PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer.

Prostate cancer has the second highest incidence among cancers in men worldwide and is the second leading cause of cancer deaths of men in the United States. Although androgen deprivation can initially lead to remission, the disease often progresses to castration-resistant prostate cancer (CRPC), which is still reliant on androgen receptor (AR) signaling and is associated with a poor prognosis. Some success against CRPC has been achieved by drugs that target AR signaling, but secondary resistance invariably emerges, and new therapies are urgently needed. Recently, inhibitors of bromodomain and extra-terminal (BET) family proteins have shown growth-inhibitory activity in preclinical models of CRPC. Here, we demonstrate that ARV-771, a small-molecule pan-BET degrader based on proteolysis-targeting chimera (PROTAC) technology, demonstrates dramatically improved efficacy in cellular models of CRPC as compared with BET inhibition. Unlike BET inhibitors, ARV-771 results in suppression of both AR signaling and AR levels and leads to tumor regression in a CRPC mouse xenograft model. This study is, to our knowledge, the first to demonstrate efficacy with a small-molecule BET degrader in a solid-tumor malignancy and potentially represents an important therapeutic advance in the treatment of CRPC.

Discovery of GS-9973, a selective and orally efficacious inhibitor of spleen tyrosine kinase.

Spleen tyrosine kinase (Syk) is an attractive drug target in autoimmune, inflammatory, and oncology disease indications. The most advanced Syk inhibitor, R406, 1 (or its prodrug form fostamatinib, 2), has shown efficacy in multiple therapeutic indications, but its clinical progress has been hampered by dose-limiting adverse effects that have been attributed, at least in part, to the off-target activities of 1. It is expected that a more selective Syk inhibitor would provide a greater therapeutic window. Herein we report the discovery and optimization of a novel series of imidazo[1,2-a]pyrazine Syk inhibitors. This work culminated in the identification of GS-9973, 68, a highly selective and orally efficacious Syk inhibitor which is currently undergoing clinical evaluation for autoimmune and oncology indications.

PF-03814735, an orally bioavailable small molecule aurora kinase inhibitor for cancer therapy.

The Aurora family of highly related serine/threonine kinases plays a key role in the regulation of mitosis. Aurora1 and Aurora2 play important but distinct roles in the G(2) and M phases of the cell cycle and are essential for proper chromosome segregation and cell division. Overexpression and amplification of Aurora2 have been reported in different tumor types, including breast, colon, pancreatic, ovarian, and gastric cancer. PF-03814735 is a novel, potent, orally bioavailable, reversible inhibitor of both Aurora1 and Aurora2 kinases that is currently in phase I clinical trials for the treatment of advanced solid tumors. In intact cells, the inhibitory activity of PF-03814735 on the Aurora1 and Aurora2 kinases reduces levels of phospho-Aurora1, phosphohistone H3, and phospho-Aurora2. PF-03814735 produces a block in cytokinesis, resulting in inhibition of cell proliferation and the formation of polyploid multinucleated cells. Although PF-03814735 produces significant inhibition of several other protein kinases, the predominant biochemical effects in cellular assays are consistent with inhibition of Aurora kinases. Once-daily oral administration of PF-03814735 to mice bearing human xenograft tumors produces a reduction in phosphohistone H3 in tumors at doses that are tolerable and that result in significant inhibition of tumor growth. The combination of PF-03814735 and docetaxel in xenograft mouse tumor models shows additive tumor growth inhibition. These results support the clinical evaluation of PF-03814735 in cancer patients. Mol Cancer Ther; 9(4); 883-94. (c)2010 AACR.

Discovery and pharmacologic characterization of CP-724,714, a selective ErbB2 tyrosine kinase inhibitor.

Amplification and overexpression of erbB2 (Her-2/neu) proto-oncogene has been linked to human malignancies including tumors of the breast, ovary, and stomach. It has been implicated in tumor growth, sensitivity to standard chemotherapy, prognosis of patients, and disease-free survival. Although the clinical use of trastuzumab (Herceptin) has prolonged the survival of breast cancer patients with erbB2-overexpressing tumors, there is an urgent need for more potent and orally bioavailable small-molecule inhibitors. CP-724,714 is a potent inhibitor of erbB2 receptor autophosphorylation in intact cells and is currently undergoing phase I clinical trials. Here, we describe the effects of CP-724,714 in vitro and in vivo in human breast cancer models. CP-724,714 is selective for inhibiting growth of HER2-driven cell lines. In addition, we show that it induces G1 cell cycle block in erbB2-overexpressing BT-474 human breast carcinoma cells and inhibits erbB2 autophosphorylation in xenografts when administered p.o. to athymic mice. It induces a marked reduction of extracellular signal-regulated kinase and Akt phosphorylation, tumor cell apoptosis, and release of caspase-3. P.o. administration (q.d. or b.i.d.) of CP-724,714 inhibits the growth of erbB2-overexpressing tumors in athymic mice without overt adverse effects.

Combination therapy enhances the inhibition of tumor growth with the fully human anti-type 1 insulin-like growth factor receptor monoclonal antibody CP-751,871.

