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Background: Endothelial cell activation is tightly controlled by the balance between VEGF (vascular endothelial cell growth factor) and Notch signaling pathway. VEGF destabilizes blood vessels and promotes neovascularization, which are common features of sight-threatening ocular vascular disorders. Here, we show that BCL6B (B-cell CLL/lymphoma 6 member B protein), also known as BAZF, ZBTB28, and ZNF62, plays a pivotal role in the development of retinal edema and neovascularization. Methods: The pathophysiological physiological role of BCL6B was investigated in cellular and animal models mimicking 2 pathological conditions: retinal vein occlusion and choroidal neovascularization. An in vitro experimental system was used in which human retinal microvascular endothelial cells were supplemented with VEGF. Choroidal neovascularization cynomolgus monkey model was generated to investigate the involvement of BCL6B in the pathogenesis. Mice lacking BCL6B or treated with BCL6B-targeting small-interfering ribose nucleic acid were examined for histological and molecular phenotypes. Results: In retinal endothelial cells, the BCL6B expression level was increased by VEGF. BCL6B-deficient endothelial cells showed Notch signal activation and attenuated cord formation via blockage of the VEGF-VEGFR2 signaling pathway. Optical coherence tomography images showed that choroidal neovascularization lesions were decreased by BCL6B-targeting small-interfering ribose nucleic acid. Although BCL6B mRNA expression was significantly increased in the retina, BCL6B-targeting small-interfering ribose nucleic acid suppressed ocular edema in the neuroretina. The increase in proangiogenic cytokines and breakdown of the inner blood-retinal barrier were abrogated in BCL6B knockout (KO) mice via Notch transcriptional activation by CBF1 (C promotor-binding factor 1) and its activator, the NICD (notch intracellular domain). Immunostaining showed that Müller cell activation, a source of VEGF, was diminished in BCL6B-KO retinas. Conclusions: These data indicate that BCL6B may be a novel therapeutic target for ocular vascular diseases characterized by ocular neovascularization and edema.

Validation of predictive response biomarkers

Structures of compounds used in the study

Workflow for filtering cell line genomic data

Additional reproducibility data

Extracellular signal-regulated kinase 2 (ERK2) controls vital physiological processes involving proliferation and differentiation and is a drug target molecule for many diseases such as cancers. In silico screening focusing on an allosteric site that plays a crucial role in substrate anchoring conferred an ERK2 inhibitor (compound 1). However, a competitive binding assay indicated that compound 1 did not bind to the allosteric site. Here, the crystal structure of ERK2 in complex with compound 1 revealed a novel binding site. This finding demonstrates the feasibility of developing new types of ERK2 inhibitors.

Although Bruton's tyrosine kinase (BTK) has been recognized as a validated drug target for the treatment of B-cell malignances, the emergence of clinical resistance to the first-generation covalent BTK inhibitors is becoming a serious concern. As a part of our effort to develop noncovalent BTK inhibitors, a series of novel pyrrolopyrimidines was identified as noncovalent inhibitors of both the wild-type and C481S mutant BTKs. Subsequent lead optimization led to the identification of an orally available, potent, and selective BTK inhibitor 13f (AS-1763) as a next-generation noncovalent BTK inhibitor. With significant efficacies in vivo tumor xenograft models, AS-1763 has advanced to phase 1 clinical trials.

Casein kinase 2 (CK2) is a vital protein kinase that consists of two catalytic subunits (CK2α1 and/or CK2α2) and two regulatory subunits (CK2β). CK2α1 is a drug target for nephritis and cancers, while CK2α2 is a serious off-target because its inhibition causes testicular toxicity. High similarity between the isozymes CK2α1 and CK2α2 make it difficult to design CK2α1-specific inhibitors. Herein, the crystal structures of CK2α1 and CK2α2 complexed with a 3-amino-pyrazole inhibitor revealed the remarkable differences in the protein-inhibitor interaction modes. This inhibitor bound to the ATP binding sites of both isozymes in apparently distinct orientations. In addition, another molecule of this inhibitor bound to CK2α1, but not to CK2α2, at the CK2β protein-protein interface. Binding energy calculations and biochemical experiments suggested that this inhibitor possesses the conventional ATP-competitive characteristics with moderate allosteric function in a molecular glue mechanism. These results will assist the potential design of potent and selective CK2α1 inhibitors.

Background Metastasis is the primary cause of death in cancer patients, and its management is still a major challenge. Epithelial to mesenchymal transition (EMT) has been implicated in the process of cancer metastasis, and its pharmacological interference holds therapeutic promise. Methods Traf2- and Nck-interacting kinase (TNIK) functions as a transcriptional coregulator of Wnt target genes. Given the convergence of Wnt and transforming growth factor-β (TGFβ) signalling, we examined the effects of a small-molecule TNIK inhibitor (named NCB-0846) on the TGFβ1-induced EMT of lung cancer cells. Results NCB-0846 inhibited the TGFβ1-induced EMT of A549 cells. This inhibition was associated with inhibition of Sma- and Mad-Related Protein-2/3 (SMAD2/3) phosphorylation and nuclear translocation. NCB-0846 abolished the lung metastasis of TGFβ1-treated A549 cells injected into the tail veins of immunodeficient mice. The inhibition of EMT was mediated by suppression of the TGFβ receptor type-I (TGFBR1) gene, at least partly through the induction of microRNAs targeting the TGFBR1 transcript [miR-320 (a, b and d) and miR-186]. Conclusions NCB-0846 pharmacologically blocks the TGFβ/SMAD signalling and EMT induction of lung cancer cells by transcriptionally downregulating TGFBRI expression, representing a potentially promising approach for prevention of metastasis in lung cancer patients.

