Fused imidazoles as potential chemical scaffolds for inhibition of heat shock protein 70 and induction of apoptosis. Synthesis and biological evaluation of phenanthro[9,10-d]imidazoles and imidazo[4,5-f][1,10]phenanthrolines.

The imidazole ring is widespread in biologically active compounds, and hence imidazole-containing scaffolds are useful starting points for drug discovery programmes. We report the synthesis of a series of novel imidazole-containing compounds fused with either phenanthrene or phenanthroline, which show enhanced growth inhibitory potency against human colon, breast and melanoma cancer cell lines, as well as evidence of inhibition of the molecular chaperone heat shock protein 70 (Hsp70) pathway in cells, as shown by depletion of downstream oncogenic client proteins of the Hsp90 chaperone pathway, and induction of apoptosis.

HSF1 Pathway Inhibitor Clinical Candidate (CCT361814/NXP800) Developed from a Phenotypic Screen as a Potential Treatment for Refractory Ovarian Cancer and Other Malignancies.

CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies.

Targeting the Hsp90 chaperone: synthesis of novel resorcylic acid macrolactone inhibitors of Hsp90.

A series of benzo-macrolactones has been prepared by chemical synthesis, and evaluated as inhibitors of heat shock protein 90 (Hsp90), an emerging attractive target for novel cancer therapeutic agents. A new synthesis of these resorcylic acid macrolactone analogues of the natural product radicicol is described in which the key steps are the acylation and ring opening of a homophthalic anhydride to give an isocoumarin, followed by a ring-closing metathesis to form the macrocycle. The methodology has been extended to a novel series of macrolactones incorporating a 1,2,3-triazole ring.

Inhibition of Hsp90 with resorcylic acid macrolactones: synthesis and binding studies.

A series of resorcylic acid macrolactones, analogues of the natural product radicicol has been prepared by chemical synthesis, and evaluated as inhibitors of heat shock protein 90 (Hsp90), an emerging attractive target for novel cancer therapeutic agents. The synthesis involves acylation of an ortho-toluic acid dianion, esterification, followed by a ring-closing metathesis to form the macrocycle. Subsequent manipulation of the protected hydroxymethyl side chain allows access to a range of new analogues following deprotection of the two phenolic groups. Co-crystallization of one of the new macrolactones with the N-terminal domain of yeast Hsp90 confirms that it binds in a similar way to the natural product radicicol and to our previous synthetic analogues, but that the introduction of the additional hydroxymethyl substituent appears to result in an unexpected change in conformation of the macrocyclic ring. As a result of this conformational change, the compounds bound less favorably to Hsp90.

Solution structure of the Hop TPR2A domain and investigation of target druggability by NMR, biochemical and in silico approaches.

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an important role in tumour biology by promoting the stabilisation and activity of oncogenic 'client' proteins. Inhibition of Hsp90 by small-molecule drugs, acting via its ATP hydrolysis site, has shown promise as a molecularly targeted cancer therapy. Owing to the importance of Hop and other tetratricopeptide repeat (TPR)-containing cochaperones in regulating Hsp90 activity, the Hsp90-TPR domain interface is an alternative site for inhibitors, which could result in effects distinct from ATP site binders. The TPR binding site of Hsp90 cochaperones includes a shallow, positively charged groove that poses a significant challenge for druggability. Herein, we report the apo, solution-state structure of Hop TPR2A which enables this target for NMR-based screening approaches. We have designed prototype TPR ligands that mimic key native 'carboxylate clamp' interactions between Hsp90 and its TPR cochaperones and show that they block binding between Hop TPR2A and the Hsp90 C-terminal MEEVD peptide. We confirm direct TPR-binding of these ligands by mapping 1H-15N HSQC chemical shift perturbations to our new NMR structure. Our work provides a novel structure, a thorough assessment of druggability and robust screening approaches that may offer a potential route, albeit difficult, to address the chemically challenging nature of the Hop TPR2A target, with relevance to other TPR domain interactors.

In vitro biological characterization of a novel, synthetic diaryl pyrazole resorcinol class of heat shock protein 90 inhibitors.

