Martin J. Drysdale

NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

We describe the biological properties of NVP-AUY922, a novel resorcinylic isoxazole amide heat shock protein 90 (HSP90) inhibitor. NVP-AUY922 potently inhibits HSP90 (K(d) = 1.7 nmol/L) and proliferation of human tumor cells with GI(50) values of approximately 2 to 40 nmol/L, inducing G(1)-G(2) arrest and apoptosis. Activity is independent of NQO1/DT-diaphorase, maintained in drug-resistant cells and under hypoxic conditions. The molecular signature of HSP90 inhibition, comprising induced HSP72 and depleted client proteins, was readily demonstrable. NVP-AUY922 was glucuronidated less than previously described isoxazoles, yielding higher drug levels in human cancer cells and xenografts. Daily dosing of NVP-AUY922 (50 mg/kg i.p. or i.v.) to athymic mice generated peak tumor levels at least 100-fold above cellular GI(50). This produced statistically significant growth inhibition and/or regressions in human tumor xenografts with diverse oncogenic profiles: BT474 breast tumor treated/control, 21%; A2780 ovarian, 11%; U87MG glioblastoma, 7%; PC3 prostate, 37%; and WM266.4 melanoma, 31%. Therapeutic effects were concordant with changes in pharmacodynamic markers, including induction of HSP72 and depletion of ERBB2, CRAF, cyclin-dependent kinase 4, phospho-AKT/total AKT, and hypoxia-inducible factor-1alpha, determined by Western blot, electrochemiluminescent immunoassay, or immunohistochemistry. NVP-AUY922 also significantly inhibited tumor cell chemotaxis/invasion in vitro, WM266.4 melanoma lung metastases, and lymphatic metastases from orthotopically implanted PC3LN3 prostate carcinoma. NVP-AUY922 inhibited proliferation, chemomigration, and tubular differentiation of human endothelial cells and antiangiogenic activity was reflected in reduced microvessel density in tumor xenografts. Collectively, the data show that NVP-AUY922 is a potent, novel inhibitor of HSP90, acting via several processes (cytostasis, apoptosis, invasion, and angiogenesis) to inhibit tumor growth and metastasis. NVP-AUY922 has entered phase I clinical trials.

NVP-AUY922: a small molecule HSP90 inhibitor with potent antitumor activity in preclinical breast cancer models

IntroductionHeat shock protein 90 (HSP90) is a key component of a multichaperone complex involved in the post-translational folding of a large number of client proteins, many of which play essential roles in tumorigenesis. HSP90 has emerged in recent years as a promising new target for anticancer therapies.MethodsThe concentrations of the HSP90 inhibitor NVP-AUY922 required to reduce cell numbers by 50% (GI50 values) were established in a panel of breast cancer cell lines and patient-derived human breast tumors. To investigate the properties of the compound in vivo, the pharmacokinetic profile, antitumor effect, and dose regimen were established in a BT-474 breast cancer xenograft model. The effect on HSP90-p23 complexes, client protein degradation, and heat shock response was investigated in cell culture and breast cancer xenografts by immunohistochemistry, Western blot analysis, and immunoprecipitation.ResultsWe show that the novel small molecule HSP90 inhibitor NVP-AUY922 potently inhibits the proliferation of human breast cancer cell lines with GI50 values in the range of 3 to 126 nM. NVP-AUY922 induced proliferative inhibition concurrent with HSP70 upregulation and client protein depletion – hallmarks of HSP90 inhibition. Intravenous acute administration of NVP-AUY922 to athymic mice (30 mg/kg) bearing subcutaneous BT-474 breast tumors resulted in drug levels in excess of 1,000 times the cellular GI50 value for about 2 days. Significant growth inhibition and good tolerability were observed when the compound was administered once per week. Therapeutic effects were concordant with changes in pharmacodynamic markers, including HSP90-p23 dissociation, decreases in ERBB2 and P-AKT, and increased HSP70 protein levels.ConclusionNVP-AUY922 is a potent small molecule HSP90 inhibitor showing significant activity against breast cancer cells in cellular and in vivo settings. On the basis of its mechanism of action, preclinical activity profile, tolerability, and pharmaceutical properties, the compound recently has entered clinical phase I breast cancer trials.