Introduction: Transforming growth factor β (TGF-β) plays a key role in promoting tumor progression mainly through facilitating angiogenesis, epithelial-mesenchymal transition (EMT) and also inducing immunosuppression in tumor microenvironment, allowing cancer cells to escape immune surveillance. Activin receptor-like kinases (ALKs) consisting of seven type I receptor serine/threonine kinases are responsible for TGF-β family signal transduction by phosphorylating Smad proteins, which can activate target gene expression. Therefore, ALKs are attractive drug targets for intervention in TGF- β signaling pathways as cancer immunotherapy. Materials and methods: We have produced seven human recombinant ALK proteins (ALK1-7). The in vitro kinase assays for all ALKs have been developed using TR-FRET system. To evaluated cellular potency, TGF-β-dependent Smad3 phosphorylation assays in A549 (human lung adenocarcinoma cell line) and NMuMG (murine normal mammary gland cell line) cells were established. A TGF-β-mediated Treg differentiation assay was performed as an ex-vivo cellular assay using naive CD4+ T cell isolated from mouse spleen. In vivo anti-tumor efficacies of selected compounds, alone or in combination with anti-PD-1 antibody were assessed in a CT26.WT murine colon carcinoma syngeneic model. Results: We have implemented a high-throughput screening (HTS) against human ALK5 over our in-house compound library. Hit compound 1 was identified from the HTS, and the subsequent Hit-to-Lead study led to a novel thiazole analog 2 as the starting point of lead optimization. Our lead optimization efforts led to advanced lead compound AL1, which exhibited strong inhibitory potency for ALK5 with an IC50 value of single digit nanomolar. Unfortunately AL1 showed very low oral bioavailability in dogs, we continued the optimization study and identified compound AL2 having improved ADME property. In cellular assays, AL2 strongly suppressed the TGF-β-dependent Smad3 phosphorylation in A549 cells. In addition, treatment with AL2 decreased in the percentage of mouse CD4+/CD25+/Foxp3+ Treg cells dose-dependently in the ex vivo TGF-β-mediated Treg differentiation assay. As a monotherapy and combined with an anti-PD-1 antibody, oral administration of AL2 demonstrated robust in vivo antitumor efficacy in the CT26.WT murine colon carcinoma syngeneic model. Conclusions: On the basis of its in vitro potencies, PK profiles, and in vivo efficacies, AL2 is currently being further evaluated in preclinical studies. Citation Format: Mai Arai, Mitsuharu Hanada, Hideki Moriyama, Hiroshi Ohmoto, Kazuhito Naka, Masaaki Sawa. Discovery of a novel activin receptor-like kinases (ALKs) inhibitor targeting TGF-β signaling pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5336.

Eukaryotic protein kinases contain an Asp-Phe-Gly (DFG) motif, the conformation of which is involved in controlling the catalytic activity, at the N-terminus of the activation segment. The motif can be switched between active-state (DFG-in) and inactive-state (DFG-out) conformations: however, the mechanism of conformational change is poorly understood, partly because there are few reports of the DFG-out conformation. Here, a novel crystal structure of nonphosphorylated human mitogen-activated protein kinase kinase 1 (MEK1; amino acids 38–381) complexed with ATP-γS is reported in which MEK1 adopts the DFG-out conformation. The crystal structure revealed that the structural elements (the αC helix and HRD motif) surrounding the active site are involved in the formation/stabilization of the DFG-out conformation. The ATP-γS molecule was bound to the canonical ATP-binding site in a different binding mode that has never been found in previously determined crystal structures of MEK1. This novel ATP-γS binding mode provides a starting point for the design of high-affinity inhibitors of nonphosphorylated inactive MEK1 that adopts the DFG-out conformation.

Mitogen-activated protein kinase kinase 4 (MAP2K4) plays a critical role in regulating the stress-activated protein kinase signaling cascade. A small angle X-ray scattering experiment, a powerful technique for analyzing a solution structure cleared from the structural artifacts due to crystal packing, provided the ensemble structures of human non-phosphorylated MAP2K4 in three states involving the apo form, the binary complex with an ATP analogue, and the ternary complex with the ATP analogue and substrate peptide. These ensemble structures provided more detailed mechanisms for regulating MAP2K4 in addition to those delineated only by the crystal structures in three states.

Protein kinase CK2a1 is a serine/threonine kinase that plays a crucial role in the growth, proliferation and survival of cells and is a well known target for tumour and glomerulonephritis therapies. Here, the crystal structure of the kinase domain of CK2a1 complexed with 5-iodotubercidin (5IOD), an ATP-mimetic inhibitor, was determined at 1.78 Å resolution. The structure shows distinct structural features and, in combination with a comparison of the crystal structures of five off-target kinases complexed with 5IOD, provides valuable information for the development of highly selective inhibitors.

Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA from virus or self-DNA from mitochondria/nuclei. In response to emergence of such DNAs in the cytosol, STING translocates from the endoplasmic reticulum (ER) to the Golgi, and activates TANK-binding kinase 1 (TBK1) at the trans-Golgi network (TGN). Activated TBK1 then phosphorylates STING at Ser365, generating an interferon regulatory factor 3 (IRF3)-docking site on STING. How this reaction proceeds specifically at the TGN remains poorly understood. Here we report a cell-free reaction in which endogenous STING is phosphorylated by TBK1. The reaction utilizes microsomal membrane fraction prepared from TBK1-knockout (KO) cells and recombinant TBK1. We observed agonist-, TBK1-, “ER-to-Golgi” traffic-, and palmitoylation-dependent phosphorylation of STING at Ser365, mirroring the nature of STING phosphorylation in vivo. Treating the microsomal membrane fraction with sphingomyelinase or methyl-β-cyclodextrin, an agent to extract cholesterol from membranes, suppressed the phosphorylation of STING by TBK1. Given the enrichment of sphingomyelin and cholesterol in the TGN, these results may provide the molecular basis underlying the specific phosphorylation reaction of STING at the TGN.