The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target. Derivatives of the natural product geldanamycin, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), were the first HSP90 ATPase inhibitors to enter clinical trial. Synthetic small-molecule HSP90 inhibitors have potential advantages. Here, we describe the biological properties of the lead compound of a new class of 3,4-diaryl pyrazole resorcinol HSP90 inhibitor (CCT018159), which we identified by high-throughput screening. CCT018159 inhibited human HSP90beta with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH(2)-terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI(50) value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 mumol/L. Unlike 17-AAG, the in vitro antitumor activity of the pyrazole resorcinol analogues is independent of NQO1/DT-diaphorase and P-glycoprotein expression. The molecular signature of HSP90 inhibition, comprising increased expression of HSP72 protein and depletion of ERBB2, CDK4, C-RAF, and mutant B-RAF, was shown by Western blotting and quantified by time-resolved fluorescent-Cellisa in human cancer cell lines treated with CCT018159. CCT018159 caused cell cytostasis associated with a G(1) arrest and induced apoptosis. CCT018159 also inhibited key endothelial and tumor cell functions implicated in invasion and angiogenesis. Overall, we have shown that diaryl pyrazole resorcinols exhibited similar cellular properties to 17-AAG with potential advantages (e.g., aqueous solubility, independence from NQO1 and P-glycoprotein). These compounds form the basis for further structure-based optimization to identify more potent inhibitors suitable for clinical development.

4,5-diarylisoxazole Hsp90 chaperone inhibitors: potential therapeutic agents for the treatment of cancer.

Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential chemotherapeutic agents for cancer. Here, we describe the structure-based design, synthesis, structure-activity relationships and pharmacokinetics of potent small-molecule inhibitors of Hsp90 based on the 4,5-diarylisoxazole scaffold. Analogues from this series have high affinity for Hsp90, as measured in a fluorescence polarization (FP) competitive binding assay, and are active in cancer cell lines where they inhibit proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Compound 40f (VER-52296/NVP-AUY922) is potent in the Hsp90 FP binding assay (IC50 = 21 nM) and inhibits proliferation of various human cancer cell lines in vitro, with GI50 averaging 9 nM. Compound 40f is retained in tumors in vivo when administered i.p., as evaluated by cassette dosing in tumor-bearing mice. In a human colon cancer xenograft model, 40f inhibits tumor growth by approximately 50%.

An in vitro and in vivo study of the combination of the heat shock protein inhibitor 17-allylamino-17-demethoxygeldanamycin and carboplatin in human ovarian cancer models.

PURPOSE: To study the interactions of the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) and carboplatin in vitro and in vivo. EXPERIMENTAL DESIGN: The combination of 17-AAG and carboplatin on the growth inhibition of A2780, SKOV-3, IGROV-1 and HX62 human ovarian cancer cells was studied in vitro by MTT assays. The effect of the sequence of administration of both drugs was further investigated in A2780 cells by sulforhodamine B assays. The ability of 17-AAG to deplete HSP90 client proteins either alone or in combination with carboplatin was evaluated by western blotting. Tumor concentrations of 17-AAG and carboplatin alone or in combination in vivo were determined by validated liquid chromatography with ultraviolet detection and atomic absorption spectroscopy methods. The growth inhibitory effects of 17-AAG, carboplatin and the combination were studied in the A2780 xenograft model. RESULTS: The combination index (CI) at fu(0.5) for 17-AAG plus carboplatin was 0.97 (+/-0.12 SD) when A2780 cells were exposed to carboplatin followed by 17-AAG indicating additivity. The addition of carboplatin did not alter the ability of 17-AAG to cause C-RAF, CDK4 and p-AKT depletion or HSP70 induction. Tumor 17-AAG and carboplatin concentrations were not significantly different in the single agent and combination arms. Tumor weights relative to controls on day 6 (T/C) were 67% for the carboplatin, 64% for the 17-AAG and 22% for the combination. CONCLUSION: In the specified sequences of drug exposure, 17-AAG and carboplatin have additive growth inhibitory effects in vitro and beneficial effects were seen with the combination in vivo. These findings form the basis for the possible evaluation of 17-AAG and carboplatin in a clinical trial.

Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues.

Although the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) shows clinical promise, potential limitations encourage development of alternative chemotypes. We discovered the 3,4-diarylpyrazole resorcinol CCT018159 by high-throughput screening and used structure-based design to generate more potent pyrazole amide analogues, exemplified by VER-49009. Here, we describe the detailed biological properties of VER-49009 and the corresponding isoxazole VER-50589. X-ray crystallography showed a virtually identical HSP90 binding mode. However, the dissociation constant (K(d)) of VER-50589 was 4.5 +/- 2.2 nmol/L compared with 78.0 +/- 10.4 nmol/L for VER-49009, attributable to higher enthalpy for VER-50589 binding. A competitive binding assay gave a lower IC(50) of 21 +/- 4 nmol/L for VER-50589 compared with 47 +/- 9 nmol/L for VER-49009. Cellular uptake of VER-50589 was 4-fold greater than for VER-49009. Mean cellular antiproliferative GI(50) values for VER-50589 and VER-49009 for a human cancer cell line panel were 78 +/- 15 and 685 +/- 119 nmol/L, respectively, showing a 9-fold potency gain for the isoxazole. Unlike 17-AAG, but as with CCT018159, cellular potency of these analogues was independent of NAD(P)H:quinone oxidoreductase 1/DT-diaphorase and P-glycoprotein expression. Consistent with HSP90 inhibition, VER-50589 and VER-49009 caused induction of HSP72 and HSP27 alongside depletion of client proteins, including C-RAF, B-RAF, and survivin, and the protein arginine methyltransferase PRMT5. Both caused cell cycle arrest and apoptosis. Extent and duration of pharmacodynamic changes in an orthotopic human ovarian carcinoma model confirmed the superiority of VER-50589 over VER-49009. VER-50589 accumulated in HCT116 human colon cancer xenografts at levels above the cellular GI(50) for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as HSP90 inhibitors.

