Gisele Nishiguchi

Discovery of novel 3,5-disubstituted indole derivatives as potent inhibitors of Pim-1, Pim-2, and Pim-3 protein kinases.

A series of novel 3,5-disubstituted indole derivatives as potent and selective inhibitors of all three members of the Pim kinase family is described. High throughput screen identified a pan-Pim kinase inhibitor with a promiscuous scaffold. Guided by structure-based drug design, SAR of the series afforded a highly selective indole chemotype that was further developed into a potent set of compounds against Pim-1, 2, and 3 (Pim-1 and Pim-3: IC(50)≤2nM and Pim-2: IC(50)≤100nM).

Structure Guided Optimization, in Vitro Activity, and in Vivo Activity of Pan-PIM Kinase Inhibitors.

Proviral insertion of Moloney virus (PIM) 1, 2, and 3 kinases are serine/threonine kinases that normally function in survival and proliferation of hematopoietic cells. As high expression of PIM1, 2, and 3 is frequently observed in many human malignancies, including multiple myeloma, non-Hodgkins lymphoma, and myeloid leukemias, there is interest in determining whether selective PIM inhibition can improve outcomes of these human cancers. Herein, we describe our efforts toward this goal. The structure guided optimization of a singleton high throughput screening hit in which the potency against all three PIM isoforms was increased >10,000-fold to yield compounds with pan PIM K is < 10 pM, nanomolar cellular potency, and in vivo activity in an acute myeloid leukemia Pim-dependent tumor model is described.

Identification of N-(4-((1R,3S,5S)-3-Amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide (PIM447), a Potent and Selective Proviral Insertion Site of Moloney Murine Leukemia (PIM) 1, 2, and 3 Kinase Inhibitor in Clinical Trials for Hematological Malignancies.

Pan proviral insertion site of Moloney murine leukemia (PIM) 1, 2, and 3 kinase inhibitors have recently begun to be tested in humans to assess whether pan PIM kinase inhibition may provide benefit to cancer patients. Herein, the synthesis, in vitro activity, in vivo activity in an acute myeloid leukemia xenograft model, and preclinical profile of the potent and selective pan PIM kinase inhibitor compound 8 (PIM447) are described. Starting from the reported aminopiperidyl pan PIM kinase inhibitor compound 3, a strategy to improve the microsomal stability was pursued resulting in the identification of potent aminocyclohexyl pan PIM inhibitors with high metabolic stability. From this aminocyclohexyl series, compound 8 entered the clinic in 2012 in multiple myeloma patients and is currently in several phase 1 trials of cancer patients with hematological malignancies.

Design and Discovery of N-(2-Methyl-5′-morpholino-6′-((tetrahydro-2H-pyran-4-yl)oxy)-[3,3′-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (RAF709): A Potent, Selective, and Efficacious RAF Inhibitor Targeting RAS Mutant Cancers

RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [ Aversa , Biaryl amide compounds as kinase inhibitors and their preparation . WO 2014151616, 2014 ], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.

Design, synthesis and structure activity relationship of potent pan-PIM kinase inhibitors derived from the pyridyl carboxamide scaffold

The Pim proteins (1, 2 and 3) are serine/threonine kinases that have been found to be upregulated in many hematological malignancies and solid tumors. As a result of overlapping functions among the three isoforms, inhibition of all three Pim kinases has become an attractive strategy for cancer therapy. Herein we describe our efforts in identifying potent pan-PIM inhibitors that are derived from our previously reported pyridyl carboxamide scaffold as part of a medicinal chemistry strategy to address metabolic stability.

Antitumor Properties of RAF709, a Highly Selective and Potent Inhibitor of RAF Kinase Dimers, in Tumors Driven by Mutant RAS or BRAF

Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAFV600 alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in in vivo tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor-derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors.Significance: In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and in vivo properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling. Cancer Res; 78(6); 1537-48. ©2018 AACR.

Abstract 330: Development of a highly selective B/CRAF kinase inhibitor that exhibits antitumor activities in RAS and BRAF mutant tumors with minimal paradoxical activation

The mitogen-activated protein kinase (MAPK) signaling pathway is frequently activated in human cancers due to genetic alterations that can occur at multiple nodes in the pathway, the most prevalent of which are mutations in RAS or BRAF. While BRAFV600 mutant tumors are effectively treated with existing RAF inhibitors, RAS mutant cancers and tumors expressing atypical BRAF mutants remain an unmet medical need. Emerging biology has demonstrated that the CRAF kinase functions as a critical mediator of mutant KRAS-driven cell proliferation and tumor development. CRAF was also shown to be the mediator of feedback-mediated pathway reactivation following MEK inhibitor treatment in KRAS mutant cancers. Hence selective inhibitors that potently inhibit the activity of CRAF could be both effective in blocking mutant RAS-driven tumorigenesis and in alleviating feedback activation. We have developed a type II ATP-competitive inhibitor that inhibits both B- and CRAF kinase activities at picomolar IC50 values in biochemical assays with high selectivity profile against a panel of 456 human kinases. The inhibitor not only inhibits MAPK signaling activity in tumor models harboring BRAFV600 mutation, but also inhibits mutant N- and KRAS-driven signaling with minimum paradoxical activation, likely due to its activity in inhibiting both RAF monomers and dimers with similar potencies. Correspondingly, profiling data of the inhibitor in a panel of 480 human cancer cell lines shows that it has higher antitumor activities in cell lines harboring BRAF or RAS mutations as compared to those that are wild-type. The inhibitor is orally bioavailable, it demonstrates a direct PK/PD relationship and causes tumor regression in multiple cell line and primary human tumor derived xenograft models that have BRAF, NRAS or KRAS mutations with good tolerability. Thus, we have developed a next generation RAF inhibitor with unique biochemical and cellular properties that enables its antitumor activities in RAS mutant tumors. Citation Format: Wenlin Shao, Yuji Mishina, Yun Feng, Giordano Caponigro, Savithri Ramurthy, Vesselina Cooke, Lesley Griner, Gisele Nishiguchi, Alice Rico, Ben Taft, Matthew Burger, Huw Tanner, Valery Polyakov, Brent Appleton, John Tellew, Richard Zang, Mohammad Hekmat-Nejad, Payman Amiri, Mallika Singh, Darrin Stuart. Development of a highly selective B/CRAF kinase inhibitor that exhibits antitumor activities in RAS and BRAF mutant tumors with minimal paradoxical activation. [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 330.