Background: Covalent Bruton’s tyrosine kinase (BTK) inhibitors such as ibrutinib are approved for treating patients with B-cell malignancies, but their long-term efficacy is limited due to their toxicity (i.e., on-target and off-target inhibition of kinases) and acquired resistance (i.e., BTK C481 mutation). AS-1763 is a potent, highly selective, orally active, non-covalent inhibitor of both wild-type and C481S-mutant BTK. AS-1763 strongly inhibits the proliferation of B cell lymphoma cell line OCI-LY10 carrying wild-type and C481S-mutatant BTK in vitro and in vivo. AS-1763, therefore, is expected to be a promising BTK inhibitor for patients who have failed or are intolerant to a covalent BTK inhibitor. This study reports the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) in healthy volunteers who received single, ascending doses of AS-1763 in an alternating cohort design. Methods: This is a first-in-human, double-blind, placebo-controlled, randomized, single-center, single-ascending dose Phase I study of AS-1763. Two cohorts of 8 healthy male and female subjects were alternately dosed with AS-1763 (5, 25, 100, 300, 500, and 600 mg) or placebo orally under fasted condition. Intensive blood sampling for PK (up to 72 h) and PD (up to 24 h) was conducted before and post-dosing to allow a successful PK/PD correlation. For the PD assessment, whole blood was stimulated ex vivo with an anti-IgD antibody, and CD69 upregulation on B cells was assessed by flow cytometry. Results: Single doses of up to 600 mg AS-1763 were well-tolerated. No serious adverse events (AE) were reported during the trial. All treatment-emerged AEs reported were of mild intensity and showed no apparent dose-relationship in frequency. No clinically relevant changes from baseline were observed in all other safety parameters assessed (clinical laboratory, ECGs, vital signs, or physical examinations). AS-1763 was rapidly absorbed with a median time to maximum plasma concentrations (tmax) of 0.5-2.0 h post-dose and thereafter gradually decreased with mean elimination half-time (t1/2) of 8.5-12 h. Overall, mean plasma concentrations of AS-1763 increased with dose up to 500 mg. AS-1763 at 100 mg or higher induced >80% inhibition of B cell activation (%CD69 upregulation in naïve B cells normalized to baseline) at 1-2 h post-dose and the duration of inhibitory effect increased with dose. PK/PD analysis demonstrated that the IC50 and IC90 (90% CI) values for inhibition of CD69 upregulation in naïve B cells were 10.5 ng/mL (9.7-11.2 ng/mL) and 34.6 ng/mL (28.8-40.4 ng/mL), respectively. Conclusions: Single doses of AS-1763 up to 600 mg were well-tolerated and showed encouraging pharmacodynamics (inhibition of CD69 upregulation) with a favorable safety profile. The data support continued development of AS-1763 for patients with B-cell malignancies. Citation Format: Akinori Arimura, Kyoko Miyamoto, Maria Velinova, Marieke van den Dobbelsteen, Katsuhiro Mihara, Robert M. Miller, Masaaki Sawa. Safety, pharmacokinetics, and pharmacodynamics of AS-1763, a highly selective, orally bioavailable, non-covalent BTK inhibitor, in healthy volunteers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT137.

Introduction: Bruton's tyrosine kinase (BTK) has been recently recognized as a validated drug target for the treatment of B-cell malignances. The emergence of clinical resistance to the first-generation covalent BTK inhibitors is, however, becoming a serious concern. Patients are reported to develop resistance during the treatment due to substitution of cysteine residue at position 481 with serine (C481S mutation) in BTK, which prevents the covalent binding of the first-generation irreversible BTK inhibitors, resulting in the loss of inhibitory activities. Therefore, there is a high unmet medical need for new therapeutic approaches to overcome the BTK C481S-mediated resistance. Material and methods: Compounds were tested in biochemical assays using recombinant human wildtype (WT) and C481S mutant BTKs. Cellular activity of AS-1763 was evaluated in human diffuse large B cell lymphoma (DLBCL) cell lines, OCI-Ly10 and its BTK[C481S] knock-in cells. The in vivo antitumor activity of AS-1763 was assessed in two tumor xenograft mouse models bearing WT or BTK [C481S] knock-in OCI-Ly10 cells. Results: We have discovered AS-1763 as an orally available, highly potent, selective and reversible BTK inhibitor. AS-1763 showed potent inhibitory activities for WT BTK (an activated form, BTK[A]) and the C481S mutant with sub-nanomolar IC50s (IC50 = 0.85 and 0.99 nM for BTK[A] and BTK[C481S], respectively). In cellular assays, AS-1763 exhibited strong antiproliferative activities for WT and BTK [C481S] knock-in OCI-Ly10 cells. AS-1763 demonstrated significant tumor growth inhibition and tumor regression in WT as well as BTK[C481S] knock-in OCI-Ly10 xenograft model. Conclusions: The preclinical data support the clinical development of AS-1763 in patients with B-cell malignancies both having WT and C481S mutation in BTK. The first-in-human Phase 1 clinical trial of AS-1763 in healthy volunteers (EudraCT 2020-005599-37) is currently ongoing, and the Phase 1b study in patients is expected to begin in 2022. Citation Format: Wataru Kawahata, Tokiko Asami, Takao Kiyoi, Takayuki Irie, Shigeki Kashimoto, Hatsuo Furuichi, Masaaki Sawa. AS-1763: a highly potent, noncovalent and next generation BTK inhibitor for the treatment of patients with B-cell malignancies having C481S mutation in BTK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1400.