Correction to: An in vitro and in vivo study of the combination of the heat shock protein inhibitor 17-allylamino-17-demethoxygeldanamycin and carboplatin in human ovarian cancer models.

The corresponding author of this article has informed us of concerns about the immunoblots in Fig. 2 which were carried out in the collaborating laboratory of Professor Ann Jackman.

Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766).

Demonstrating intracellular protein target engagement is an essential step in the development and progression of new chemical probes and potential small molecule therapeutics. However, this can be particularly challenging for poorly studied and noncatalytic proteins, as robust proximal biomarkers are rarely known. To confirm that our recently discovered chemical probe 1 (CCT251236) binds the putative transcription factor regulator pirin in living cells, we developed a heterobifunctional protein degradation probe. Focusing on linker design and physicochemical properties, we generated a highly active probe 16 (CCT367766) in only three iterations, validating our efficient strategy for degradation probe design against nonvalidated protein targets.

Inhibition of Hsp90 with synthetic macrolactones: synthesis and structural and biological evaluation of ring and conformational analogs of radicicol.

A series of benzo-macrolactones of varying ring size and conformation has been prepared by chemical synthesis and evaluated by structural and biological techniques. Thus, 12- to 16-membered lactones were obtained by concise routes, involving ring-closing metathesis as a key step. In enzyme assays, the 13-, 15-, and 16-membered analogs are good inhibitors, suggesting that they can adopt the required conformation to fit in the ATP-binding site. This was confirmed by cocrystallization of 13-, 14-, and 15-membered lactones with the N-terminal domain of yeast Hsp90, showing that they bind similarly to the "natural" 14-membered radicicol. The most active compounds in the ATPase assays also showed the greatest growth-inhibitory potency in HCT116 human colon cancer cells and the established molecular signature of Hsp90 inhibition, i.e., depletion of client proteins with upregulation of Hsp70.

Discovery of a Chemical Probe Bisamide (CCT251236): An Orally Bioavailable Efficacious Pirin Ligand from a Heat Shock Transcription Factor 1 (HSF1) Phenotypic Screen.

Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather than affinity for individual recombinant proteins, have recently attracted renewed interest as an efficient strategy for drug discovery. In this article, we describe the discovery of a new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model. By developing cell-based SAR and using chemical proteomics, we identified pirin as a high affinity molecular target, which was confirmed by SPR and crystallography.

Discovery of 4,6-disubstituted pyrimidines as potent inhibitors of the heat shock factor 1 (HSF1) stress pathway and CDK9.

Heat shock factor 1 (HSF1) is a transcription factor that plays key roles in cancer, including providing a mechanism for cell survival under proteotoxic stress. Therefore, inhibition of the HSF1-stress pathway represents an exciting new opportunity in cancer treatment. We employed an unbiased phenotypic screen to discover inhibitors of the HSF1-stress pathway. Using this approach we identified an initial hit (1) based on a 4,6-pyrimidine scaffold (2.00 µM). Optimisation of cellular SAR led to an inhibitor with improved potency (25, 15 nM) in the HSF1 phenotypic assay. The 4,6-pyrimidine 25 was also shown to have high potency against the CDK9 enzyme (3 nM).

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.

Effects of HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on NEU/HER2 overexpressing mammary tumours in MMTV-NEU-NT mice monitored by Magnetic Resonance Spectroscopy.

