Purinylpyridinylamino-based DFG-in/αC-helix-out B-Raf inhibitors: Applying mutant versus wild-type B-Raf selectivity indices for compound profiling.

One of the challenges for targeting B-Raf(V600E) with small molecule inhibitors had been achieving adequate selectivity over the wild-type protein B-Raf(WT), as inhibition of the latter has been associated with hyperplasia in normal tissues. Recent studies suggest that B-Raf inhibitors inducing the 'DFG-in/αC-helix-out' conformation (Type IIB) likely will exhibit improved selectivity for B-Raf(V600E). To explore this hypothesis, we transformed Type IIA inhibitor (1) into a series of Type IIB inhibitors (sulfonamides and sulfamides 4-6) and examined the SAR. Three selectivity indices were introduced to facilitate the analyses: the B-Raf(V600E)/B-Raf(WT) biochemical ((b)S), cellular ((c)S) selectivity, and the phospho-ERK activation ((p)A). Our data indicates that α-branched sulfonamides and sulfamides show higher selectivities than the linear derivatives. We rationalized this finding based on analysis of structural information from the literature and provided evidence for a monomeric B-Raf-inhibitor complex previously hypothesized to be responsible for the desired B-Raf(V600E) selectivity.

An ALK2 inhibitor, BLU-782, prevents heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease driven by gain-of-function variants in activin receptor-like kinase 2 (ALK2), the most common variant being ALK2R206H. In FOP, ALK2 variants display increased and dysregulated signaling through the bone morphogenetic protein (BMP) pathway resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of BLU-782 (IPN60130), a small-molecule ALK2R206H inhibitor developed for the treatment of FOP. A small-molecule library was screened in a biochemical ALK2 binding assay to identify potent ALK2 binding compounds. Iterative rounds of structure-guided drug design were used to optimize compounds for ALK2R206H binding, ALK2 selectivity, and other desirable pharmacokinetic properties. BLU-782 preferentially bound to ALK2R206H with high affinity, inhibiting signaling from ALK2R206H and other rare FOP variants in cells in vitro without affecting signaling of closely related homologs ALK1, ALK3, and ALK6. In vivo efficacy of BLU-782 was demonstrated using a conditional knock-in ALK2R206H mouse model, where prophylactic oral dosing reduced edema and prevented cartilage and heterotopic ossification (HO) in both muscle and bone injury models. BLU-782 treatment preserved the normal muscle-healing response in ALK2R206H mice. Delayed dosing revealed a short 2-day window after injury when BLU-782 treatment prevented HO in ALK2R206H mice, but dosing delays of 4 days or longer abrogated HO prevention. Together, these data suggest that BLU-782 may be a candidate for prevention of HO in FOP.

Discovery and optimization of potent and selective imidazopyridine and imidazopyridazine mTOR inhibitors.

mTOR is a critical regulator of cellular signaling downstream of multiple growth factors. The mTOR/PI3K/AKT pathway is frequently mutated in human cancers and is thus an important oncology target. Herein we report the evolution of our program to discover ATP-competitive mTOR inhibitors that demonstrate improved pharmacokinetic properties and selectivity compared to our previous leads. Through targeted SAR and structure-guided design, new imidazopyridine and imidazopyridazine scaffolds were identified that demonstrated superior inhibition of mTOR in cellular assays, selectivity over the closely related PIKK family and improved in vivo clearance over our previously reported benzimidazole series.

Discovery of triazine-benzimidazoles as selective inhibitors of mTOR.

mTOR is part of the PI3K/AKT pathway and is a central regulator of cell growth and survival. Since many cancers display mutations linked to the mTOR signaling pathway, mTOR has emerged as an important target for oncology therapy. Herein, we report the discovery of triazine benzimidazole inhibitors that inhibit mTOR kinase activity with up to 200-fold selectivity over the structurally homologous kinase PI3Kα. When tested in a panel of cancer cell lines displaying various mutations, a selective inhibitor from this series inhibited cellular proliferation with a mean IC(50) of 0.41 µM. Lead compound 42 demonstrated up to 83% inhibition of mTOR substrate phosphorylation in a murine pharmacodynamic model.