p38α mitogen activated protein kinase (MAPK) plays important roles in multiple cellular functions by phosphorylating a wide variety of substrates, and therefore, p38α MAPK has been considered as an important drug target. In this study, we designed peptide-based inhibitors for p38α MAPK, which can only inhibit the Smad3 phosphorylation specifically, by targeting the KIM binding site of p38α MAPK. Peptide 6 showed a significant inhibitory potency for the Smad3 phosphorylation by p38α MAPK. Peptide 6 showed no ATP dependency, and did not inhibit the phosphorylation of other substrates by p38α MAPK. The discovery of peptide 6 by targeting the KIM binding site likely provide an opportunity for the discovery of a novel class of allosteric and substrate-specific p38α MAPK inhibitors.

Kinase inhibitors form the largest class of precision medicine. From 2013 to 2017, 17 have been approved, with 8 different mechanisms. We present a comprehensive profiling study of all 17 inhibitors on a biochemical assay panel of 280 kinases and proliferation assays of 108 cancer cell lines. Drug responses of the cell lines were related to the presence of frequently recurring point mutations, insertions, deletions, and amplifications in 15 well-known oncogenes and tumor-suppressor genes. In addition, drug responses were correlated with basal gene expression levels with a focus on 383 clinically actionable genes. Cell lines harboring actionable mutations defined in the FDA labels, such as mutant BRAF(V600E) for cobimetinib, or ALK gene translocation for ALK inhibitors, are generally 10 times more sensitive compared with wild-type cell lines. This sensitivity window is more narrow for markers that failed to meet endpoints in clinical trials, for instance CDKN2A loss for CDK4/6 inhibitors (2.7-fold) and KRAS mutation for cobimetinib (2.3-fold). Our data underscore the rationale of a number of recently opened clinical trials, such as ibrutinib in ERBB2- or ERBB4-expressing cancers. We propose and validate new response biomarkers, such as mutation in FBXW7 or SMAD4 for EGFR and HER2 inhibitors, ETV4 and ETV5 expression for MEK inhibitors, and JAK3 expression for ALK inhibitors. Potentially, these new markers could be combined to improve response rates. This comprehensive overview of biochemical and cellular selectivities of approved kinase inhibitor drugs provides a rich resource for drug repurposing, basket trial design, and basic cancer research.

Bruton's tyrosine kinase (BTK) is a promising drug target for the treatment of multiple diseases, such as B-cell malignances, asthma, and rheumatoid arthritis. A series of novel aminotriazines were identified as highly selective inhibitors of BTK by a scaffold-hopping approach. Subsequent SAR studies of this series using two conformationally different BTK proteins, an activated form of BTK and an unactivated form of BTK, led to the discovery of a highly selective BTK inhibitor, 4b. With significant efficacy in models in vivo and good ADME and safety profiles, 4b was advanced into preclinical studies.

G protein-coupled receptors (GPCRs) are seven-transmembrane domain receptors that interact with the β-arrestin family, particularly β-arrestin 1 (ARRB1). GPCRs interact with 33% of small molecule drugs. Ligand screening is promising for drug discovery concerning GPCR-related diseases. Luciferase complementation assay (LCA) enables detection of protein-protein complementation via bioluminescence following complementation of N- and C-terminal luciferase fragments (NEluc and CEluc) fused to target proteins, but it is necessary to co-express the two genes. Here, we developed LCAs with mouse artificial chromosomes (MACs) that have unique characteristics such as stable maintenance and a substantial insert-carrying capacity. First, an NEluc-ARRB1 was inserted into MAC4 by Cre-loxP recombination in CHO cells, named ARRB1-MAC4. Second, a parathyroid hormone receptor 2 (PTHR2)-CEluc or prostaglandin EP4 receptor (hEP4)-CEluc were inserted into ARRB1-MAC4, named ARRB1-PTHR2-MAC4 and ARRB1-hEP4-MAC4, respectively, via the sequential integration of multiple vectors (SIM) system. Each MAC was transferred into HEK293 cells by microcell-mediated chromosome transfer (MMCT). LCAs using the established HEK293 cell lines resulted in 35,000 photon counts upon somatostatin stimulation for ARRB1-MAC4 with transient transfection of the somatostatin receptor 2 (SSTR2) expression vector, 1800 photon counts upon parathyroid hormone stimulation for ARRB1-PTHR2-MAC4, and 35,000 photon counts upon prostaglandin E2 stimulation for ARRB1-hEP4-MAC4. These MACs were maintained independently from host chromosomes in CHO and HEK293 cells. HEK293 cells containing ARRB1-PTHR2-MAC4 showed a stable reaction for long-term. Thus, the combination of gene loading by the SIM system into a MAC and an LCA targeting GPCRs provides a novel and useful platform to discover drugs for GPCR-related diseases.

Mitogen-activated protein kinase kinase 7 (MAP2K7) regulates stress and inflammatory responses, and is an attractive drug discovery target for several diseases including arthritis and cardiac hypertrophy. Intracellular proteins such as MAP2K7 are prone to aggregation due to cysteine-driven oxidation in in vitro experiments. MAP2K7 instability due to the four free cysteine residues on the molecular surface abrogated the crystal growth and led to a low-resolution structure with large residual errors. To acquire a higher resolution structure for promoting rational drug discovery, we explored stable mutants of MAP2K7 by replacing the surface cysteine residues, Cys147, Cys218, Cys276 and Cys296. Single-site mutations, except for Cys147, maintained the specific activity and increased the protein yield, while all the multi-site mutations massively reduced the activity. The C218S mutation drastically augmented the protein production and crystallographic resolution. Furthermore, the C218S crystals grown under microgravity in a space environment yielded a 1.3 Å resolution structure, providing novel insights for drug discovery: the precisely assigned water molecules in the active site, the double conformations in the flexible region and the C-terminal extension bound to the N-terminal region of the adjacent molecules. The latter insight is likely to promote the production of allosteric MAP2K7 inhibitors.

