Spirocyclic Thiohydantoin Antagonists of F877L and Wild-Type Androgen Receptor for Castration-Resistant Prostate Cancer.

Androgen receptor (AR) transcriptional reactivation plays a key role in the development and progression of lethal castration-resistant prostate cancer (CRPC). Recurrent alterations in the AR enable persistent AR pathway signaling and drive resistance to the treatment of second-generation antiandrogens. AR F877L, a point mutation in the ligand binding domain of the AR, was identified in patients who acquired resistance to enzalutamide or apalutamide. In parallel to our previous structure-activity relationship (SAR) studies of compound 4 (JNJ-pan-AR) and clinical stage compound 5 (JNJ-63576253), we discovered additional AR antagonists that provide opportunities for future development. Here we report a highly potent series of spirocyclic thiohydantoins as AR antagonists for the treatment of the F877L mutant and wild-type CRPC.

Discovery of JNJ-63576253, a Next-Generation Androgen Receptor Antagonist Active Against Wild-Type and Clinically Relevant Ligand Binding Domain Mutations in Metastatic Castration-Resistant Prostate Cancer.

Numerous mechanisms of resistance arise in response to treatment with second-generation androgen receptor (AR) pathway inhibitors in metastatic castration-resistant prostate cancer (mCRPC). Among these, point mutations in the ligand binding domain can transform antagonists into agonists, driving the disease through activation of AR signaling. To address this unmet need, we report the discovery of JNJ-63576253, a next-generation AR pathway inhibitor that potently abrogates AR signaling in models of human prostate adenocarcinoma. JNJ-63576253 is advancing as a clinical candidate with potential effectiveness in the subset of patients who do not respond to or are progressing while on second-generation AR-targeted therapeutics.

Discovery of N-(4-(3-(2-aminopyrimidin-4-yl)pyridin-2-yloxy)phenyl)-4-(4-methylthiophen-2-yl)phthalazin-1-amine (AMG 900), a highly selective, orally bioavailable inhibitor of aurora kinases with activity against multidrug-resistant cancer cell lines.

Efforts to improve upon the physical properties and metabolic stability of Aurora kinase inhibitor 14a revealed that potency against multidrug-resistant cell lines was compromised by increased polarity. Despite its high in vitro metabolic intrinsic clearance, 23r (AMG 900) showed acceptable pharmacokinetic properties and robust pharmacodynamic activity. Projecting from in vitro data to in vivo target coverage was not practical due to disjunctions between enzyme and cell data, complex and apparently contradictory indicators of binding kinetics, and unmeasurable free fraction in plasma. In contrast, it was straightforward to relate pharmacokinetics to pharmacodynamics and efficacy by following the time above a threshold concentration. On the basis of its oral route of administration, a selectivity profile that favors Aurora-driven pharmacology and its activity against multidrug-resistant cell lines, 23r was identified as a potential best-in-class Aurora kinase inhibitor. In phase 1 dose expansion studies with G-CSF support, 23r has shown promising single agent activity.

AMG 900, a potent inhibitor of aurora kinases causes pharmacodynamic changes in p-Histone H3 immunoreactivity in human tumor xenografts and proliferating mouse tissues.

BACKGROUND: The Aurora family of serine-threonine kinases are essential regulators of cell division in mammalian cells. Aurora-A and -B expression and kinase activity is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis. AMG 900 is a highly potent and selective pan-aurora kinase inhibitor that has entered clinical evaluation in adult patients with advanced cancers. In mice, oral administration of AMG 900 blocks the phosphorylation of histone H3 on serine-10 (p-Histone H3), a proximal substrate of aurora-B and inhibits the growth of multiple human tumor xenografts, including multidrug-resistant models. METHODS: In order to establish a preclinical pharmacokinetic-pharmacodynamic (PK-PD) relationship for AMG 900 that could be translated to the clinic, we used flow cytometry and laser scanning cytometry detection platforms to assess the effects on p-Histone H3 inhibition in terms of sensitivity, precision, and specificity, in human tumor xenografts in conjunction with mouse skin and bone marrow tissues. Mice with established COLO 205 tumors were administered AMG 900 at 3.75, 7.5, and 15 mg/kg and assessed after 3 hours. RESULTS: Significant suppression of p-Histone H3 in mouse skin was only observed at 15 mg/kg (p <0.0001), whereas in mouse bone marrow and in tumor a dose-dependent inhibition was achieved at all three doses (p ≤ 0.00015). These studies demonstrate that AMG 900 inhibits p-Histone H3 in tumors and surrogate tissues (although tissues such as skin may be less sensitive for assessing PD effects). To further extend our work, we evaluated the feasibility of measuring p-Histone H3 using fine-needle aspirate (FNA) tumor xenograft biopsies. Treatment with AMG 900 significantly inhibited p-Histone H3 (>99% inhibition, p <0.0001) in COLO 205 tumors. Lastly, we illustrate this LSC-based approach can detect p-Histone H3 positive cells using mock FNAs from primary human breast tumor tissues. CONCLUSION: Phosphorylation of histone H3 is a useful biomarker to determine the pharmacodynamics (PD) activity of AMG 900. FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic.

