Eli M. Wallace

Biological Characterization of ARRY-142886 (AZD6244), a Potent, Highly Selective Mitogen-Activated Protein Kinase Kinase 1/2 Inhibitor

Purpose: The Ras-Raf-mitogen-activated protein kinase kinase (MEK) pathway is overactive in many human cancers and is thus a target for novel therapeutics. We have developed a highly potent and selective inhibitor of MEK1/2. The purpose of these studies has been to show the biological efficacy of ARRY-142886 (AZD6244) in enzymatic, cellular, and animal models. Experimental Design: The ability of ARRY-142886 to inhibit purified MEK1 as well as other kinases was evaluated. Its effects on extracellular signal-regulated kinase (ERK) phosphorylation and proliferation in several cell lines were also determined. Finally, the inhibitor was tested in HT-29 (colorectal) and BxPC3 (pancreatic) xenograft tumor models. Results: The IC50 of ARRY-142886 was determined to be 14 nmol/L against purified MEK1. This activity is not competitive with ATP, which is consistent with the high specificity of compound for MEK1/2. Basal and epidermal growth factor–induced ERK1/2 phosphorylation was inhibited in several cell lines as well as 12-O-tetradecanoylphorbol-13-acetate–induced ERK1/2 phosphorylation in isolated peripheral blood mononuclear cells. Treatment with ARRY-142886 resulted in the growth inhibition of several cell lines containing B-Raf and Ras mutations but had no effect on a normal fibroblast cell line. When dosed orally, ARRY-142886 was capable of inhibiting both ERK1/2 phosphorylation and growth of HT-29 xenograft tumors in nude mice. Tumor regressions were also seen in a BxPC3 xenograft model. In addition, tumors remained responsive to growth inhibition after a 7-day dosing holiday. Conclusions: ARRY-142886 is a potent and selective MEK1/2 inhibitor that is highly active in both in vitro and in vivo tumor models. This compound is currently being investigated in clinical studies.

A Small-Molecule Antagonist of HIF2α Is Efficacious in Preclinical Models of Renal Cell Carcinoma

More than 90% of clear cell renal cell carcinomas (ccRCC) exhibit inactivation of the von Hippel-Lindau (pVHL) tumor suppressor, establishing it as the major underlying cause of this malignancy. pVHL inactivation results in stabilization of the hypoxia-inducible transcription factors, HIF1α and HIF2α, leading to expression of a genetic program essential for the initiation and progression of ccRCC. Herein, we describe the potent, selective, and orally active small-molecule inhibitor PT2385 as a specific antagonist of HIF2α that allosterically blocks its dimerization with the HIF1α/2α transcriptional dimerization partner ARNT/HIF1β. PT2385 inhibited the expression of HIF2α-dependent genes, including VEGF-A, PAI-1, and cyclin D1 in ccRCC cell lines and tumor xenografts. Treatment of tumor-bearing mice with PT2385 caused dramatic tumor regressions, validating HIF2α as a pivotal oncogenic driver in ccRCC. Notably, unlike other anticancer agents that inhibit VEGF receptor signaling, PT2385 exhibited no adverse effect on cardiovascular performance. Thus, PT2385 represents a novel class of therapeutics for the treatment of RCC with potent preclincal efficacy as well as improved tolerability relative to current agents that target the VEGF pathway. Cancer Res; 76(18); 5491-500. ©2016 AACR.

Potent non-peptidic dual inhibitors of endothelin-converting enzyme and neutral endopeptidase 24.11 ☆

Abstract Structural modifications of CGS 26303, a non-peptidic α-aminophosphonate dual ECE/NEP inhibitor lead, were performed to maximize inhibition of recombinant human ECE-1, while maintaining strong NEP inhibition. Specifically, substitution of the α-aminophosphonate moiety with aryl ethyl side-chains led to the discovery of a new class of potent, non-peptidic, dual inhibitors, such as CGS 31447, which blocked ECE-1 and NEP activities with IC50s of 17 and 5 nM, respectively.

Synthesis of tetrazole analogs of α-amino acids by alkylation of a Schiff base of α-aminomethyltetrazole

Abstract A benzophenone imine of N-1 protected α-aminomethyltetrazoles was found to undergo alkylation with organic halides in good yields. Deprotection afforded tetrazole analogs of α-amino acids.

