitors had no preferential effects on AhR signaling in strains that lacked p23, suggesting that the inhibitors exert their effects through a p23-independent mechanism. In contrast, overexpression of p23 buffered the effects of radicicol and herbimycin A, but not novobiocin, on AhR signaling. The data collectively suggest preferential use or function of the Hsc82 isoprotein in AhR signaling and provide new insight into the effects of three structurally unrelated Hsp90 inhibitors.

Array technology in the molecular pharmacology of anticancer agents

Exposure of cells to anticancer agents can result in altered gene expression. In general, only a single gene or a small number of genes are studied. To improve the efficiency and value of such experiments, we are interested in using DNA array technology to identify, on a genome-wide scale, drug-responsive genes which might serve both as surrogate markers for drug action and as future targets for drug development. As a means to establish this approach in our Centre we have carried out two exploratory projects. In the first project, we examined gene expression in p53 wild-type and mutant ovarian cell lines established from two patients with ovarian cancer who achieved a complete remission following treatment. Neither line can grow in vitro and were passaged as subcutaneous xenografts in nude mice. In the xenograft model both tumours were curable following multiple dosing with cisplatin. Growth-delay experiments following a single dose of 6 mg/kg cisplatin demonstrated that the p53 mutant PXN65 line was more sensitive to cisplatin than the wild-type PXN100 line. Northern blot, RNase protection and western blot demonstrated the induction of p21, MDM-2 and Bax expression in PXN100, but not PXN65. This was consistent with the p53 status of these tumours. These studies were extended to two commercial cDNA arrays, one encompassing genes involved in signal transduction, cell cycle and apoptosis regulation and the other with ESTs of known identity. There was very little difference in the overall constitutive expression pattern between the two cell lines. Following cisplatin treatment the expression of a number of p53-responsive genes was increased in the wild-type PXN100 cell line. There were also changes in the expression of a number of cell-cycle regulators and regulators of the insulin-like growth/survival factor pathway. In contrast, no major changes in gene expression were detected in the p53 mutant PXN65 line. In the second project, we have looked at the effects of a novel agent, the benzoquinone ansamycin 17-allylamino geldanamycin (17-AAG), in two colon adenocarcinoma cancer lines in vitro. This agent inhibits hsp-90 activity, a protein required to maintain the stability of a number of signalling proteins, including c-Raf-1, and is about to enter clinical trial in our centre and elsewhere. Treatment of HT29 and HCT116 cells rapidly reduced the levels of c-Raf-1 detected by western blot and inhibited the high levels of constitutive MAP kinase phosphorylation detected in HCT116 cells. This occurred as early as 24 hours and resulted in death by apoptosis 72 hours after treatment. Data detailing changes in gene expression in response to 17-AAG treatment of both HT29 and HCT 116 cells will be presented.