Cathy Bull

Discovery of 3-(3-(4-(1-Aminocyclobutyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine (ARQ 092): An Orally Bioavailable, Selective, and Potent Allosteric AKT Inhibitor

The work in this paper describes the optimization of the 3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine chemical series as potent, selective allosteric inhibitors of AKT kinases, leading to the discovery of ARQ 092 (21a). The cocrystal structure of compound 21a bound to full-length AKT1 confirmed the allosteric mode of inhibition of this chemical class and the role of the cyclobutylamine moiety. Compound 21a demonstrated high enzymatic potency against AKT1, AKT2, and AKT3, as well as potent cellular inhibition of AKT activation and the phosphorylation of the downstream target PRAS40. Compound 21a also served as a potent inhibitor of the AKT1-E17K mutant protein and inhibited tumor growth in a human xenograft mouse model of endometrial adenocarcinoma.

Abstract 2517: Pharmacodynamic biomarkers for ARQ 736, a small molecule BRAF inhibitor

The MAPK/ERK pathway is a cascade of serine/threonine protein kinases activated by various extracellular factors to control cell growth, differentiation, migration and death. A number of somatic mutations occur that lead to constitutive activation of this pathway in multiple human cancers. BRAF mutations were detected in over 60% of melanomas and may account for a significant proportion of colon cancer patients whose tumors are resistant to anti-EGFR therapy. ARQ 736 is a novel inhibitor of BRAF and is selectively potent in killing cancer cell lines harboring at least one mutated BRAF allele. The objective of this study is to identify potential protein biomarkers for use in connection with ARQ 736 treatment. A number of proteins released by BRAF (V600E) mutant A375 human melanoma cells into conditioned media detected by antibody arrays (RD 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2517.

Abstract A278: ARQ 087, a multi-tyrosine kinase inhibitor with potent in vitro and in vivo activity in FGFR2 driven models.

Fibroblast growth factors (FGF) and their receptors (FGFR) play important roles in cell proliferation, cell differentiation, cell migration, cell survival, protein synthesis, and angiogenesis. The FGFR family consists of four genes encoding tyrosine kinase receptors (FGFR1, FGFR2, FGFR3, and FGFR4). Dysregulation of FGFR signaling has been implicated in a number of developmental syndromes as well as cancers, e.g., squamous non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric, liver, breast, ovarian, endometrial, and bladder carcinomas, fueling significant interest in FGFRs as targets for therapeutic intervention. ArQule, Inc. has discovered a novel, ATP competitive class of FGFR inhibitors from which ARQ 087 emerged as a candidate for advancement into clinical development. ARQ 087 is a potent multi-kinase inhibitor with pan-FGFR activity against FGFR1, FGFR2, mutant FGFR2 (N549H), FGFR3, and FGFR4 kinases, all exhibiting IC 50 values in the low nanomolar range in biochemical assays. Ki versus FGFR1 and FGFR2 were 2.8nM and 0.68nM, respectively. ARQ 087 inhibited ectopically expressed FGFR1, 2, and 3 in COS-1 cells as well as, to a varying extent, the proliferation of BaF3 cells expressing the FGFR family of receptors (FGFR2≫FGFR1/FGFR3≫FGFR4). Cell proliferation studies suggested a correlation of FGFR2 mRNA amplification in gastric and other cancers with associated sensitivity to treatment with ARQ 087. Along these lines, ARQ 087 demonstrated potent inhibition of FGFR2 phosphorylation in NCI-H716, Kato III, SNU-16 and MFM223 cells; all demonstrated to be driven by FGFR2. The inhibition of cell growth was associated with an ARQ 087-induced G1 cell cycle arrest and subsequent induction in apoptosis that appears to be related to the levels of FGFR2 protein. Cell lines driven by FGFR2 activating mutations did not undergo apoptosis but did accumulate in G1 following ARQ 087 treatment. In vivo, ARQ 087 induced regressions in FGFR2-driven xenograft models (SNU-16, NCI-H716 and BaF3/FGFR2) and inhibited tumor progression in a model harboring an FGFR2-activating mutation (AN3CA). In addition, concentration-dependent inhibition of phosphorylation of FGFR2 and the downstream FGFR pathway signals (FRS2α, MEK, ERK, and AKT) was evident in response to ARQ 087 treatment in both in vitro and in vivo pharmacodynamic assays. In summary, ARQ 087 is an orally bioavailable kinase inhibitor with potent in vitro and in vivo activity in FGFR2 driven models, possessing good drug-like properties. A clinical development plan including a patient selection strategy is defined and the drug is currently in Phase I clinical studies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A278. Citation Format: Daniel Dransfield, Jennifer Lee, Carol Waghorne, Cathy Bull, Ronald E. Savage, Xiaolan Zhao, Shipeng Yuan, Edward Chang, Enkeleda Nakuci, Sudharshan Eathiraj, Susan Cornell-Kennon, Xiubin Gu, Syed Ali, Chang-Rung Chen. ARQ 087, a multi-tyrosine kinase inhibitor with potent in vitro and in vivo activity in FGFR2 driven models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A278.