Apo2L/TRAIL and the death receptor 5 agonist antibody AMG 655 cooperate to promote receptor clustering and antitumor activity.

Death receptor agonist therapies have exhibited limited clinical benefit to date. Investigations into why Apo2L/TRAIL and AMG 655 preclinical data were not predictive of clinical response revealed that coadministration of Apo2L/TRAIL with AMG 655 leads to increased antitumor activity in vitro and in vivo. The combination of Apo2L/TRAIL and AMG 655 results in enhanced signaling and can sensitize Apo2L/TRAIL-resistant cells. Structure determination of the Apo2L/TRAIL-DR5-AMG 655 ternary complex illustrates how higher order clustering of DR5 is achieved when both agents are combined. Enhanced agonism generated by combining Apo2L/TRAIL and AMG 655 provides insight into the limited efficacy observed in previous clinical trials and suggests testable hypotheses to reconsider death receptor agonism as a therapeutic strategy.

AMG 900, a small-molecule inhibitor of aurora kinases, potentiates the activity of microtubule-targeting agents in human metastatic breast cancer models.

Breast cancer is the most prevalent malignancy affecting women and ranks second in cancer-related deaths, in which death occurs primarily from metastatic disease. Triple-negative breast cancer (TNBC) is a more aggressive and metastatic subtype of breast cancer that is initially responsive to treatment of microtubule-targeting agents (MTA) such as taxanes. Recently, we reported the characterization of AMG 900, an orally bioavailable, potent, and highly selective pan-Aurora kinase inhibitor that is active in multidrug-resistant cell lines. In this report, we investigate the activity of AMG 900 alone and in combination with two distinct classes of MTAs (taxanes and epothilones) in multidrug-resistant TNBC cell lines and xenografts. In TNBC cells, AMG 900 inhibited phosphorylation of histone H3 on Ser(10), a proximal substrate of Aurora-B, and induced polyploidy and apoptosis. Furthermore, AMG 900 potentiated the antiproliferative effects of paclitaxel and ixabepilone at low nanomolar concentrations. In mice, AMG 900 significantly inhibited the growth of MDA-MB-231 (F(11); parental), MDA-MB-231 (F(11)) PTX-r (paclitaxel-resistant variant), and DU4475 xenografts. The combination of AMG 900 with docetaxel enhanced tumor inhibition in MDA-MB-231 (F(11)) xenografts compared with either monotherapy. Notably, combining AMG 900 with ixabepilone resulted in regressions of MDA-MB-231 (F(11)) PTX-r xenografts, in which more than 50% of the tumors failed to regrow 75 days after the cessation of drug treatment. These findings suggest that AMG 900, alone and in combination with MTAs, may be an effective intervention strategy for the treatment of metastatic breast cancer and provide potential therapeutic options for patients with multidrug-resistant tumors.

Discovery and optimization of potent and selective imidazopyridine and imidazopyridazine mTOR inhibitors.

mTOR is a critical regulator of cellular signaling downstream of multiple growth factors. The mTOR/PI3K/AKT pathway is frequently mutated in human cancers and is thus an important oncology target. Herein we report the evolution of our program to discover ATP-competitive mTOR inhibitors that demonstrate improved pharmacokinetic properties and selectivity compared to our previous leads. Through targeted SAR and structure-guided design, new imidazopyridine and imidazopyridazine scaffolds were identified that demonstrated superior inhibition of mTOR in cellular assays, selectivity over the closely related PIKK family and improved in vivo clearance over our previously reported benzimidazole series.

Discovery of triazine-benzimidazoles as selective inhibitors of mTOR.

mTOR is part of the PI3K/AKT pathway and is a central regulator of cell growth and survival. Since many cancers display mutations linked to the mTOR signaling pathway, mTOR has emerged as an important target for oncology therapy. Herein, we report the discovery of triazine benzimidazole inhibitors that inhibit mTOR kinase activity with up to 200-fold selectivity over the structurally homologous kinase PI3Kα. When tested in a panel of cancer cell lines displaying various mutations, a selective inhibitor from this series inhibited cellular proliferation with a mean IC(50) of 0.41 µM. Lead compound 42 demonstrated up to 83% inhibition of mTOR substrate phosphorylation in a murine pharmacodynamic model.

Preclinical evaluation of AMG 900, a novel potent and highly selective pan-aurora kinase inhibitor with activity in taxane-resistant tumor cell lines.