BACKGROUND: The importance of ERBB2/NEU/HER2 in the response of breast tumours to the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG; tanespimycin) has been demonstrated in the clinic. ERBB2 is an oncoprotein client that is highly dependent on HSP90. This and other oncogenic client proteins (e.g. B-RAF, C-RAF, ALK and CDK4) are depleted by 17-AAG in both animal tumours and patients. Here we investigate by Magnetic Resonance Spectroscopy (MRS) the metabolic response of 17-AAG in spontaneous, NEU/HER2 driven mammary tumours in transgenic MMTV-NEU-NT mice and in cells isolated and cultured from these tumours. METHODS: Mammary tumours were monitored by 31P MRS in vivo and in tumour extracts, comparing control and 17-AAG treated mice. A cell line derived from NEU/HER2 mammary tumours was also cultured and the effect of 17-AAG was measured by 31P MRS in cell extracts. Molecular biomarkers were assessed by immunoblotting in extracts from cells and tumours. For comparison of tumour volume, metabolite concentrations and Western blot band intensities, two-tailed unpaired t-tests were used. RESULTS: The NEU/HER2 mammary tumours were very sensitive to 17-AAG and responded in a dose-dependent manner to 3 daily doses of 20, 40 and 80mg/kg of 17-AAG, all of which caused significant regression. At the higher doses, 31P MRS of tumour extracts showed significant decreases in phosphocholine (PC) and phosphoethanolamine (PE) whereas no significant changes were seen at the 20mg/kg dose. Extracts of isolated cells cultured from the mammary carcinomas showed a significant decrease in viable cell number and total PME after 17-AAG treatment. Western blots confirmed the expected action of 17-AAG in inducing HSP72 and significantly depleting HSP90 client proteins, including NEU/HER2 both in tumours and in isolated cells. CONCLUSIONS: The data demonstrate the high degree of sensitivity of this clinically relevant NEU/HER2-driven tumour model to HSP90 inhibition by 17-AAG, consistent with the clinical data, and suggest that the metabolic signature of choline phospholipids obtained by MRS could be useful both as a preclinical and clinical tool for investigating surrogate markers of response to treatment.

Targeting the Hsp90 molecular chaperone with novel macrolactams. Synthesis, structural, binding, and cellular studies.

A series of resorcylic acid macrolactams, nitrogen analogues of the naturally occurring macrolactone radicicol, have been prepared by chemical synthesis and evaluated as inhibitors of heat shock protein 90 (Hsp90), an emerging attractive target for novel cancer therapeutic agents. The synthesis involves, as key steps, ring opening of an isocoumarin intermediate, followed by a ring-closing metathesis reaction to form the macrocycle. Subsequent manipulation of the ester group into a range of amides allows access to a range of new macrolactams following deprotection of the two phenolic groups. These new resorcylic acid lactams exhibit metabolic stability greater than that of related lactone counterparts, while co-crystallization of three macrolactams with the N-terminal domain ATP site of Hsp90 confirms that they bind in a similar way to the natural product radicicol and to our previous synthetic lactone analogues. Interestingly, however, in the case of the N-benzylamide, additional binding to a hydrophobic pocket of the protein was observed. In biological assays, the new macrocyclic lactams exhibit a biological profile equivalent or superior to that of the related lactones and show the established molecular signature of Hsp90 inhibitors in human colon cancer cells.

Mechanistic evaluation of the novel HSP90 inhibitor NVP-AUY922 in adult and pediatric glioblastoma.

The dismal prognosis of glioblastoma (GB) indicates the urgent need for new therapies for these tumors. Heat shock protein 90 (HSP90) inhibitors induce the proteasome-mediated degradation of many oncogenic client proteins involved in all of the hallmark characteristics of cancer. Here, we explored the mechanistic potential of the potent synthetic diarylisoxazole amide resorcinol HSP90 inhibitor, NVP-AUY922, in adult and pediatric GB. In vitro antiproliferative potency (nanomolar range) was seen in both adult and pediatric human GB cell lines with different molecular pathologies. A cytostatic effect was observed in all GB lines; more apoptosis was observed at lower concentrations in the SF188 pediatric GB line and at 144 hours in the slower growing KNS42 pediatric GB line, as compared with the adult GB lines U87MG and SF268. In vitro combination studies with inhibitors of phosphoinositide 3-kinase/mammalian target of rapamycin (PI-103) or mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (PD-0325901) supported the hypothesis that sustained inhibition of ERK up to 72 hours and at least temporary inhibition of AKT were necessary to induce apoptosis in GB lines. In athymic mice bearing established s.c U87MG GB xenografts, NVP-AUY922 (50 mg/kg i.p x 3 days) caused the inhibition of ERK1/2 and AKT phosphorylation and induced apoptosis, whereas 17-AAG used at maximum tolerated dose was less effective. NVP-AUY922 antitumor activity with objective tumor regression resulted from antiproliferative, proapoptotic, and antiangiogenic effects, the latter shown by decreased microvessel density and HIF1alpha levels. Our results have established a mechanistic proof of concept for the potential of novel synthetic HSP90 inhibitors in adult and pediatric GB, alone or in combination with phosphoinositide 3-kinase/mammalian target of rapamycin and mitogen-activated protein/ERK kinase inhibitors.