Pyridyl-pyrimidine benzimidazole derivatives as potent, selective, and orally bioavailable inhibitors of Tie-2 kinase.

Selective small molecule inhibitors of Tie-2 kinase are important tools for the validation of Tie-2 signaling in pathological angiogenesis. Reported herein is the optimization of a nonselective scaffold into a potent and highly selective inhibitor of Tie-2 kinase.

Acquired On-Target Clinical Resistance Validates FGFR4 as a Driver of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide with no clinically confirmed oncogenic driver. Although preclinical studies implicate the FGF19 receptor FGFR4 in hepatocarcinogenesis, the dependence of human cancer on FGFR4 has not been demonstrated. Fisogatinib (BLU-554) is a potent and selective inhibitor of FGFR4 and demonstrates clinical benefit and tumor regression in patients with HCC with aberrant FGF19 expression. Mutations were identified in the gatekeeper and hinge-1 residues in the kinase domain of FGFR4 upon disease progression in 2 patients treated with fisogatinib, which were confirmed to mediate resistance in vitro and in vivo. A gatekeeper-agnostic, pan-FGFR inhibitor decreased HCC xenograft growth in the presence of these mutations, demonstrating continued FGF19-FGFR4 pathway dependence. These results validate FGFR4 as an oncogenic driver and warrant further therapeutic targeting of this kinase in the clinic. SIGNIFICANCE: Our study is the first to demonstrate on-target FGFR4 kinase domain mutations as a mechanism of acquired clinical resistance to targeted therapy. This further establishes FGF19-FGFR4 pathway activation as an oncogenic driver. These findings support further investigation of fisogatinib in HCC and inform the profile of potential next-generation inhibitors.See related commentary by Subbiah and Pal, p. 1646.This article is highlighted in the In This Issue feature, p. 1631.

Structure-based design of novel 2-amino-6-phenyl-pyrimido[5',4':5,6]pyrimido[1,2-a]benzimidazol-5(6H)-ones as potent and orally active inhibitors of lymphocyte specific kinase (Lck): synthesis, SAR, and in vivo anti-inflammatory activity.

Lck, or lymphocyte specific kinase, is a cytoplasmic tyrosine kinase of the Src family expressed in T-cells and NK cells. Genetic evidence from knockout mice and human mutations demonstrates that Lck kinase activity is critical for T-cell receptor-mediated signaling, leading to normal T-cell development and activation. A small molecule inhibitor of Lck is expected to be useful in the treatment of T-cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection. In this paper, we describe the structure-guided design, synthesis, structure-activity relationships, and pharmacological characterization of 2-amino-6-phenylpyrimido[5',4':5,6]pyrimido[1,2- a]benzimidazol-5(6 H)-ones, a new class of compounds that are potent inhibitors of Lck. The most promising compound of this series, 6-(2,6-dimethylphenyl)-2-((4-(4-methyl-1-piperazinyl)phenyl)amino)pyrimido[5',4':5,6]pyrimido-[1,2- a]benzimidazol-5(6 H)-one ( 25), exhibits potent inhibition of Lck kinase activity. This activity translates into inhibition of in vitro cell-based assays and in vivo models of T-cell activation and arthritis, respectively.

Structure-guided design of aminopyrimidine amides as potent, selective inhibitors of lymphocyte specific kinase: synthesis, structure-activity relationships, and inhibition of in vivo T cell activation.

The lymphocyte-specific kinase (Lck), a member of the Src family of cytoplasmic tyrosine kinases, is expressed in T cells and natural killer (NK) cells. Genetic evidence, including knockout mice and human mutations, demonstrates that Lck kinase activity is critical for normal T cell development, activation, and signaling. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. With the aid of X-ray structure-based analysis, aminopyrimidine amides 2 and 3 were designed from aminoquinazolines 1, which had previously been demonstrated to exhibit potent inhibition of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminopyrimidine amides 3 possessing improved cellular potency and selectivity profiles relative to their aminoquinazoline predecessors 1. Orally bioavailable compound 13b inhibited the anti-CD3-induced production of interleukin-2 (IL-2) in mice in a dose-dependent manner (ED 50 = 9.4 mg/kg).