Abstract A230: Discovery and characterization of ARQ 092, an ATP-independent, potent and selective inhibitor of AKT kinases.

The AKT family of serine-threonine kinases is a critical signal transduction node serving a variety of cellular functions including survival, proliferation, protein synthesis, and glucose metabolism. Small molecule inhibitors of AKT are emerging as potential targeted agents to treat cancers that exhibit aberrant AKT pathway signaling. A screening strategy was employed to identify inhibitors which utilize the intrinsic negative regulatory function of hydrophobic clusters in the ATP-binding cleft to inhibit the kinase activity of AKT1. ARQ 092 was identified through this screening paradigm following in vitro and in vivo optimization and was selected for further development. Crystallographic studies provided evidence that this mechanism of inhibition of AKT had been achieved. ARQ 092 binds to inactive, unphosphorylated AKT1 with sub-nanomolar affinity (Kd = 0.28 nM measured by surface plasma resonance) and biochemically inhibits all three isoforms (IC50 values of 3 nM, 4.5 nM, and 5.5 nM respectively for AKT1, AKT2, and AKT3). ARQ 092 displayed inhibition kinetics against AKT1 which were not affected by ATP concentrations up to 1000 M. When screened against a panel of over 300 kinases, ARQ 092 inhibited only three kinases within 100-fold of its IC50 against AKT1. ARQ 092 potently inhibited AKT phosphorylation (Ser473 & Thr308) in AN3CA human endometrial carcinoma cells (EC50 values of 39 nM and 61 nM, respectively for p-Ser473 & p-Thr308) and demonstrated concentration-dependent inhibition of phosphorylation of the downstream AKT substrate PRAS40. ARQ 092 inhibited growth of AN3CA cells (GI50 = 555 nM), A2780 cells (GI50 = 400 nM), and IGROV-1 cells (GI50 = 66 nM), in addition to LNCaP, ZR-75–1, and BT-474 human cancer cell lines (GI50 values ranging from 1 to 4 μM). Following a single oral dose of ARQ 092, inhibition of AKT and PRAS40 phosphorylation was documented in vivo in both AN3CA human endometrial tumor and BT474 human breast tumor xenograft models. The growth of AN3CA tumor xenografts was markedly suppressed after daily oral administration of ARQ 092 for 10 days. Finally, ARQ 092 was shown to have good pharmaceutical properties and was advanced into preclinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A230.

Abstract 3571: Exploratory biomarker discovery for clinical development of ARQ 087, a potent pan-FGFR kinase inhibitor