In mammalian cells, the aurora kinases (aurora-A, -B, and -C) play essential roles in regulating cell division. The expression of aurora-A and -B is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis, making them attractive targets for anticancer therapy. AMG 900 is an orally bioavailable, potent, and highly selective pan-aurora kinase inhibitor that is active in taxane-resistant tumor cell lines. In tumor cells, AMG 900 inhibited autophosphorylation of aurora-A and -B as well as phosphorylation of histone H3 on Ser(10), a proximal substrate of aurora-B. The predominant cellular response of tumor cells to AMG 900 treatment was aborted cell division without a prolonged mitotic arrest, which ultimately resulted in cell death. AMG 900 inhibited the proliferation of 26 tumor cell lines, including cell lines resistant to the antimitotic drug paclitaxel and to other aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358), at low nanomolar concentrations. Furthermore, AMG 900 was active in an AZD1152-resistant HCT116 variant cell line that harbors an aurora-B mutation (W221L). Oral administration of AMG 900 blocked the phosphorylation of histone H3 in a dose-dependent manner and significantly inhibited the growth of HCT116 tumor xenografts. Importantly, AMG 900 was broadly active in multiple xenograft models, including 3 multidrug-resistant xenograft models, representing 5 tumor types. AMG 900 has entered clinical evaluation in adult patients with advanced cancers and has the potential to treat tumors refractory to anticancer drugs such as the taxanes.

Discovery of a potent, selective, and orally bioavailable pyridinyl-pyrimidine phthalazine aurora kinase inhibitor.

The discovery of aurora kinases as essential regulators of cell division has led to intense interest in identifying small molecule aurora kinase inhibitors for the potential treatment of cancer. A high-throughput screening effort identified pyridinyl-pyrimidine 6a as a moderately potent dual inhibitor of aurora kinases -A and -B. Optimization of this hit resulted in an anthranilamide lead (6j) that possessed improved enzyme and cellular activity and exhibited a high level of kinase selectivity. However, this anthranilamide and subsequent analogues suffered from a lack of oral bioavailability. Converting the internally hydrogen-bonded six-membered pseudo-ring of the anthranilamide to a phthalazine (8a-b) led to a dramatic improvement in oral bioavailability (38-61%F) while maintaining the potency and selectivity characteristics of the anthranilamide series. In a COLO 205 tumor pharmacodynamic assay measuring phosphorylation of the aurora-B substrate histone H3 at serine 10 (p-histone H3), oral administration of 8b at 50 mg/kg demonstrated significant reduction in tumor p-histone H3 for at least 6 h.

Motesanib inhibits Kit mutations associated with gastrointestinal stromal tumors.

BACKGROUND: Activating mutations in Kit receptor tyrosine kinase or the related platelet-derived growth factor receptor (PDGFR) play an important role in the pathogenesis of gastrointestinal stromal tumors (GIST). METHODS: This study investigated the activity of motesanib, an inhibitor of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3; PDGFR; and Kit, against primary activating Kit mutants and mutants associated with secondary resistance to imatinib. Single- and double-mutant isoforms of Kit were evaluated for their sensitivity to motesanib or imatinib in autophosphorylation assays and in Ba/F3 cell proliferation assays. RESULTS: Motesanib inhibited Kit autophosphorylation in CHO cell lines expressing primary activating mutations in exon 9 (AYins503-504, IC50 = 18 nM) and exon 11 (V560 D, IC50 = 5 nM; Delta552-559, IC50 = 1 nM). Motesanib also demonstrated activity against kinase domain mutations conferring imatinib resistance (V560D/V654A, IC50 = 77 nM; V560D/T670I, IC50 = 277 nM; Y823 D, IC50 = 64 nM) but failed to inhibit the imatinib-resistant D816V mutant (IC50 > 3000 nM). Motesanib suppressed the proliferation of Ba/F3 cells expressing Kit mutants with IC50 values in good agreement with those observed in the autophosphorylation assays. CONCLUSIONS: In conclusion, our data suggest that motesanib possesses inhibitory activity against primary Kit mutations and some imatinib-resistant secondary mutations.

Discovery and evaluation of dual CDK1 and CDK2 inhibitors.

In eukaryotic cells, cyclin-dependent kinase (CDK) complexes regulate the temporal progression of cells through the cell cycle. Deregulation in the cell cycle is an essential component in the evolution of cancer. Here, we validate CDK1 and CDK2 as potential therapeutic targets using novel selective small-molecule inhibitors of cyclin B1/CDK1 and cyclin E2/CDK2 enzyme complexes (CDKi). Flow cytometry-based methods were developed to assess intracellular retinoblastoma (Rb) phosphorylation to show inhibition of the CDK pathway. Tumor cells treated with CDK inhibitors showed an overall decrease in cell proliferation, accumulation of cells in G1 and G2, and apoptosis in a cell line-specific manner. Although CDK inhibitors activate p53, the inhibitors were equipotent in arresting the cell cycle in isogenic breast and colon tumor cells lacking p53, suggesting the response is independent of p53. In vivo, the CDK inhibitors prevented the growth of colon and prostate tumors, blocked proliferation of tumor cells, and inhibited Rb phosphorylation. The discovery and evaluation of novel potent and selective CDK1 and CDK2 inhibitors will help delineate the role that CDK complexes play in regulating tumorigenesis.