Targeting kinases with precision.

Cancer genomics and mechanistic studies have revealed that heterogeneous mutations within a single kinase can result in a variety of activation mechanisms. The challenge has been to match these insights with tailored drug discovery strategies to yield potent, highly selective drugs. With optimized drugs in hand, physicians could apply the principles of personalized medicine with an increasing number of options to treat patients with improved precision according to their tumor's molecular genotype.

Precision Targeted Therapy with BLU-667 for RET-Driven Cancers.

The receptor tyrosine kinase rearranged during transfection (RET) is an oncogenic driver activated in multiple cancers, including non-small cell lung cancer (NSCLC), medullary thyroid cancer (MTC), and papillary thyroid cancer. No approved therapies have been designed to target RET; treatment has been limited to multikinase inhibitors (MKI), which can have significant off-target toxicities and limited efficacy. BLU-667 is a highly potent and selective RET inhibitor designed to overcome these limitations. In vitro, BLU-667 demonstrated ≥10-fold increased potency over approved MKIs against oncogenic RET variants and resistance mutants. In vivo, BLU-667 potently inhibited growth of NSCLC and thyroid cancer xenografts driven by various RET mutations and fusions without inhibiting VEGFR2. In first-in-human testing, BLU-667 significantly inhibited RET signaling and induced durable clinical responses in patients with RET-altered NSCLC and MTC without notable off-target toxicity, providing clinical validation for selective RET targeting.Significance: Patients with RET-driven cancers derive limited benefit from available MKIs. BLU-667 is a potent and selective RET inhibitor that induces tumor regression in cancer models with RET mutations and fusions. BLU-667 attenuated RET signaling and produced durable clinical responses in patients with RET-altered tumors, clinically validating selective RET targeting. Cancer Discov; 8(7); 836-49. ©2018 AACR.See related commentary by Iams and Lovly, p. 797This article is highlighted in the In This Issue feature, p. 781.

Design, synthesis, and evaluation of orally active benzimidazoles and benzoxazoles as vascular endothelial growth factor-2 receptor tyrosine kinase inhibitors.

Inhibition of the VEGF signaling pathway has become a valuable approach in the treatment of cancers. Guided by X-ray crystallography and molecular modeling, a series of 2-aminobenzimidazoles and 2-aminobenzoxazoles were identified as potent inhibitors of VEGFR-2 (KDR) in both enzymatic and HUVEC cellular proliferation assays. In this report we describe the synthesis and structure-activity relationship of a series of 2-aminobenzimidazoles and benzoxazoles, culminating in the identification of benzoxazole 22 as a potent and selective VEGFR-2 inhibitor displaying a good pharmacokinetic profile. Compound 22 demonstrated efficacy in both the murine matrigel model for vascular permeability (79% inhibition observed at 100 mg/kg) and the rat corneal angiogenesis model (ED(50) = 16.3 mg/kg).

Evolution of a highly selective and potent 2-(pyridin-2-yl)-1,3,5-triazine Tie-2 kinase inhibitor.

Inhibition of angiogenesis is a promising and clinically validated approach for limiting tumor growth and survival. The receptor tyrosine kinase Tie-2 is expressed almost exclusively in the vascular endothelium and is required for developmental angiogenesis and vessel maturation. However, the significance of Tie-2 signaling in tumor angiogenesis is not well understood. In order to evaluate the therapeutic utility of inhibiting Tie-2 signaling, we developed a series of potent and orally bioavailable small molecule Tie-2 kinase inhibitors with selectivity over other kinases, especially those that are believed to be important for tumor angiogenesis. Our earlier work provided pyridinyl pyrimidine 6 as a potent, nonselective Tie-2 inhibitor that was designed on the basis of X-ray cocrystal structures of KDR inhibitors 34 (triazine) and 35 (nicotinamide). Lead optimization resulted in pyridinyl triazine 63, which exhibited >30-fold selectivity over a panel of kinases, good oral exposure, and in vivo inhibition of Tie-2 phosphorylation.