Abstract 2515: Preclinical Development of ARRY-162, A Potent and Selective MEK 1/2 Inhibitor

Activation of the Ras/Raf/MEK/MAP kinase pathway is implicated in uncontrolled cell proliferation and tumor growth. Inappropriate activation of the RAS pathway can occur through several distinct mechanisms, including activating mutations in Ras and B-raf, or activated growth factor-signaling, cytokines and stress responses. Mutated, oncogenic forms of Ras are found in 50% of colon, 90% of pancreatic, and 30% of lung cancers. Also, B-Raf mutations have been identified in more than 60% of malignant melanomas and from 40-70% of papillary thyroid cancers. MEK, a dual specific kinase, is a key player in this pathway; it is downstream of both Ras and Raf and activates ERK1/2 through phosphorylation of key tyrosine and threonine residues. These data suggest that targeting MEK can inhibit cancer cell signaling mediated by a wide variety of signals, making MEK an attractive target for the treatment of cancer. We have discovered ARRY-162, a novel ATP-uncompetitive inhibitor of MEK 1/2, which is un-competitive with respect to ATP. ARRY-162 has nanomolar activity against purified MEK enzyme (IC50 = 12 nM) and is highly selective. It has been evaluated against 220 serine/threonine and tyrosine kinases with no inhibitory activity observed up to 20 μM. ARRY-162 inhibits both basal and induced levels of ERK phosphorylation in numerous cancer cell lines with IC50s as low as 5 nM. ARRY-162 is especially potent at inhibiting the cell proliferation of mutant B-Raf and Ras cell lines such as HT29, Malme-3M, SK-MEL-2, COLO 205, SK-MEL-28 and A375 (IC50s from 30-250 nM). In vivo, ARRY-162 has demonstrated efficacy in several xenograft tumor models in mice, including HT29, BxPC3, MIA PaCa2, A549, LoVo, Calu6, DU145 and COLO 205. In the HT29 and in the COLO 205 colon carcinoma models, dose-dependent inhibition of tumor growth (up to 75% TGI) was observed at doses ranging from 3 to 30 mg/kg, QD, PO for 21 days. In the Colo-205 colon carcinoma model, significant tumor regressions were observed with 50% partial responses and 13% complete responses at 30 mg/kg, PO, QD. In the BxPC3 pancreatic carcinoma model (which does not harbor either Ras or Raf mutations), tumor growth inhibition (∼70% TGI) and 13% partial responses were seen at doses of 30 mg/kg, QD, PO for 21 days. Consistent with ARRY-1629s mechanism of action, tumor growth inhibition correlates with decreased phospho-ERK levels in tumor xenografts. In addition to its potency against MEK, this compound demonstrates other desirable attributes for development including good physical chemical characteristics, low clearance, medium-to-high Caco-2 permeability and minimal predicted drug-drug interactions. With preclinical efficacy and safety studies on ARRY-162 completed, this compound has entered clinical development for treatment of cancer. 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 2515.

Discovery of spirocyclic sulfonamides as potent Akt inhibitors with exquisite selectivity against PKA.

We describe the design and synthesis of novel bicyclic spiro sulfonamides as potent Akt inhibitors. Through structure-based rational design, we have successfully improved PKA selectivity of previously disclosed spirochromanes. Representative compounds showed favorable Akt potency while exhibiting up to 1000-fold selectivity against PKA.

Abstract A143: ARRY-380, a selective HER2 inhibitor: From drug design to clinical evaluation.

