Jean-Marc Lapierre

Discovery and optimization of a series of 3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amines: orally bioavailable, selective, and potent ATP-independent Akt inhibitors.

This paper describes the implementation of a biochemical and biophysical screening strategy to identify and optimize small molecule Akt1 inhibitors that act through a mechanism distinct from that observed for kinase domain ATP-competitive inhibitors. With the aid of an unphosphorylated Akt1 cocrystal structure of 12j solved at 2.25 Å, it was possible to confirm that as a consequence of binding these novel inhibitors, the ATP binding cleft contained a number of hydrophobic residues that occlude ATP binding as expected. These Akt inhibitors potently inhibit intracellular Akt activation and its downstream target (PRAS40) in vitro. In vivo pharmacodynamic and pharmacokinetic studies with two examples, 12e and 12j, showed the series to be similarly effective at inhibiting the activation of Akt and an additional downstream effector (p70S6) following oral dosing in mice.

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.

Preclinical Activity of ARQ 087, a Novel Inhibitor Targeting FGFR Dysregulation.

Dysregulation of Fibroblast Growth Factor Receptor (FGFR) signaling through amplifications, mutations, and gene fusions has been implicated in a broad array of cancers (e.g. liver, gastric, ovarian, endometrial, and bladder). ARQ 087 is a novel, ATP competitive, small molecule, multi-kinase inhibitor with potent in vitro and in vivo activity against FGFR addicted cell lines and tumors. Biochemically, ARQ 087 exhibited IC50 values of 1.8 nM for FGFR2, and 4.5 nM for FGFR1 and 3. In cells, inhibition of FGFR2 auto-phosphorylation and other proteins downstream in the FGFR pathway (FRS2α, AKT, ERK) was evident by the response to ARQ 087 treatment. Cell proliferation studies demonstrated ARQ 087 has anti-proliferative activity in cell lines driven by FGFR dysregulation, including amplifications, fusions, and mutations. Cell cycle studies in cell lines with high levels of FGFR2 protein showed a positive relationship between ARQ 087 induced G1 cell cycle arrest and subsequent induction of apoptosis. In addition, ARQ 087 was effective at inhibiting tumor growth in vivo in FGFR2 altered, SNU-16 and NCI-H716, xenograft tumor models with gene amplifications and fusions. ARQ 087 is currently being studied in a phase 1/2 clinical trial that includes a sub cohort for intrahepatic cholangiocarcinoma patients with confirmed FGFR2 gene fusions (NCT01752920).

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 3565: Abrogation of the paradoxical activation of the MAPK pathway following RAF inhibition by ARQ 680, a potent inhibitor of the RAF family of serine/threonine kinases

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Recent reports have documented the paradoxical activation of the MAPK pathway in response to pharmacological RAF inhibition in cells bearing either wild-type (wt) or mutant RAS, as evidenced by hyperphosphorylation of ERK in human tumor cells exposed to BRAF inhibitors in vitro. ARQ 680 is a pan-RAF kinase inhibitor (IC50 values of 2.6, 2.7, & 7.3 nM respectively for BRAF, mutant BRAF(V600E), and CRAF) which is the active moiety of the highly soluble pro-drug ARQ 736, whose biological properties and anti-tumor activities have been described previously. Here we compared the activity of ARQ 680 and PLX 4032, the latter a structurally distinct pan-RAF inhibitor currently in advanced clinical trials, in various cell lines with various RAS/RAF genotypes. ARQ 680 and PLX 4032 show comparable degrees of selectivity across the human kinome, with ARQ 680 inhibiting 11 other kinases within 100-fold of its activity against BRAF. Interestingly, there is no discernable overlap between the off-target kinases of ARQ 680 and PLX 4032. In human melanoma cell lines (A375, SK-MEL-28) bearing mutant BRAF(V600E), both compounds exhibited comparable concentration-dependent inhibition of phospho-ERK as shown by quantitative western blotting (EC50 ∼ 10 – 30 nM). In 3 colon cancer cell lines harboring a mutant kras gene (HCT-116, SW480, & DLD-1), both ARQ 680 and PLX 4032 induced robust phosphorylation of ERK (1.8 to 5-fold increases) at varying concentrations, with ARQ 680 exhibiting the phenomenon at lower concentrations than PLX 4032. Importantly, ARQ 680 induced a concentration-dependent decrease in, and ultimately a nearly complete inhibition of, ERK phosphorylation at concentrations of 3 μM and above in all KRAS-mutant cell lines. In contrast, PLX 4032 exerted no inhibition of ERK phosphorylation below untreated controls at concentrations up to 100 μM, with the exception of the SW480 cell line, where the elevated p-ERK levels decreased to control levels at 10 to 100 μM. In an NRAS-mutant melanoma cell line (SK-MEL-2), ARQ 680 exerted a concentration-dependent inhibition of ERK phosphorylation (EC50 ∼ 400 nM) with no paradoxical activation of p-ERK at any concentration, while PLX 4032 induced an increase in p-ERK at concentrations up to 10 μM. Finally, in a wt RAS/RAF colon epithelial cell line (NCM-460), both ARQ 680 and PLX 4032 doubled p-ERK levels at sub-micromolar concentrations, with ARQ 680 exhibiting a concentration-dependent abrogation of this effect at concentrations between 1 and 10 μM while PLX 4032 sustained a 2-fold increase in p-ERK at concentrations as high as 100 μM. In summary, we have delineated the pharmacodynamic profile for a novel RAF inhibitor, ARQ 680. The molecular mechanisms by which ARQ 680 inhibits pERK in KRAS and NRAS mutant cell lines are under investigation and may be due in part to the suite of kinases inhibited by ARQ 680 in cells. 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 3565. doi:10.1158/1538-7445.AM2011-3565

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 C83: Nonclinical in vitro ADME, disposition, and pharmacokinetic assessment of ARQ 092, a selective AKT inhibitor.