Alkynylpyrimidine amide derivatives as potent, selective, and orally active inhibitors of Tie-2 kinase.

The recognition that aberrant angiogenesis contributes to the pathology of inflammatory diseases, cancer, and myocardial ischemia has generated considerable interest in the molecular mechanisms that regulate blood vessel growth. The receptor tyrosine kinase Tie-2 is expressed primarily by vascular endothelial cells and is critical for embryonic vasculogenesis. Interference with the Tie-2 pathway by diverse blocking agents such as soluble Tie-2 receptors, anti-Tie-2 intrabodies, anti-Ang-2 antibodies, and peptide-Fc conjugates has been shown to suppress tumor growth in xenograft studies. An alternative strategy for interfering with the Tie-2 signaling pathway involves direct inhibition of the kinase functions of the Tie-2 receptor. Herein we describe the development of alkynylpyrimidine amide derivatives as potent, selective, and orally available ATP-competitive inhibitors of Tie-2 autophosphorylation.

A precision therapy against cancers driven by KIT/PDGFRA mutations.

Targeting oncogenic kinase drivers with small-molecule inhibitors can have marked therapeutic benefit, especially when administered to an appropriate genomically defined patient population. Cancer genomics and mechanistic studies have revealed that heterogeneous mutations within a single kinase can result in various mechanisms of kinase activation. Therapeutic benefit to patients can best be optimized through an in-depth understanding of the disease-driving mutations combined with the ability to match these insights to tailored highly selective drugs. This rationale is presented for BLU-285, a clinical stage inhibitor of oncogenic KIT and PDGFRA alterations, including activation loop mutants that are ineffectively treated by current therapies. BLU-285, designed to preferentially interact with the active conformation of KIT and PDGFRA, potently inhibits activation loop mutants KIT D816V and PDGFRA D842V with subnanomolar potency and also inhibits other well-characterized disease-driving KIT mutants both in vitro and in vivo in preclinical models. Early clinical evaluation of BLU-285 in a phase 1 study has demonstrated marked activity in patients with diseases associated with KIT (aggressive systemic mastocytosis and gastrointestinal stromal tumor) and PDGFRA (gastrointestinal stromal tumor) activation loop mutations.

Discovery of aminoquinazolines as potent, orally bioavailable inhibitors of Lck: synthesis, SAR, and in vivo anti-inflammatory activity.

The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and natural killer (NK) cells. Genetic evidence in both mice and humans demonstrates that Lck kinase activity is critical for signaling mediated by the T cell receptor (TCR), which leads to normal T cell development and activation. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. Screening of our kinase-preferred collection identified aminoquinazoline 1 as a potent, nonselective inhibitor of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminoquinazolines possessing in vitro mechanism-based potency. Optimized, orally bioavailable compounds 32 and 47 exhibit anti-inflammatory activity (ED(50) of 22 and 11 mg/kg, respectively) in the anti-CD3-induced production of interleukin-2 (IL-2) in mice.

First Selective Small Molecule Inhibitor of FGFR4 for the Treatment of Hepatocellular Carcinomas with an Activated FGFR4 Signaling Pathway.