Combining Hit Identification Strategies: Fragment- Based and in Silico Approaches to Orally Active 2-Aminothieno[2,3-D]Pyrimidine Inhibitors of the Hsp90 Molecular Chaperone.

Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential molecular therapeutic agents for the treatment of cancer. Here we describe novel 2-aminothieno[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors, which were designed by combining structural elements of distinct low affinity hits generated from fragment-based and in silico screening exercises in concert with structural information from X-ray protein crystallography. Examples from this series have high affinity (IC50 = 50-100 nM) for Hsp90 as measured in a fluorescence polarization (FP) competitive binding assay and are active in human cancer cell lines where they inhibit cell proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Several examples (34a, 34d and 34i) caused tumor growth regression at well tolerated doses when administered orally in a human BT474 human breast cancer xenograft model.

Novel adenosine-derived inhibitors of 70 kDa heat shock protein, discovered through structure-based design

The design and synthesis of novel adenosine-derived inhibitors of HSP70, guided by modeling and X-ray crystallographic structures of these compounds in complex with HSC70/BAG-1, is described. Examples exhibited submicromolar affinity for HSP70, were highly selective over HSP90, and some displayed potency against HCT116 cells. Exposure of compound 12 to HCT116 cells caused significant reduction in cellular levels of Raf-1 and Her2 at concentrations similar to that which caused cell growth arrest.

Discovery and development of pyrazole-scaffold Hsp90 inhibitors.

This review explains why the chaperone Hsp90 is an exciting protein target for the discovery of new drugs to treat cancer in the clinic, and summarises the properties of natural product derived inhibitors before relating the discovery and current state of development of synthetic pyrazole compounds. Blockade of Hsp90 results in reduced cellular levels of several proteins implicated in cancer including CDK4, ERBB2 and C-RAF, and causes simultaneous inhibition of cancer cell proliferation in culture and of tumor xenograft growth in vivo. Hsp90 has an ATPase domain that is necessary for its Hsp chaperone function, and X-ray crystallography has shown that natural product inhibitors (geldanamycin, radicicol) of Hsp90 function bind to this domain. High throughput assays focusing on the ATPase activity of Hsp90 were developed and used to discover novel chemical starting points for cancer drug discovery. The discovery, synthesis and SAR of 3,4-diaryl pyrazoles is described. X-Ray crystallography of protein-inhibitor complexes revealed important interactions involving the resorcinol substituent at C-3, and these X-ray structures strongly influenced subsequent medicinal chemistry research that has resulted in highly potent inhibitors with sub-micromolar activity in cells. SAR and X-ray data are summarised for analogues in which the 4-phenyl substituent is replaced by amides or piperazine derivatives. Prospects for the pyrazoles as they progress towards clinical development are discussed in relation to current Phase I trials with derivatives of geldanamycin.

Design and characterization of libraries of molecular fragments for use in NMR screening against protein targets

We have designed four generations of a low molecular weight fragment library for use in NMR-based screening against protein targets. The library initially contained 723 fragments which were selected manually from the Available Chemicals Directory. A series of in silico filters and property calculations were developed to automate the selection process, allowing a larger database of 1.79 M available compounds to be searched for a further 357 compounds that were added to the library. A kinase binding pharmacophore was then derived to select 174 kinase-focused fragments. Finally, an additional 61 fragments were selected to increase the number of different pharmacophores represented within the library. All of the fragments added to the library passed quality checks to ensure they were suitable for the screening protocol, with appropriate solubility, purity, chemical stability, and unambiguous NMR spectrum. The successive generations of libraries have been characterized through analysis of structural properties (molecular weight, lipophilicity, polar surface area, number of rotatable bonds, and hydrogen-bonding potential) and by analyzing their pharmacophoric complexity. These calculations have been used to compare the fragment libraries with a drug-like reference set of compounds and a set of molecules that bind to protein active sites. In addition, an analysis of the overall results of screening the library against the ATP binding site of two protein targets (HSP90 and CDK2) reveals different patterns of fragment binding, demonstrating that the approach can find selective compounds that discriminate between related binding sites.

