Hanne Merritt

Identification of NVP-BKM120 as a Potent, Selective, Orally Bioavailable Class I PI3 Kinase Inhibitor for Treating Cancer

Phosphoinositide-3-kinases (PI3Ks) are important oncology targets due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein we describe the structure guided optimization of a series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines where the pharmacokinetic properties were improved by modulating the electronics of the 6-position heterocycle, and the overall druglike properties were fine-tuned further by modification of the 4-position substituent. The resulting 2,4-bismorpholino 6-heterocyclic pyrimidines are potent class I PI3K inhibitors showing mechanism modulation in PI3K dependent cell lines and in vivo efficacy in tumor xenograft models with PI3K pathway deregulation (A2780 ovarian and U87MG glioma). These efforts culminated in the discovery of 15 (NVP-BKM120), currently in Phase II clinical trials for the treatment of cancer.

RAF Inhibitors Activate the MAPK Pathway by Relieving Inhibitory Autophosphorylation

ATP competitive inhibitors of the BRAF(V600E) oncogene paradoxically activate downstream signaling in cells bearing wild-type BRAF (BRAF(WT)). In this study, we investigate the biochemical mechanism of wild-type RAF (RAF(WT)) activation by multiple catalytic inhibitors using kinetic analysis of purified BRAF(V600E) and RAF(WT) enzymes. We show that activation of RAF(WT) is ATP dependent and directly linked to RAF kinase activity. These data support a mechanism involving inhibitory autophosphorylation of RAFs phosphate-binding loop that, when disrupted either through pharmacologic or genetic alterations, results in activation of RAF and the mitogen-activated protein kinase (MAPK) pathway. This mechanism accounts not only for compound-mediated activation of the MAPK pathway in BRAF(WT) cells but also offers a biochemical mechanism for BRAF oncogenesis.

Abstract 3790: Preclinical profile of LGX818: A potent and selective RAF kinase inhibitor

Selective RAF inhibitors have significant activity in patients with metastatic melanoma whose tumors express BRAFV600E. However, not all patients respond equally well to treatment and the duration of response is often limited to less than 6 months. LGX818 was developed with the hypothesis that a more potent inhibitor with excellent pharmacological properties would maximize the degree and duration of patient response. LGX818 is a highly potent RAF inhibitor with selective anti-proliferative and apoptotic activity in cells expressing BRAFV600E. In the A375 (BRAFV600E) human melanoma cell line LGX818 suppresses phospho-ERK (EC50 = 3 nM) leading to potent inhibition of proliferation (EC50 = 4 nM). No significant activity was observed against a panel of 100 kinases (IC50 > 900 nM) and LGX818 did not inhibit proliferation of > 400 cell lines expressing wild-type BRAF. Contributing to the high potency of LGX818 is the extremely slow off-rate from BRAFV600E which is not observed with other RAF inhibitors. In biochemical assays the dissociation half-life was >24 hours which translated into sustained target inhibition in cells following drug wash-out. Single dose PK/PD studies in human melanoma xenograft models (BRAFV600E) indicated that LGX818 treatment at oral doses as low as 6 mg/kg resulted in strong (75%) and sustained (>24 hours) decrease in phospho-MEK, even following clearance of drug from circulation. Decreases in phospho-ERK were consistent with phospho-MEK but markers of downstream transcriptional output (DUSP6 and SPRY4) appeared to provide a more sensitive measure of pathway activation. LGX818 induced tumor regression in multiple BRAF mutant human tumor xenograft models grown in immune compromised mice and rats at doses as low as 1 mg/kg. Consistent with the in vitro data, LGX818 was inactive against BRAF wild-type tumors at doses up to 300 mg/kg bid, with good tolerability and linear increase in exposure. Efficacy was also achieved in a more disease-relevant spontaneous metastatic melanoma and a model of melanoma brain metastasis. LGX818 is a potent and selective RAF kinase inhibitor with unique biochemical properties that contribute to an excellent pharmacological profile. A Phase I clinical trial in patients with BRAF mutant tumors is ongoing. 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 3790. doi:1538-7445.AM2012-3790

Identification and structure-activity relationship of 2-morpholino 6-(3-hydroxyphenyl) pyrimidines, a class of potent and selective PI3 kinase inhibitors.