UNLABELLED: Aberrant signaling through the fibroblast growth factor 19 (FGF19)/fibroblast growth factor receptor 4 (FGFR 4) signaling complex has been shown to cause hepatocellular carcinoma (HCC) in mice and has been implicated to play a similar role in humans. We have developed BLU9931, a potent and irreversible small-molecule inhibitor of FGFR4, as a targeted therapy to treat patients with HCC whose tumors have an activated FGFR4 signaling pathway. BLU9931 is exquisitely selective for FGFR4 versus other FGFR family members and all other kinases. BLU9931 shows remarkable antitumor activity in mice bearing an HCC tumor xenograft that overexpresses FGF19 due to amplification as well as a liver tumor xenograft that overexpresses FGF19 mRNA but lacks FGF19 amplification. Approximately one third of patients with HCC whose tumors express FGF19 together with FGFR4 and its coreceptor klotho ß (KLB) could potentially respond to treatment with an FGFR4 inhibitor. These findings are the first demonstration of a therapeutic strategy that targets a subset of patients with HCC. SIGNIFICANCE: This article documents the discovery of BLU9931, a novel irreversible kinase inhibitor that specifically targets FGFR4 while sparing all other FGFR paralogs and demonstrates exquisite kinome selectivity. BLU9931 is efficacious in tumors with an intact FGFR4 signaling pathway that includes FGF19, FGFR4, and KLB. BLU9931 is the first FGFR4-selective molecule for the treatment of patients with HCC with aberrant FGFR4 signaling.

The landscape of kinase fusions in cancer.

Human cancer genomes harbour a variety of alterations leading to the deregulation of key pathways in tumour cells. The genomic characterization of tumours has uncovered numerous genes recurrently mutated, deleted or amplified, but gene fusions have not been characterized as extensively. Here we develop heuristics for reliably detecting gene fusion events in RNA-seq data and apply them to nearly 7,000 samples from The Cancer Genome Atlas. We thereby are able to discover several novel and recurrent fusions involving kinases. These findings have immediate clinical implications and expand the therapeutic options for cancer patients, as approved or exploratory drugs exist for many of these kinases.

Discovery of potent and highly selective thienopyridine Janus kinase 2 inhibitors.

Developing Janus kinase 2 (Jak2) inhibitors has become a significant focus for small molecule drug discovery programs in recent years due to the identification of a Jak2 gain-of-function mutation in the majority of patients with myeloproliferative disorders (MPD). Here, we describe the discovery of a thienopyridine series of Jak2 inhibitors that culminates with compounds showing 100- to >500-fold selectivity over the related Jak family kinases in enzyme assays. Selectivity for Jak2 was also observed in TEL-Jak cellular assays, as well as in cytokine-stimulated peripheral blood mononuclear cell (PBMC) and whole blood assays. X-ray cocrystal structures of 8 and 19 bound to the Jak2 kinase domain aided structure-activity relationship efforts and, along with a previously reported small molecule X-ray cocrystal structure of the Jak1 kinase domain, provided structural rationale for the observed high levels of Jak2 selectivity.

Selective and potent Raf inhibitors paradoxically stimulate normal cell proliferation and tumor growth.

Raf inhibitors are under clinical investigation, specifically in patients with tumor types harboring frequent activating mutations in B-Raf. Here, we show that cell lines and tumors harboring mutant B-Raf were sensitive to a novel series of Raf inhibitors (e.g., (V600E)B-Raf A375, IC(50) on cells = 2 nmol/L; ED(50) on tumor xenografts = 1.3 mg/kg). However, in cells and tumors with wild-type B-Raf, exposure to Raf inhibitors resulted in a dose-dependent and sustained activation of mitogen-activated protein kinase signaling. In some of these cell lines, Raf inhibition led to entry into the cell cycle, enhanced proliferation, and significantly stimulated tumor growth in vivo. Inhibition with structurally distinct Raf inhibitors or isoform-specific small interfering RNA knockdown of Raf showed that these effects were mediated directly through Raf. Either A-Raf or C-Raf mediated the Raf inhibitor-induced mitogen-activated protein kinase pathway activation in an inhibitor-specific manner. These paradoxical effects of Raf inhibition were seen in malignant and normal cells in vitro and in vivo. Hyperplasia of normal epithelial cells in the esophagus and the stomach was evident in mice with all efficacious Raf inhibitors (n = 8) tested. An implication of these results is that Raf inhibitors may induce unexpected normal cell and tumor tissue proliferation in patients.