Aihe Zhou

Discovery of Potent and Selective Pyrazolopyrimidine Janus Kinase 2 Inhibitors

The discovery of somatic Jak2 mutations in patients with chronic myeloproliferative neoplasms has led to significant interest in discovering selective Jak2 inhibitors for use in treating these disorders. A high-throughput screening effort identified the pyrazolo[1,5-a]pyrimidine scaffold as a potent inhibitor of Jak2. Optimization of lead compounds 7a-b and 8 in this chemical series for activity against Jak2, selectivity against other Jak family kinases, and good in vivo pharmacokinetic properties led to the discovery of 7j. In a SET2 xenograft model that is dependent on Jak2 for growth, 7j demonstrated a time-dependent knock-down of pSTAT5, a downstream target of Jak2.

A Restricted Role for TYK2 Catalytic Activity in Human Cytokine Responses Revealed by Novel TYK2-Selective Inhibitors

TYK2 is a JAK family protein tyrosine kinase activated in response to multiple cytokines, including type I IFNs, IL-6, IL-10, IL-12, and IL-23. Extensive studies of mice that lack TYK2 expression indicate that the IFN-α, IL-12, and IL-23 pathways, but not the IL-6 or IL-10 pathways, are compromised. In contrast, there have been few studies of the role of TYK2 in primary human cells. A genetic mutation at the tyk2 locus that results in a lack of TYK2 protein in a single human patient has been linked to defects in the IFN-α, IL-6, IL-10, IL-12, and IL-23 pathways, suggesting a broad role for TYK2 protein in human cytokine responses. In this article, we have used a panel of novel potent TYK2 small-molecule inhibitors with varying degrees of selectivity against other JAK kinases to address the requirement for TYK2 catalytic activity in cytokine pathways in primary human cells. Our results indicate that the biological processes that require TYK2 catalytic function in humans are restricted to the IL-12 and IL-23 pathways, and suggest that inhibition of TYK2 catalytic activity may be an efficacious approach for the treatment of select autoimmune diseases without broad immunosuppression.

A pentacyclic aurora kinase inhibitor (AKI-001) with high in vivo potency and oral bioavailability.

Aurora kinase inhibitors have attracted a great deal of interest as a new class of antimitotic agents. We report a novel class of Aurora inhibitors based on a pentacyclic scaffold. A prototype pentacyclic inhibitor 32 (AKI-001) derived from two early lead structures improves upon the best properties of each parent and compares favorably to a previously reported Aurora inhibitor, 39 (VX-680). The inhibitor exhibits low nanomolar potency against both Aurora A and Aurora B enzymes, excellent cellular potency (IC50 < 100 nM), and good oral bioavailability. Phenotypic cellular assays show that both Aurora A and Aurora B are inhibited at inhibitor concentrations sufficient to block proliferation. Importantly, the cellular activity translates to potent inhibition of tumor growth in vivo. An oral dose of 5 mg/kg QD is well tolerated and results in near stasis (92% TGI) in an HCT116 mouse xenograft model.

Metabolic plasticity underpins innate and acquired resistance to LDHA inhibition

Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.

Discovery of (S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one (GDC-0994), an Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibitor in Early Clinical Development.

The extracellular signal-regulated kinases ERK1/2 represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Described herein is the discovery and characterization of GDC-0994 (22), an orally bioavailable small molecule inhibitor selective for ERK kinase activity.

Discovery of imidazo[1,5-a]pyridines and -pyrimidines as potent and selective RORc inverse agonists.

The nuclear receptor (NR) retinoic acid receptor-related orphan receptor gamma (RORγ, RORc, or NR1F3) is a promising target for the treatment of autoimmune diseases. RORc is a critical regulator in the production of the pro-inflammatory cytokine interleukin-17. We discovered a series of potent and selective imidazo[1,5-a]pyridine and -pyrimidine RORc inverse agonists. The most potent compounds displayed >300-fold selectivity for RORc over the other ROR family members, PPARγ, and NRs in our cellular selectivity panel. The favorable potency, selectivity, and physiochemical properties of GNE-0946 (9) and GNE-6468 (28), in addition to their potent suppression of IL-17 production in human primary cells, support their use as chemical biology tools to further explore the role of RORc in human biology.