Cancer cell line panels are important tools to characterize the in vitro activity of new investigational drugs. Here, we present the inhibition profiles of 122 anticancer agents in proliferation assays with 44 or 66 genetically characterized cancer cell lines from diverse tumor tissues (Oncolines). The library includes 29 cytotoxics, 68 kinase inhibitors, and 11 epigenetic modulators. For 38 compounds this is the first comparative profiling in a cell line panel. By strictly maintaining optimized assay protocols, biological variation was kept to a minimum. Replicate profiles of 16 agents over three years show a high average Pearson correlation of 0.8 using IC50 values and 0.9 using GI50 values. Good correlations were observed with other panels. Curve fitting appears a large source of variation. Hierarchical clustering revealed 44 basic clusters, of which 26 contain compounds with common mechanisms of action, of which 9 were not reported before, including TTK, BET and two clusters of EZH2 inhibitors. To investigate unexpected clusterings, sets of BTK, Aurora and PI3K inhibitors were profiled in biochemical enzyme activity assays and surface plasmon resonance binding assays. The BTK inhibitor ibrutinib clusters with EGFR inhibitors, because it cross-reacts with EGFR. Aurora kinase inhibitors separate into two clusters, related to Aurora A or pan-Aurora selectivity. Similarly, 12 inhibitors in the PI3K/AKT/mTOR pathway separated into different clusters, reflecting biochemical selectivity (pan-PI3K, PI3Kβγδ-isoform selective or mTOR-selective). Of these, only allosteric mTOR inhibitors preferentially targeted PTEN-mutated cell lines. This shows that cell line profiling is an excellent tool for the unbiased classification of antiproliferative compounds. Mol Cancer Ther; 15(12); 3097–109. ©2016 AACR.

Background: The treatment of patients with metastatic synovial sarcoma is still challenging, and the development of new molecular therapeutics is desirable. Dysregulation of Wnt signaling has been implicated in synovial sarcoma. Traf2-and-Nck-interacting kinase (TNIK) is an essential transcriptional co-regulator of Wnt target genes. We examined the efficacy of a small interfering RNA (siRNA) to TNIK and a small-molecule TNIK inhibitor, NCB-0846, for synovial sarcoma. Methods: The expression of TNIK was determined in 20 clinical samples of synovial sarcoma. The efficacy of NCB-0846 was evaluated in four synovial sarcoma cell lines and a mouse xenograft model. Results: We found that synovial sarcoma cell lines with Wnt activation were highly dependent upon the expression of TNIK for proliferation and survival. NCB-0846 induced apoptotic cell death in synovial sarcoma cells through blocking of Wnt target genes including MYC, and oral administration of NCB-846 induced regression of xenografts established by inoculation of synovial sarcoma cells. Discussion: It has become evident that activation of Wnt signaling is causatively involved in the pathogenesis of synovial sarcoma, but no molecular therapeutics targeting the pathway have been approved. This study revealed for the first time the therapeutic potential of TNIK inhibition in synovial sarcoma.

The BRAF inhibitors dabrafenib and vemurafenib induce remarkable clinical responses in patients with BRAF-mutated melanomas. However, adverse events, including the emergence of secondary tumors and drug resistance, have been reported. Studies have revealed that undesirable RAF dimerization induced by inhibitors promotes these adverse effects. Here, we developed highly sensitive biosensors of RAF dimerization in cells utilizing the split enhanced click beetle luciferase (Emerald Luc, ELuc) complementation technique. We demonstrated that our biosensor system works effectively for high-throughput screens in the microplate format. A comprehensive analysis of commercially available RAF inhibitors performed using this assay system revealed that the inhibitors exhibit various potencies in inducing the dimerization of RAF isoforms, and their dimerization potencies do not always correlate with the RAF enzyme inhibition. This sensitive assay system will become a powerful tool to discover next-generation BRAF inhibitors with safer profiles.

The genetic and epigenetic alterations occurring during the course of multistage colorectal carcinogenesis have been extensively studied in the last few decades. One of the most notable findings is that the great majority of colorectal cancers (>80%) have mutations in the adenomatous polyposis coli (APC) tumor suppressor gene. Loss of functional APC protein results in activation of canonical Wnt/β-catanin signaling and initiates intestinal carcinogenesis. Mutational inactivation of APC is the first genetic event, but colorectal cancer cells retain their dependency on constitutive Wnt signal activation even after accumulation of other genetic events. Accordingly, pharmacological blocking of Wnt signaling has been considered an attractive therapeutic approach for colorectal cancer. Several therapeutics targeting various molecular components of the Wnt signaling pathway, including porcupine, frizzled receptors and co-receptor, tankyrases, and cAMP response element binding protein (CREB)-binding protein (CBP), have been developed, and some of those are currently being evaluated in early-phase clinical trials. Traf2- and Nck-interacting protein kinase (TNIK) has been identified as a regulatory component of the T-cell factor-4 and β-catenin transcriptional complex independently by two research groups. TNIK regulates Wnt signaling in the most downstream part of the pathway, and its inhibition is expected to block the signal even in colorectal cancer cells with APC gene mutation. Here we discuss some of the TNIK inhibitors under preclinical development.