Introduction: ARQ 092 is a potent and selective pan-AKT inhibitor currently in Phase 1 clinical studies for the treatment of advanced solid tumors. Here, we present preclinical in vitro drug-drug interaction, metabolism, disposition, and pharmacokinetic studies with ARQ 092. Methods: ARQ 092 was assessed in vitro for cross-species metabolic stability; CYP450 (cytochrome P450) and UGT (UDP-glucuronosyltransferase) reaction phenotyping; CYP450 inhibition/induction; Caco-2 absorption and efflux; and P-gp (P-glycoprotein), BCRP (Breast Cancer Resistance Protein), and MRP2 (Multidrug Resistance-associated Protein 2) mediated ATPase stimulation. Distribution of ARQ 092 to various tissues was measured after single and repeat dosing to mice as well as pharmacokinetics in mice, rats, and monkeys. Results: Cross-species NADPH-dependent metabolism studies revealed that ARQ 092 metabolism was highly species dependent with ARQ 092 being most stable in rat and human liver microsomes (t1/2 > 52 min) and least stable in mouse, dog, and monkey liver microsomes (t1/2 > 13-33 min). Reaction phenotyping with CYP450 and UGT isozymes and CYP450 inhibitors/antibodies indicated that CYP2D6, CYP3A, UGT1A4, and possibly CYP2C9 are involved in ARQ 092 metabolism. ARQ 092 inhibited CYP2D6, 2C9, and 2C19 in HLM with IC50 values of 10.2, 3.0, and 4.0 µM, respectively, but had little activity against other isozymes tested; and no induction of CYP3A, 2B6, or 1A2 was observed. Based on these in vitro results, interactions with CYP3A4, 2D6, CYP2C9, or CYP2C19 substrates and/or inhibitors in the clinic are possible. Caco-2 and ATPase transporter studies showed that ARQ 092 was highly permeable with little efflux in Caco-2 monolayers but may be an inhibitor/substrate of P-gp and mutant BCRP. Oral bioavailability values of 23, 62, and 50% were determined in mice, rats, and monkeys, respectively, with half-lives ranging widely from 1.6 to 13.6 hrs with iv dosing and 4.3 to 16.6 hrs with po dosing. In mice, ARQ 092 was highly distributed to tissues with liver, lung, and kidney having the highest concentrations. This result correlates with the high volume of distribution of the compound in mice of 24.5 L/kg. In toxicokinetic studies, increases in Cmax and AUC were generally greater than dose proportional with repeat dosing and accumulation was observed in rats but not monkeys. Conclusion: Collectively, these data indicate that ARQ 092 has sufficient oral bioavailability to advance into clinical testing. However, the potential for drug-drug interactions with ARQ 092 in the clinic will need to be investigated further. Additionally, careful selection of dosing regimens for ARQ 092 may be crucial based on the high volume of distribution and accumulation observed preclinically. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C83. Citation Format: Laurie P. Volak, Karen R. Bresciano, Terence Hall, David Vensel, Jean-Marc Lapierre, Inese Smukste, David K. McKearn, Ronald E. Savage. Nonclinical in vitro ADME, disposition, and pharmacokinetic assessment of ARQ 092, a selective AKT inhibitor. [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 C83.

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

Abstract 3231: In vitro cross species metabolism of a novel RAF kinase inhibitor phosphate prodrug, ARQ 736

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL ARQ 736 is a highly-soluble phosphate prodrug that readily undergoes conversion to ARQ 680 in the presence of phosphatases. The active drug, ARQ 680, is a pan-RAF inhibitor that is effective, both in vitro and in vivo, in inhibiting proliferation of cancer cell lines expressing mutant-BRAF (V600E). During preclinical development, the metabolic fate of ARQ 736 and ARQ 680 was evaluated in human, rat, and dog in vitro systems. Based on the data generated, metabolic pathways were proposed for ARQ 736 and ARQ 680. Cross-species metabolic stability studies were conducted in microsomes and hepatocytes. CYP450 reaction phenotyping using 6 recombinant CYP isozymes was conducted to identify the CYP isozyme(s) responsible for the phase one metabolism of ARQ 736 and ARQ 680. ARQ 736 and ARQ 680 were incubated in hepatocytes and liver fractions to evaluate the phase one and phase two metabolites produced in humans rats, and dogs. Additional studies in microsomes and hepatocytes were conducted to evaluate the potential for ARQ 736 or ARQ 680 to inhibit or induce various CYP450 isozymes. Cross-species NADPH-dependent metabolism studies revealed that ARQ 736 and 680 were highly stable in, rat, dog, and human liver microsomes with half-life values >30 min. CYP450 reaction phenotyping indicated that CYP3A4 is the predominant CYP450 isozyme responsible for phase one metabolism of ARQ 736 and 680. Detailed metabolite ID work in hepatocytes identified multiple phase one and two metabolites as a result of oxidations, demethylation and glucuronidation. Metabolic pathways appeared to be similar across all species tested. In conclusion, this research is the first characterization of the metabolism of ARQ 736, a novel pan-RAF inhibitor currently being tested in phase one clinical trial in cancer patients. 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 3231. doi:10.1158/1538-7445.AM2011-3231