Abstract DDT02-03: Discovery of GDC-0994, a potent and selective ERK1/2 inhibitor in early clinical development

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The extracellular-signal-regulated kinases (ERK1 and ERK2) represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that commonly is activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling, such as receptor tyrosine kinase (RTK) activation. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Here, we present the discovery and characterization of GDC-0994, an orally bioavailable, small molecule inhibitor of ERK kinase activity. GDC-0994 is highly selective for ERK1 and ERK2, with biochemical potency of 1.1 nM and 0.3 nM, respectively. Daily, oral dosing of GDC-0994 results in significant single-agent activity in multiple in vivo cancer models, including KRAS-mutant and BRAF-mutant human xenograft tumors in mice. PD biomarker inhibition of phospho-p90RSK in these tumors correlates with potency in vitro and in vivo. In contrast to other published ERK inhibitors, GDC-0994 neither increases nor decreases phospho-ERK, suggesting that different ERK inhibitors have alternative mechanisms of action with respect to feedback signaling. Furthermore, we demonstrate a novel approach for targeting the oncogenic signaling through the RAS pathway by combining ERK and MEK inhibitors. GDC-0994 is currently in Phase I clinical development. Citation Format: Kirk Robarge, Jacob Schwarz, Jim Blake, Michael Burkard, Jocelyn Chan, Huifen Chen, Kang-Jye Chou, Dolores Diaz, John Gaudino, Stephen Gould, Jonas Grina, Xin Linghu, Lichuan Liu, Matthew Martinson, David A. Moreno, Christine Orr, Patricia Pacheco, Ann Qin, Kevin Rasor, Li Ren, Sheerin Shahidi-Latham, Jeffrey Stults, Francis Sullivan, Weiru Wang, Peter Yin, Aihe Zhou, Marcia Belvin, Mark Merchant, John G. Moffat. Discovery of GDC-0994, a potent and selective ERK1/2 inhibitor in early clinical development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr DDT02-03. doi:10.1158/1538-7445.AM2014-DDT02-03

Abstract A20: Development and validation of a novel acute myeloid leukemia xenograft model that is dependent on the JAK2V617F mutation for growth in vivo

Background: The Janus‐associated kinase (JAK) proteins are a family of non‐receptor tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) that play an important role in cellular survival, proliferation, and differentiation. A gain of function mutation (V617F) in the pseudo‐kinase domain of JAK2 is detected at high frequency in Philadelphia‐negative myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. To further elucidate the role of JAK2V617F on cell growth in vivo we established and validated a novel xenograft model using the acute myeloid leukemia cell line, SET2, which is heterozygous for the mutation. Materials and Methods: SET2 cells containing stable doxycycline‐inducible JAK2V617F shRNA clones were generated by lentiviral transduction. Cell proliferation and viability were assessed by incorporation of 3H‐Thymidine or by ATP quantitation. Cell‐cycle and apoptosis markers were evaluated by Western blotting using antibodies against cyclin D1, PIM1, cleaved caspase 3 and 7 and PARP. Phospho‐STAT5 and total STAT5 were measured using both western blotting and ELISA. A SET2 shRNA clone was selected for in vivo growth in SCID beige female mice and treated with doxycycline or a JAK2 inhibitor (JAK2i). Plasma and tumor drug levels were measured by LC/MS. Results: Genetic knockdown using a doxycycline‐inducible JAK2V617F shRNA confirmed SET2 cells are dependent on this gain of function mutation for growth based on inhibition of cell proliferation in the presence of doxycycline. The latter was corroborated with a selective JAK2i (Ki= 4nM) that potently inhibited SET2 cell viability (EC50 = 355nM). The JAK2i rapidly suppressed phosphorylation of STAT5 (EC50 = 313nM) and induced cycle arrest at the G0–G1 phase based on downregulation of cyclin D1 expression. The JAK2i also effectively inhibited expression of PIM‐1 kinase, a STAT5 target gene, and activated cleavage of procaspase 3 and 7 and DNA‐repair enzyme PARP to induce apoptosis in a time‐ and dose‐dependent manner. A SET2 xenograft model was established by in vivo selection of a variant of the doxycycline‐ JAK2V617F shRNA parental cell line. Its dependence on JAK2V617F was confirmed by treatment with doxcycline and oral administration of a JAK2i, which resulted in significant tumor growth inhibition in vivo. Investigation of the PK/PD relationship underlying the activity of JAK2i provided direct evidence of phospho‐STAT5 suppression and induction of apoptosis for its antitumor effect in vivo. Plasma drug levels correlated with the duration and magnitude of suppression. Conclusion: A JAK2V617F dependent SET2 xenograft model has been established and validated by shRNA gene knockdown and pharmacologically with a JAK2i in vivo. JAK2V617F inhibition results in suppression of STAT5 phosphorylation, cell‐cycle arrest and induction of apoptosis in vitro and in vivo. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A20.