The majority of kinase inhibitors have been developed as ATP competitors to interact with a hinge region in ATP binding sites of kinases. 7-Azaindole has been found as an excellent hinge binding motif by making two hydrogen bonds with the kinase hinge region. Vemurafenib, a B-RAF kinase (serine-threonine kinase [STK]) inhibitor approved by the U.S. Food and Drug Administration (FDA) for the treatment of melanoma, was created from this simple 7-azaindole fragment by successful use of structure-based drug design techniques. The huge potential of 7-azaindole as a hinge-binding motif has encouraged many researchers to employ it as a kinase privileged fragment. This paper will review recent examples of 7-azaindole-based kinase inhibitors, and discusses their binding interactions with the kinase hinge regions.

5Z-7-Oxozeaenol (5Z7O) is a covalent bonding inhibitor against the several protein kinases (e.g., ERK2 and TAK1) that possess a free cysteine at the gatekeeper-2 position. In addition to this cysteine, MAP2K7 has three other cysteine residues that are candidate for covalent bonding by the inhibitor 5Z7O. The crystal structure of the MAP2K7/5Z7O complex revealed that the inhibitor binds to MAP2K7 at a cysteine residue located at the end of the hinge region and not at the gatekeeper-2 residue. The structural insights into the interaction of 5Z7O with MAP2K7 should aid the development of 5Z7O derivatives with improved potency and selectivity.

The conserved serine-threonine kinase, Cdc7, plays a crucial role in initiation of DNA replication by facilitating the assembly of an initiation complex. Cdc7 is expressed at a high level and exhibits significant kinase activity not only during S-phase but also during G2/M-phases. A conserved mitotic kinase, Aurora B, is activated during M-phase by association with INCENP, forming the chromosome passenger complex with Borealin and Survivin. We show that Cdc7 phosphorylates and stimulates Aurora B kinase activity in vitro. We identified threonine-236 as a critical phosphorylation site on Aurora B that could be a target of Cdc7 or could be an autophosphorylation site stimulated by Cdc7-mediated phosphorylation elsewhere. We found that threonines at both 232 (that has been identified as an autophosphorylation site) and 236 are essential for the kinase activity of Aurora B. Cdc7 down regulation or inhibition reduced Aurora B activity in vivo and led to retarded M-phase progression. SAC imposed by paclitaxel was dramatically reversed by Cdc7 inhibition, similar to the effect of Aurora B inhibition under the similar situation. Our data show that Cdc7 contributes to M-phase progression and to spindle assembly checkpoint most likely through Aurora B activation.

Cancer cells often survive by getting addicted to specific genes. In most cases, such genes function as an enzyme, especially kinases that are specifically activated in cancers by genetic aberrations; e.g., BCR-ABL fusion kinase found in chronic myelogenous leukemias and epidermal growth factor receptor (EGFR) with activating mutations found in non-small cell lung cancers (NSCLCs). These activated kinases would be an ideal target for cancer therapy and indeed imatinib and gefitinib displayed good clinical benefits to the patients in these diseases. However, only a limited population of cancer cells harbor such well-characterized driver genes which could be attacked by targeted drugs developed so far and there remain unmet medical needs to identify novel therapeutic targets and drugs for the rest of cancers. To this end, we exploited our original database of drug efficacy of >100 kinase inhibitors across a panel of 39 human cancer cell lines (JFCR39) to characterize each of the cell line in terms of susceptibility to these kinase inhibitors. Expectedly, cell lines harboring PIK3CA hotspot mutation and PTEN mutation were susceptible to PI3K and mTOR inhibitors, whereas those harboring KRAS or BRAF mutation to MEK inhibitors, respectively. Of note, we found four of the cells responding to a series of EGFR family tyrosine kinase inhibitors (TKIs) including gefitinib and lapatinib. Gefitinib exert selective effect on NSCLCs addicted to activated EGFR mutant; however, none of the four gefitinib-sensitive cell lines possessed such EGFR mutation. Of these, an NSCLC cell line NCI-H522 hardly expressed EGFR but overexpressed ERBB4 instead, while an ovarian cancer cell line SK-OV3 overexpressed ERBB2 as reported previously. Actually, treatment with a certain TKI in this class efficiently dephosphorylated ERBB4 in NCI-H522 along with inhibition of cell growth and suppression of downstream factor ribosomal S6 protein. Moreover, knockdown of ERBB family kinases revealed that cell growth was dependent on overexpressed ERBB4 in NCI-H522 cells. These results indicate that NCI-H522 cells exhibited susceptibility to EGFR family TKIs via inhibition of ERBB4. Of note, NCI-H522 cells exhibited hypersensitivity to three of these TKIs, sapitinib, PF-7775 and GW583340, as compared to gefitinib. Especially, GW583340 suppressed growth of NCI-H522 cells at the same or lower concentration than in HCC827 cells harboring an EGFR active mutation. Consistent with this observation, in vitro kinase assay revealed that sapitinib and PF-7775 exhibited ~10-fold higher ERBB4-inhibitory effect than gefitinib did while GW583340 displayed selective inhibition on ERBB4, with ~10-fold higher activity than that on activated EGFR. In conclusion, we identified NCI-H522 as an ERBB4-dependent cell line, and found ERBB4 as a novel therapeutic cancer target that could be attacked by ERBB4-directing TKIs. Citation Format: Noritaka Tanaka, Kanami Yamazaki, Yuko Uno, Yoshimi Ohashi, Yumiko Nishimura, Masaaki Sawa, Shingo Dan. Identification of a lung adenocarcinoma cell line addicted to ERBB4, an actionable target for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 384.