Fibroblast growth factor receptor (FGFR) tyrosine kinase family members have gained increasing attention as potential therapeutic targets. Gene amplification, or gain-of-function translocations and mutations of these receptors, have been implicated in a variety of cancers including breast, gastric, bladder, ovarian, endometrial, lymphoma and myeloma. We have previously described the biological profiling of ARQ 087, a potent small-molecule pan-FGFR kinase inhibitor that shows anti-tumor activity in FGFR dysregulated tumor cells in vitro and in vivo. A number of cell lines with genetically-altered FGF receptors were identified that were sensitive in anti-proliferative assays to ARQ 087, including FGFR2 amplified gastric carcinoma lines KATO III and SNU-16, and endometrial lines AN3CA, MFE-296 and MFE-280 harboring FGFR2 mutants, as well as a breast cancer cell line with increased FGFR1 gene copy (MDA MB-157). Western blot pharmacodynamic analysis demonstrates that ARQ 087 inhibits FGFR phosphorylation and downstream pathway markers in these cell lines in a concentration-dependent manner. Using antibody arrays specific for known cytokines as well as regulatory proteins involved in angiogenesis and apoptosis, we have identified a number of protein biomarkers in cell lysates and in conditioned media of sensitive cell lines that changed in response to ARQ 087 exposure. Three proteins (cytochrome c, clusterin, and HTRA2) decreased in SNU-16 (ARQ 087-sensitive, FGFR amplified) but not DLD1 (ARQ 087-resistant, non FGFR expressing) cells. VEGF decreased and IL-1rα increased in SNU-16 but not in DLD1 conditioned media in response to ARQ 087. Furthermore, analysis of mouse plasma from animals bearing AN3CA tumors treated with ARQ 087 revealed a decrease in human VEGF, CXCL16, MMP-9, Serpin E1 and MIF and an increase in RANTES, IL-1β, IP-10, SDF-1, IL-1rα. TNFα, GM-CSF, IL-2, sTREM-1, sICAM-1, MIP-1β, IL-8. In summary, we have identified a number of cell lines from a broad cell line screening campaign that exhibited dysregulated FGFR pathway function and are sensitive to the anti-proliferative effects of ARQ 087. These cell lines, in turn, have been used as models in which to identify candidate biomarkers that may greatly facilitate subsequent clinical evaluation of this molecularly targeted anti-tumor agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3571. doi:10.1158/1538-7445.AM2011-3571

Abstract 4501: Development and biological annotation of a novel RAF inhibitor amenable for clinical evaluation against BRAF (V600E)-harboring human tumors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC An activating somatic mutation in BRAF kinase (V600E) is found in approximately 70% of melanomas, 50% of papillary thyroid cancers and 10% of colon cancers, fueling interest in BRAF (V600E) as a therapeutic target for cancer treatment. We have previously described a novel class of RAF inhibitors with initial pre-clinical profiling. This compound class has been optimized for solubility, eADME properties, and oral bioavailability. ARQ 736 has been selected as a candidate to advance into clinical testing. ARQ 736 is a potent RAF inhibitor (IC50 values of 2.6, 2.7, and 7.3 nM against BRAF, BRAF (V600E) and c-Raf-1, respectively) and extremely selective biochemically, with only 11 of 272 human kinases being inhibited within 100-fold of the IC50 against BRAF. Worthy of note, ARQ 736 was also found to be inactive against vascular endothelial growth factor receptor 2 (VEGFR2 or KDR). Despite being a pan-RAF inhibitor, at the cellular level, ARQ 736 showed a high degree of selectivity against cancer cells harboring at least one allele of BRAF (V600E) (IC50 values in cytotoxicity assays of 200-300 nM), while sparing normal cells with wild-type (wt) BRAF and wt ras as well as cancer cells harboring k-ras mutations (IC50 values in cytotoxicity assays ranging from 4 μM to greater than 10 μM). In cellular pharmacodynamic assays, ARQ 736 inhibited the MAPK pathway potently in a rapid and durable fashion (78, 65, and 11 nM respectively for inhibition of ERK phosphorylation in the human melanoma lines A375, SK-MEL-28, and Colo-205). Acute pharmacodynamic studies in vivo showed a nearly complete reduction of phospho-MEK and phospho-ERK in A375 human melanoma tumors grown in athymic xenograft models and significant tumor growth inhibition was shown in the same model with daily dosing of ARQ 736. ARQ 736 was found to be well-tolerated and orally bioavailable in dogs, and has been advanced into full pre-clinical toxicology studies, data from which will be discussed. In summary, ARQ 736 is the advanced candidate of a novel class of BRAF kinase inhibitors with excellent drug-like properties that may have clinical utility for human cancers driven by oncogenic BRAF (V600E). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4501.

Abstract B270: Discovery and preliminary characterization of novel tyrosine kinase inhibitors with selectivity to BTK.