In recent years, there has been discussion on the relative benefits of drugs that are kinase selective and those targeting multiple kinases, both in terms of clinical activity and safety. While the value of selectivity may be target dependent, there is now evidence that HER2 can be effectively targeted with a selective small molecule. Designing a selective HER2 inhibitor is challenging, as the ATP-binding pockets of EGFR and HER2 differ by only 2 amino acids and only 1 of these is involved with inhibitor binding (Cys775 vs. Ser783). While obtaining this selectivity was difficult, the goal of developing a drug that would inhibit HER2 without EGFR-related side effects was deemed advantageous, especially since it appears that inhibition of EGFR does not improve the efficacy of HER2-targeted therapy in patients (pts) with HER2+ metastatic breast cancer (MBC) (Arteaga, et al. Clin Cancer Res. 2008; 14(19):6277–83). ARRY-380 is an orally active, potent, small-molecule tyrosine kinase inhibitor of HER2. The compound is a reversible, ATP-competitive inhibitor with nanomolar potency in enzymatic and cellular assays. In cell-based assays, ARRY-380 is ∼500-fold selective for HER2 versus EGFR and is equipotent against truncated p95-HER2. ARRY-380 is currently undergoing evaluation in a first-inhuman Phase 1 dose-escalation and expansion study designed to identify the maximum tolerated dose (MTD) and to assess the safety, pharmacokinetics (PK) and preliminary efficacy in pts with advanced solid tumors that express the HER2 target. In the dose-escalation phase, pts with HER2+ MBC or other documented HER2+ cancers were treated with ARRY-380 as a single oral dose on Cycle (C)1 Day (D)1 followed by twice daily (BID) dosing, beginning on C1 D3, in 28-day dosing cycles. Safety was assessed by adverse events (AEs), clinical laboratory test results, physical examinations, vital signs and ECGs. Tumor response was assessed every 2 cycles. As of August 31, 2011, 50 pts (43 with MBC) have received ARRY-380. In the completed dose-escalation phase, 33 pts were enrolled and treated at doses ranging from 25 mg to 800 mg BID; of these, 26 had HER2+ MBC and all were previously treated with trastuzumab and 81% with lapatinib. The MTD was determined to be 600 mg BID. Of 19 evaluable pts with HER2+ MBC receiving doses ≥ 200 mg BID, 6 (32%) had a partial response (PR) or stable disease (SD) ≥ 6 months; 10 pts had regression of tumor lesions, of these, 1 pt (5%) had a PR and 9 pts (47%) had SD. Treatment-related AEs included Grade 2 events of increased ALT/AST (5), constipation (1), fatigue (3), hyperbilirubinemia (1) and nausea (1) and Grade 3 AEs of increased ALT/AST (3), night sweats (1) and rash (1). No Grade 4 treatment-related AEs have been reported, nor has any treatment-related AE led to study drug discontinuation. An expansion cohort to confirm safety and further examine activity of ARRY-380 at the MTD in pts with HER2+ MBC is ongoing; enrollment is complete (N = 17) and data analysis is continuing. In conclusion, in the small number of pts treated to date, ARRY-380 is associated with few EGFR-related side effects. In heavily pre-treated MBC pts, ARRY-380 is exhibiting preliminary signs of efficacy with an acceptable safety profile. Thus, continued clinical development of ARRY-380 is warranted to further evaluate if a selective, small-molecule HER2 inhibitor may be an alternative treatment option to a multi-kinase inhibitor in pts with HER2+ cancers. 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 A143.

Design and Activity of Specific Hypoxia-Inducible Factor-2α (HIF-2α) Inhibitors for the Treatment of Clear Cell Renal Cell Carcinoma: Discovery of Clinical Candidate (S)-3-((2,2-Difluoro-1-hydroxy-7-(methylsulfonyl)-2,3-dihydro-1H-inden-4-yl)oxy)-5-fluorobenzonitrile (PT2385)

HIF-2α, a member of the HIF family of transcription factors, is a key oncogenic driver in cancers such as clear cell renal cell carcinoma (ccRCC). A signature feature of these cancers is the overaccumulation of HIF-2α protein, often by inactivation of the E3 ligase VHL (von Hippel-Lindau). Herein we disclose our structure based drug design (SBDD) approach that culminated in the identification of PT2385, the first HIF-2α antagonist to enter clinical trials. Highlights include the use of a putative n → π*Ar interaction to guide early analog design, the conformational restriction of an essential hydroxyl moiety, and the remarkable impact of fluorination near the hydroxyl group. Evaluation of select compounds from two structural classes in a sequence of PK/PD, efficacy, PK, and metabolite profiling identified 10i (PT2385, luciferase EC50 = 27 nM) as the clinical candidate. Finally, a retrospective crystallographic analysis describes the structural perturbations necessary for efficient antagonism.

Abstract 4022: PT2385, a novel HIF-2α antagonist, combines with checkpoint inhibitor antibodies to inhibit tumor growth in preclinical models by modulating myeloid cells and enhancing T cell infiltration