Cdc7 is a serine-threonine kinase and plays a conserved and important role in DNA replication, and it has been recognized as a potential anticancer target. Herein, we report the design, synthesis and structure-activity relationship of novel furanone derivatives as Cdc7 kinase inhibitors. Compound 13 was identified as a strong inhibitor of Cdc7 with an IC50 value of 0.6 nM in the presence of 1 mM ATP and showed excellent kinase selectivity. In addition, it exhibited slow off-rate characteristics, which may offer advantages over known Cdc7 inhibitors in its potential to yield prolonged inhibitory effects in vivo. Compound 13 potently inhibited Cdc7 activity in cancer cells, and effectively induced cell death.

Target residence time (τ) has been suggested to be a better predictor of the biological activity of kinase inhibitors than inhibitory potency (IC50) in enzyme assays. Surface plasmon resonance binding assays for 46 human protein and lipid kinases were developed. The association and dissociation constants of 80 kinase inhibitor interactions were determined. τ and equilibrium affinity constants (KD) were calculated to determine kinetic selectivity. Comparison of τ and KD or IC50 values revealed a strikingly different view on the selectivity of several kinase inhibitors, including the multi-kinase inhibitor ponatinib, which was tested on 10 different kinases. In addition, known pan-Aurora inhibitors resided much longer on Aurora B than on Aurora A, despite having comparable affinity for Aurora A and B. Furthermore, the γ/δ-selective PI3K inhibitor duvelisib and the δ-selective drug idelalisib had similar 20-fold selectivity for δ- over γ-isoform but duvelisib resided much longer on both targets.

Mitogen-activated protein kinase kinase 7 (MAP2K7) is an indispensable kinase of the c-Jun N-terminal kinase signal cascade and is rigorously regulated via phosphorylation. To investigate the regulatory mechanism of the inactive non-phosphorylated state of MAP2K7, the crystal structures of the wild-type and C218S mutant were solved. The wild-type apo-structure revealed an unprecedented auto-inhibition form that occluded the ATP site. This closed form was configured by the n-σ* interaction of Cys218, a non-conserved residue among the MAP2K family kinases, with Gly145 in the glycine-rich loop. The interaction was unaltered in the presence of an ATP analog, whereas the C218S mutation precluded the closed configuration. These structural insights are potentially valuable for drug discovery of highly selective MAP2K7 inhibitors.

[Introduction] Osteosarcoma (OS) is a rare malignant bone tumor and affects predominantly adolescents and young adults. There is no molecular targeted therapy for OS and breakthrough intervention has long been awaited for decades. Disturbances in differentiation towards mature osteoblast from mesenchymal stem cell are related to OS pathogenesis. Wnt signaling is a key component in managing this differentiation process. We previously reported that TNIK was a positive regulator of Wnt signaling. To develop the first molecular targeted therapy for OS patients, we focused on Wnt pathway-targeted therapy in OS and initiated this study to demonstrate TNIK as a novel therapeutic target for OS treatment. [Methods] Eleven different osteosarcoma cell lines were used in the present study. Twenty clinical samples were obtained at biopsy and surgical resection from ten OS patients. TNIK expression was assessed by western blotting, immunofluorescence and immunohistochemical staining. The generation of the novel TNIK inhibitor was previously described (Masuda et al., 2016). Cells were cultured with TNIK inhibitor or siTNIK. Cell viability was evaluated by real-time cell analysis system or ATP quantitation assay. The effect of TNIK inhibitor in vivo was also assessed by a mouse xenograft model. RNA-seq was performed using the RNA extracted from vehicle or TNIK inhibitor-treated OS cells. Cancer stem cell (CSC)-like properties were assessed by soft-agar colony-formation assay, limiting dilution assay, ALDH activities and the expression of CSC markers. Adipogenic phenotypes were qualitatively and quantitatively analyzed by Oil red O staining and the expression of adipogenesis markers. [Results] TNIK was identified in all cell lines and most of cases. Immunohistochemistry of OS tissue microarray also revealed that 52 out of 55 OS samples were positive for TNIK protein. TNIK inhibitor suppressed OS cell proliferation both in vitro and in vivo. SiTNIK also inhibited OS cell growth. Global gene expression analysis revealed a significant decrease in the expression of the genes relating to Wnt signaling pathway and maintenance of stem cell pluripotency. Soft-agar colony-formation assay and limiting dilution assay revealed that TNIK inhibitor decreased sphere formation activities in OS cells. Accordingly, TNIK inhibitor significantly suppressed ALDH activities and the expression of the proteins that are involved in the maintenance of CSCs, including NANOG, SOX2, OCT4A and MYC. Concomitantly, TNIK inhibitor drove adipogenic transdifferentiation of OS cells with PPARG activation. TNIK knock-down U2OS cells created by shRNAs showed nearly identical results. [Conclusion] We present that TNIK inhibition promotes adipogenic transdifferentiation of OS cells and eliminates CSCs. Our study thus suggests the feasibility of TNIK as a target for OS treatment. Citation Format: Toru Hirozane, Mari Masuda, Naoko Goto, Teppei Sugano, Naofumi Asano, Eisuke Kobayashi, Keisuke Horiuchi, Hideo Morioka, Akira Kawai, Masaaki Sawa, Morio Matsumoto, Masaya Nakamura, Tesshi Yamada. TRAF2 and NCK-interacting protein kinase (TNIK) regulates cancer stemness and adipogenesis of osteosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1123.