PI3 Kinases are a family of lipid kinases mediating numerous cell processes such as proliferation, migration, and differentiation. The PI3 kinase pathway is often de-regulated in cancer through PI3Kα overexpression, gene amplification, mutations, and PTEN phosphatase deletion. PI3K inhibitors represent therefore an attractive therapeutic modality for cancer treatment. Herein we describe a novel series of PI3K inhibitors sharing a pyrimidine core and showing significant potency against class I PI3 kinases in the biochemical assay and in cells. The discovery, synthesis and SAR of this chemotype are described.

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.

Structure guided optimization of a fragment hit to imidazopyridine inhibitors of PI3K.

PI3 kinases are a family of lipid kinases mediating numerous cell processes such as proliferation, migration and differentiation. The PI3 Kinase pathway is often de-regulated in cancer through PI3Kα overexpression, gene amplification, mutations and PTEN phosphatase deletion. PI3K inhibitors represent therefore an attractive therapeutic modality for cancer treatment. Herein we describe how the potency of a benzothiazole fragment hit was quickly improved based on structural information and how this early chemotype was further optimized through scaffold hopping. This effort led to the identification of a series of 2-acetamido-5-heteroaryl imidazopyridines showing potent in vitro activity against all class I PI3Ks and attractive pharmacokinetic properties.

Expression, purification and characterization of inactive and active forms of ERK2 from insect expression system.

Extracellular signal-regulated kinase 2 (ERK2) is a serine/threonine protein kinase involved in many cellular programs, such as cell proliferation, differentiation, motility and programed cell-death. It is therefore considered an important target in the treatment of cancer. In an effort to support biochemical screening and small molecule drug discovery, we established a robust system to generate both inactive and active forms of ERK2 using insect expression system. We report here, for the first time, that inactive ERK2 can be expressed and purified with 100% homogeneity in the unphosphorylated form using insect system. This resulted in a significant 20-fold yield improvement compared to that previously reported using bacterial expression system. We also report a newly developed system to generate active ERK2 in insect cells through in vivo co-expression with a constitutively active MEK1 (S218D S222D). Isolated active ERK2 was confirmed to be doubly phosphorylated at the correct sites, T185 and Y187, in the activation loop of ERK2. Both ERK2 forms, inactive and active, were well characterized by biochemical activity assay for their kinase function. Inactive and active ERK2 were the two key reagents that enabled successful high through-put biochemical assay screen and structural drug discovery studies.

Imidazo[1,2-a]pyridin-6-yl-benzamide Analogs as Potent RAF Inhibitors

A series of imidazo[1,2-a]pyridin-6-yl-benzamide analogs was designed as inhibitors of B-RAFV600E. Medicinal chemistry techniques were employed to explore the SAR for this series and improve selectivity versus P38 and VEGFR2.

Abstract LB-121: Dissecting MAPK pathway in BRAFmut melanoma: Intricacies of ERK1 and ERK2