The majority of clear cell renal cell carcinoma (ccRCC) have deficiency in the gene encoding the von Hippel Landau (VHL) protein, as a result of DNA copy loss, non-sense mutations, and epigenetic silencing. Deficiency in VHL and its E3 ligase activity results in stabilization of the transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α. In ccRCC, HIF-2α has been proposed to function as an oncogenic driver, as its depletion in tumor cells results in inhibition of tumor growth. We previously described the identification of a potent and selective small molecule antagonist of HIF-2α, PT2385 that disrupts the hetero-dimerization of HIF-2α with the aryl hydrocarbon receptor nuclear translocator. PT2385 inhibits growth of ccRCC tumor xenografts derived from cancer cell lines and from patients’ tumors. Inhibition of tumor growth is mediated by suppression of HIF-2α target genes, such as cyclin D1 and VEGFA, that promote tumor growth. In addition to a direct role in the transcription regulation of growth-promoting genes in ccRCC, HIF-2α has also been implicated in the pro-tumorigenic property of the tumor microenvironment. HIF-2α is expressed in cells of the myeloid lineage, and HIF-2α expression in tumor-associated macrophages was reported in a number of tumor types. A role for HIF-2α in the polarization of macrophages to the M2 phenotype has also been described. The availability of PT2385 provides an opportunity to confirm the involvement of HIF-2α in immune suppression by the tumor microenvironment, and to assess the therapeutic utility of HIF-2α antagonism in tumors other than ccRCC. PT2385 was evaluated for its ability to inhibit tumor growth in syngeneic mouse tumor models. The mouse tumor cell lines used in these models do not express HIF-2α and PT2385 has no single agent efficacy. However, the combination of PT2385 with antibodies to immune checkpoint control molecules (PD-1, PD-L1, and CTLA4) yielded additive or synergistic efficacy in a model-dependent manner. Immune phenotyping of the treated tumors was performed by immunohistochemistry and flow cytometry. The analyses revealed that tumor growth inhibition by the combination regimens is accompanied by an increase in T cell infiltration and changes in macrophage and myeloid-derived suppressor cell populations in the tumors. Changes in cytokine expression were also observed. Results of our studies show that combinations of PT2385 and immune checkpoint inhibitor antibodies are well tolerated and efficacious in preclinical models. The results are consistent with the hypothesis that HIF-2α plays an immunosuppressive role in the tumor microenvironment, and its inhibition provides an additional approach to reverse immune evasion by tumors. The combination of PT2385 and immune checkpoint inhibitors is being further evaluated in clinical trials. Citation Format: Guangzhou Han, Christina Stevens, Zhaodan Cao, Shanhai Xie, Melissa Maddie, Barry Goggin, Eli Wallace, John Josey, Tai W. Wong. PT2385, a novel HIF-2α antagonist, combines with checkpoint inhibitor antibodies to inhibit tumor growth in preclinical models by modulating myeloid cells and enhancing T cell infiltration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4022.

Abstract DDT01-01: PT2385: First-in-class HIF-2α antagonist for the treatment of renal cell carcinoma

Hypoxia-inducible factors (HIFs), including HIF-1α and HIF-2α, are transcription factors that mediate cellular response to changes of oxygen supply. These proteins become stabilized under hypoxia and subsequently activate the expression of genes to facilitate cell survival and proliferation. HIF proteins are activated in many types of cancers due to the tumor hypoxic microenvironment and have been implicated in cancer initiation, progression and metastasis. The oncogenic role of HIF-2α is pertinent in clear cell renal carcinoma (ccRCC). In the majority of ccRCC tumors, the von Hippel-Lindau protein (VHL) that targets HIF-2α for degradation is inactivated, leading to the accumulation of HIF-2α and the activation of genes that drive kidney cancer tumorigenesis. We have identified small molecules that bind to the PAS-B domain of HIF-2α protein and block it9s dimerization with ARNT (aryl hydrocarbon receptor nuclear translocator, HIF-1β), a prerequisite for its transcriptional activities. Specifically, we describe PT2385, a selective, orally active HIF-2α antagonist with potent anti-cancer activity in mouse models of ccRCC. PT2385 is currently under evaluation in Phase I clinical trials for the treatment of ccRCC. Citation Format: Eli M. Wallace, Zhaodan Cao, Tzuling Cheng, Robert Czerwinski, Darryl D. Dixon, Xinlin Du, Barry Goggin, Jonas Grina, Megan Halfmann, Guangzhou Han, Heli Huang, John A. Josey, Melissa A. Maddie, Sarah Olive, James Rizzi, Stephen T. Schlachter, Hui-Ling Tan, Bin Wang, Keshi Wang, Paul M. Wehn, Shanhai Xie, Rui Xu, Hanbiao Yang. PT2385: First-in-class HIF-2α antagonist for the treatment of renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr DDT01-01. doi:10.1158/1538-7445.AM2015-DDT01-01