PURPOSE: The insulin-like growth factor (IGF) signaling pathway is implicated in cellular mitogenesis, angiogenesis, tumor cell survival, and tumorigenesis. Inhibition of this pathway results in decreased cell growth, inhibition of tumor formation in animal models, and increased apoptosis in cells treated with cytotoxic chemotherapy. We generated and characterized a human monoclonal antibody that targeted the IGF receptor. EXPERIMENTAL DESIGN: By use of XenoMouse technology, we generated CP-751,871, a fully human IgG2 antibody with high affinity (K(d) = 1.5 nmol/L) for human IGF-1R and evaluated its biological, pharmacologic, and antitumor properties. RESULTS: This antibody blocks binding of IGF-1 to its receptor (IC(50) 1.8 nmol/L), IGF-1-induced receptor autophosphorylation (IC(50) 0.42 nmol/L) and induced the down-regulation of IGF-1R in vitro and in tumor xenografts. The extent of IGF-1R down-regulation in vivo was proportional to CP-751,871 concentrations in the serum of tumor-bearing mice. Pharmacokinetic profiles in cynomolgus monkeys indicated a close to linear increase of exposure following i.v. dosing of antibody in the range of 3 to 100 mg/kg. CP-751,871 showed significant antitumor activity both as a single agent and in combination with Adriamycin, 5-fluorouracil, or tamoxifen in multiple tumor models. A biomarker assay was developed to establish the relationship between circulating antibody concentrations and down-regulation of IGF-1R in peripheral blood cells. The concentration of CP-751,871 required to down-regulate 50% of IGF-1R on peripheral blood cells was 0.3 nmol/L. CONCLUSION: These data suggest that inhibition of the IGF cascade by use of this monoclonal antibody may be of clinical benefit in the treatment of human cancers.

Design and SAR of thienopyrimidine and thienopyridine inhibitors of VEGFR-2 kinase activity.

Novel classes of thienopyrimidines and thienopyridines have been identified as potent inhibitors of VEGFR-2 kinase. The synthesis and SAR of these compounds is presented, along with successful efforts to diminish EGFR activity present in the lead series.

Pharmacological characterization of CP-547,632, a novel vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor for cancer therapy.

Signaling through vascular endothelial growth factor (VEGF) receptors (VEGFRs) is a key pathway initiating endothelial cell proliferation and migration resulting in angiogenesis, a requirement for human tumor growth and metastasis. Abrogation of signaling through VEGFR by a variety of approaches has been demonstrated to inhibit angiogenesis and tumor growth. Small molecule inhibitors of VEGFR tyrosine kinase have been shown to inhibit angiogenesis, inhibit tumor growth, and prevent metastases. Our goal was to discover and characterize an p.o. active VEGFR-2 small molecule inhibitor. A novel isothiazole, CP-547,632, was identified as a potent inhibitor of the VEGFR-2 and basic fibroblast growth factor (FGF) kinases (IC(50) = 11 and 9 nM, respectively). It is selective relative to epidermal growth factor receptor, platelet-derived growth factor beta, and other related TKs. It also inhibits VEGF-stimulated autophosphorylation of VEGFR-2 in a whole cell assay with an IC(50) value of 6 nM. After oral administration of CP-547,632 to mice bearing NIH3T3/H-ras tumors, VEGFR-2 phosphorylation in tumors was inhibited in a dose-dependent fashion (EC(50) = 590 ng/ml). These plasma concentrations correlated well with the observed concentrations of the compound necessary to inhibit VEGF-induced corneal angiogenesis in BALB/c mice. A sponge angiogenesis assay was used to directly compare the inhibitory activities of CP-547,632 against FGF receptor 2 or VEGFR-2; this compound potently inhibits both basic FGF and VEGF-induced angiogenesis in vivo. The antitumor efficacy of this agent was evaluated after once daily p.o. administration to athymic mice bearing human xenografts and resulted in as much as 85% tumor growth inhibition. CP-547,632 is a well-tolerated, orally-bioavailable inhibitor presently under clinical investigation for the treatment of human malignancies.

The biological and biochemical effects of CP-654577, a selective erbB2 kinase inhibitor, on human breast cancer cells.

Aberrant expression or activity of epidermal growth factor receptor (EGFr) or the closely related p185(erbB2) can promote cell proliferation and survival and thereby contribute to tumorigenesis. Specific antibodies and low molecular-weight tyrosine kinase inhibitors of both proteins are in clinical trials for cancer treatment. CP-654577 is a potent inhibitor selective for p185(erbB2), relative to EGFr tyrosine kinase, and selectively reduces erbB2 autophosphorylation in intact cells. Treatment of SKBr3 human breast cancer cells with CP-654577 reduces the levels of the activated form of mitogen-activated protein kinase, increases the levels of cyclin-dependent kinase inhibitor p27(kip1) and reduces expression of cyclins D and E. These biochemical changes result in a reduced level of phosphorylated retinoblastoma protein and an inhibition of cell-cycle progression at G(1). Apoptosis is triggered in both SKBr3 and another high erbB2-expressing cell line, BT474, by exposure to 1 micro M CP-654577, but this effect is not observed in MCF7 cells that express low erbB2. Levels of activated Akt, an important positive regulator of cell survival, are reduced within 2 h of exposure to 250 nM CP-654577, and this may contribute to the increased apoptosis. These biochemical effects are distinct from those produced by Tarceva, a selective EGFr inhibitor. The antitumor activity of CP-654577 was investigated in athymic mice bearing s.c. tumors from Fischer rat embryo fibroblasts transfected with erbB2. CP-654577 produced a dose-dependent reduction of p185(erbB2) autophosphorylation and inhibited the growth of these tumors. CP-654577 warrants further evaluation in tumors with high expression of p185(erbB2) and may differ from selective EGFr inhibitors or nonselective dual EGFr/erbB2 inhibitors in efficacy and therapeutic index.