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The MAPK signaling cascade, comprised of the RAS GTPases, the RAF, MEK1/2 and ERK1/2 kinases is frequently deregulated in cancer. ERK1 and ERK2 transmit signals generated by mutant BRAF, Ras or by activated receptor tyrosine kinases to a wide range of nuclear and cytoplasmic substrates, resulting in signal amplification, cell growth, migration and survival. ERK1 and ERK2 have been considered as redundant because of their high homology, large number of overlapping substrates, and ability to substitute for each other in genetically engineered mouse models. Nevertheless, several investigators have identified non-redundant roles for ERK isoforms in oncogenesis; for instance, ERK2, but not ERK1, appears to be responsible for RASmut induced epithelial-to-mesenchymal transformation. Besides, each of the ERK isoforms employs spatially distinct substrate docking domains, DEF (docking site for ERK FXFP) and D (docking domain), to signal to different subsets of substrates and differentially transmit signals downstream. We set out to determine the roles of ERK isoforms as well as DEF- and D-domain dependent signaling in the survival of melanoma tumor cells expressing activating BRAF mutations which are highly sensitive to pharmacological inhibitors of RAF, MEK1/2 and ERK1/2. We designed ERK1 and ERK2 mutants resistant to ATP-competitive ERK1/2 inhibitors and employed auto-activating, MEK-independent, ERK1 and ERK2 mutants to ask if BRAFmut melanoma survival is dependent on either or both ERK isoforms. In addition, we used RNAi and zinc-finger nucleases’ to knockdown or delete each of ERK isoforms. These experimental approaches consistently demonstrated that ERK2, but not ERK1, was the sole driver of cell survival in multiple BRAFmut melanoma cell lines. Moreover, genome-wide gene expression analysis indicated that ERK2, but not ERK1, was largely responsible for transcriptional effects imposed by pharmacological RAF, MEK1/2 or ERK1/2 inhibitors. Thus, in BRAFmut melanoma, functions of ERK1 and 2 are not redundant, and ERK1 cannot substitute for a disabled ERK2. Next, we introduced DEF- and D-substrate docking domain mutations into an ERK inhibitor resistant ERK2 to investigate whether signaling through either domain is sufficient to support melanoma survival. We observed that signaling through D- or DEF- domains of ERK2 had differential effects on gene expression and substrate phosphorylation. Consequently, we have found that a subset of melanoma cell lines was sensitive to elimination of DEF- docking domain interactions, whereas another subset of cell lines tolerated mutations in the DEF-site. Interactions and signaling through ERK D-docking site were dispensable for survival of all melanoma cell lines tested. These data suggest potential novel approaches to target oncogenic MAPK pathway. Citation Format: Tatiana Zavorotinskaya, Upasana Mehra, Yumin Dai, Michel Faure, Ken Crawford, Karen Yu, Jan Marie Cheng, Xiaolei Ma, Jan Xuan, Kelly Yan, Mohammad Hekmat-Nejad, Hanne Merritt, Darrin Stuart, Charles Voliva. Dissecting MAPK pathway in BRAFmut melanoma: Intricacies of ERK1 and ERK2. [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 LB-121. doi:10.1158/1538-7445.AM2014-LB-121

Abstract LB-54: Identification of a RAF inhibitory auto-phosphorylation site.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Preclinical studies have demonstrated that BRAF wild-type cancer cells are not only refractory but paradoxically activate the MAPK pathway when treated with RAF inhibitors. Potentially through a related mechanism, multiple point mutations in the activation loop and Phosphate binding loop (P. loop) domains of BRAF have been characterized in human cancers, which often render BRAF catalytically impaired, yet stimulate phosphorylation of downstream targets in cells. However, the underlying biochemistry causing these phenomena has yet to be fully understood. In this study we investigate the biochemical mechanism of wild-type RAF activation in response to RAF inhibitor treatment and identify a novel auto-phosphorylation site within the well conserved BRAF/CRAF P. loop. Disruption of the auto-phosphorylation, either through pharmacologic or genetic alterations, results in activation of the MAPK pathway. This work offers new insight into RAF biochemistry by uncovering a previously unrecognized regulatory mechanism of RAF kinases that is bypassed by the BRAF oncoproteins. Citation Format: Matthew Holderfield, Hanne Merritt, Julie Lin, Zenhai Gao, Darrin Stuart, Frank McCormick. Identification of a RAF inhibitory auto-phosphorylation site. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-54. doi:10.1158/1538-7445.AM2013-LB-54