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 (Kd) 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 IC50 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 GI50 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 GI50 for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as HSP90 inhibitors. [Mol Cancer Ther 2007;6(4):1198–211]

The SeeDs approach: integrating fragments into drug discovery.

Finding novel compounds as starting points for optimization is a major challenge in drug discovery research. Fragment-based methods have emerged in the past ten years as an effective way to sample chemical diversity with a limited number of low molecular weight compounds. The structures of the fragments(s) binding to the protein can then be used to design new compounds with increased affinity, specificity and novelty. This article describes the Vernalis approach to fragment based drug discovery, called SeeDs (Structural exploitation of experimental Drug startpoints). The approach includes the design of a fragment library, identification of fragments that bind competitively to a target by ligand-based NMR techniques and protein crystal structures to characterize binding. Fragments that bind are then evolved to hits, either by growing the fragment or by combining structural features from a number of compounds. The process is illustrated with examples from recent medicinal chemistry programmes to discover compounds against the oncology targets Hsp90 and PDK1. In addition, we summarise our experience with using molecular docking calculations to predict fragment binding and anecdotes on the selectivity and binding modes for fragments seen against a range of targets.

Adenosine-Derived Inhibitors of 78 kDa Glucose Regulated Protein (Grp78) ATPase: Insights into Isoform Selectivity.

78 kDa glucose-regulated protein (Grp78) is a heat shock protein (HSP) involved in protein folding that plays a role in cancer cell proliferation. Binding of adenosine-derived inhibitors to Grp78 was characterized by surface plasmon resonance and isothermal titration calorimetry. The most potent compounds were 13 (VER-155008) with K(D) = 80 nM and 14 with K(D) = 60 nM. X-ray crystal structures of Grp78 bound to ATP, ADPnP, and adenosine derivative 10 revealed differences in the binding site between Grp78 and homologous proteins.

Preclinical pharmacokinetics and metabolism of a novel diaryl pyrazole resorcinol series of heat shock protein 90 inhibitors

CCT018159 was recently identified as a novel inhibitor of heat shock protein (Hsp) 90, a promising target for cancer therapy. Pharmacokinetic and metabolic properties are likely to be important for efficacy and need to be optimized during drug development. Here, we define the preclinical metabolism and pharmacokinetics of CCT018159 and some early derivatives. In addition, we assess in vitro metabolic stability screening and in vivo cassette dosing (simultaneous administration of several compounds to a single animal) as approaches to investigate these compounds. The plasma clearance following individual i.v. administration to mice was rapid (0.128–0.816 L/h), exceeding hepatic blood flow. For CCT066950 and CCT066952, this could be attributed in part to extensive (>80%) blood cell binding. Oral bioavailability ranged from 1.8% to 29.6%. Tissue distribution of CCT066952 was rapid and moderate, and renal excretion of the compounds was minimal (<1% of dose excreted). Compounds underwent rapid glucuronidation both in vivo and following incubation with mouse liver microsomes. However, whereas CCT066965 was metabolized to the greatest extent in vitro, this compound displayed the slowest plasma clearance. The rank order of the compounds from the highest to lowest area under the curve was the same following discrete and cassette dosing. Furthermore, pharmacokinetic variables were similar whether the compounds were dosed alone or in combination. We conclude that the pharmacokinetics of CCT018159 are complex. Cassette dosing is currently the best option available to assess the pharmacokinetics of this promising series of compounds in relatively high throughput and is now being applied to identify compounds with optimal pharmacokinetic properties during structural analogue synthesis. [Mol Cancer Ther 2006;5(6):1628–37]