Brutons tyrosine kinase (BTK) is a non receptor tyrosine kinase and belongs to the SRC-related TEC subfamily. The role of BTK in the B cell receptor (BCR) signaling pathway is well defined and is critical for full activation of phospholipase-C γ and MAPK as well as calcium mobilization. More than 95% of human lymphomas are B cell lymphoma and among them over 70% expresses BCR. Studies in lymphoma cell lines in vitro have demonstrated the role of BTK in BCR signaling. We have performed in vitro kinase profiling to determine percent inhibition and IC50 for kinase families of interest. An extensive cellular characterization including anti-proliferative assays, Western blot analysis for evaluation of phospho BTK (pY223) and cell cycle analysis was performed in DLBCL TMD8 cells. An in vivo tumor xenograft model was employed to further evaluate the target knockdown and efficacy against tumor growth. Compounds 1 and 2 were found to exhibit potent inhibition of BTK (IC50 0.6 nM), Src family (IC50 0.6-10 nM) and Trk isoforms (IC50 2-4 nM). Inhibition of cell proliferation was accompanied by reduction of phosphorylation of BTK. In addition to the anti-proliferative activity, following a single oral dose, inhibition of BTK phosphorylation (pY223) was observed in vivo in a TMD8 tumor xenograft model. The growth of TMD8 tumor xenografts was markedly suppressed after daily oral administration for 12 days of these two compounds. A series of potent multi non-receptor tyrosine kinase inhibitors targeting the Tec, Src and Trk family of kinases have been discovered and characterized. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B270. Citation Format: Nivedita D. Namdev, Susan Cornell-Kennon, Yi Yu, Jason Hill, Cathy Bull, Laurie Volak, Jianqiang Wang, Deirdre Lowe, Xuibin Gu, Steffi Koerner, Magdi Moussa, David Vensel, Yanbin Liu, Hui Wu, Chang-Rung Chen, Daniel T. Dransfield, Mark A. Ashwell. Discovery and preliminary characterization of novel tyrosine kinase inhibitors with selectivity to BTK. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B270.

Abstract 3905: Synthesis and structure activity relationship of substituted N,6-diphenyl-5,6-dihydrobenzo[h]quinazolin-2-amine as inhibitors of fibroblast growth factor receptors (FGFR)

Utilization of hydrophobic motifs present in auto-inhibited protein kinases has resulted in the identification of a series of 5,6-dihydrobenzo [h]quinazolin-2-amines with activity as fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors. Herein we describe the combination of a proprietary in silico design process, a new screening paradigm using an array of biochemical and biophysical technologies in conjunction with an established parallel chemistry process for the identification and optimization of a series of novel FGFR inhibitors. These potent FGFR inhibitors exhibit a preference for the inactive form of the kinase, are non-ATP competitive, and exhibit robust cellular pharmacodynamic inhibition as well as in vitro anti-proliferative effects in cells dependent on FGFR and significant anti-tumor activity in appropriate xenograft models in vivo. The design strategy, synthesis, structure activity relationships and in vitro and in vivo biology of selected inhibitors will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3905. doi:1538-7445.AM2012-3905

Abstract LB-1: Discovery and optimization of orally bioavailable, selective and potent ATP-independent Akt inhibitors

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Herein we describe the implementation of a biochemical and biophysical screening strategy to discover small molecules that inhibit Akt through a mechanism distinct from ATP-competitive inhibitors. A series of novel derivatives of the core scaffold 3H-imidazo[4,5-b]pyridine were identified and optimized. These Akt inhibitors demonstrated potent inhibition of intracellular Akt and downstream targets including PRAS40 activation in vitro. Pharmacodynamic and pharmacokinetic studies in vivo demonstrated the effectiveness of the series at inhibiting the activation of Akt and an additional downstream effector (p70S6) following oral dosing in mice. Co-crystallization studies with un-phosphorylated Akt1 revealed that as a consequence of binding these novel, potent and selective, ATP-independent inhibitors the ATP binding cleft is occupied by non-polar residues which are associated as tight clusters. The cleft is closed with a ‘hydrophobic lock’ which may function to sterically exclude the binding of both ATP and ATP-competitive inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-1. doi:1538-7445.AM2012-LB-1