Epithelial mesenchymal transition (EMT) has been implicated in cancer cell migration, invasion, and metastasis. Transforming growth factor-β (TGF-β) signal was known to be an essential EMT promotor. Development of therapeutics for targeting EMT through the TGF-β signal may be beneficial for the management of cancer metastasis. Recently, we found that a Traf2- and Nck-interacting kinase (TNIK) inhibitor, named NCB-0846, was capable of attenuating tumor-initiating cells among human colorectal cancer (1, 2). The cross link between EMT and cancer stemness has been revealed in several studies. Therefore we evaluated whether this small-molecule compound could have efficacy to inhibit TGF-β-induced EMT. NCB-0846 reduced the expression of mesenchymal markers (Vimentin and N-cadherin) and upregulated the expression of epithelial marker E-cadherin in A549 and H2228 cells. NCB-0846 suppressed the phosphorylation and nuclear translocation of Smad proteins and also inhibited migration, invasion, and metastasis. NCB-0846 inhibited TGF-β1-induced EMT through the down-regulation of TGFBR1 in mRNA levels. MiR-186-5p and miR-320 family were identified as candidate miRNAs that could target TGFBR1 and inhibited TGFBR1 protein expression.NCB-0846 might be a novel therapeutics drugs that targets the invasion and metastasis through inhibiting TGF-β-induced EMT in lung cancer. 1. Masuda M, Sawa M, Yamada T. Therapeutic targets in the Wnt signaling pathway: Feasibility of targeting TNIK in colorectal cancer. Pharmacology & therapeutics. 2015;156:1-9. Epub 2015/11/07.2. Masuda M, Uno Y, Ohbayashi N, Ohata H, Mimata A, Kukimoto-Niino M, et al. TNIK inhibition abrogates colorectal cancer stemness. Nature communications. 2016;7:12586. Epub 2016/08/27. Citation Format: Teppei Sugano, Mari Masuda, Yuko Uno, Naoko Goto, Masahiro Seike, Masaaki Sawa, Akihiko Gemma, Tesshi Yamada. Traf2-and Nck-interacting kinase (TNIK) inhibitor, NCB-0846, suppresses TGF-β1-induced epithelial-mesenchymal transition in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2034.

Most kinase inhibitors reported so far are designed to bind to a highly conserved ATP binding pocket in a competitive manner. In this case, inhibitors targeting the ATP pocket may crossreact with different kinases, as well as with other proteins that bind ATP, and this may cause undesirable side effects that would limit their clinical usefulness. In addition to the kinase selectivity issues, human ether-a-go-go-related gene (hERG) inhibition could be an obstacle to develop a kinase inhibitor as a safe drug. This paper will review the methods employed in the development of selective kinase inhibitors with several successful examples. These include medicinal chemistry efforts to conquer hERG inhibition problems as sometimes seen in kinase inhibitor programs.

Canonical Wnt/β-catenin signalling is essential for maintaining intestinal stem cells, and its constitutive activation has been implicated in colorectal carcinogenesis. We and others have previously identified Traf2- and Nck-interacting kinase (TNIK) as an essential regulatory component of the T-cell factor-4 and β-catenin transcriptional complex. Consistent with this, Tnik-deficient mice are resistant to azoxymethane-induced colon tumorigenesis, and Tnik−/−/Apcmin/+ mutant mice develop significantly fewer intestinal tumours. Here we report the first orally available small-molecule TNIK inhibitor, NCB-0846, having anti-Wnt activity. X-ray co-crystal structure analysis reveals that NCB-0846 binds to TNIK in an inactive conformation, and this binding mode seems to be essential for Wnt inhibition. NCB-0846 suppresses Wnt-driven intestinal tumorigenesis in Apcmin/+ mice and the sphere- and tumour-forming activities of colorectal cancer cells. TNIK is required for the tumour-initiating function of colorectal cancer stem cells. Its inhibition is a promising therapeutic approach., TRAF2 and NCK-interacting protein kinase (TNIK) is a key regulatory component of the TCF4 and β-catenin transcriptional complex. In this study, the authors identify a TNIK inhibitor that blocks Wnt signalling and Wnt-driven colorectal tumorigenesis in mice.

The treatment of patients with advanced colorectal cancer still remains challenging, and identification of new target molecules and therapeutic avenues remains a priority. The great majority of colorectal cancers have mutations in one of two genes involved in the Wnt signaling pathway: the adenomatous polyposis coli (APC) and β-catenin (CTNNB1) genes. Up to now, however, no therapeutics for targeting this pathway have been established.This review article begins with a brief summary of Wnt signaling from the viewpoints of genetics, cancer stem cell biology, and drug development. We then overview current attempts to develop drugs directed at various components of the Wnt signaling pathway.APC is a tumor suppressor, and therefore only downstream signal transducers of the APC protein can be considered as targets for pharmaceutical intervention. TRAF2 and NCK-interacting protein kinase (TNIK) was identified as the most downstream regulator of Wnt signaling by two independent research groups, and several classes of small-molecule inhibitors targeting this protein kinase have been developed. TNIK is a multifunctional protein with actions that extend beyond Wnt signaling regulation. Such TNIK inhibitors are expected to have a large variety of clinical applications.

Glycomics is emerging as a new field for the biology of complex glycoproteins and glycoconjugates. The lack of versatile glycan-labeling methods has presented a major obstacle to visualizing at the cellular level and studying glycoconjugates. To address this issue, we developed a fluorescent labeling technique based on the Cu(I)-catalyzed [3 + 2] cycloaddition, or click chemistry, which allows rapid, versatile, and specific covalent labeling of cellular glycans bearing azide groups. The method entails generating a fluorescent probe from a nonfluorescent precursor, 4-ethynyl- N -ethyl-1,8-naphthalimide, by clicking the fluorescent trigger, the alkyne at the 4 position, with an azido-modified sugar. Using this click-activated fluorescent probe, we demonstrate incorporation of an azido-containing fucose analog into glycoproteins via the fucose salvage pathway. Distinct fluorescent signals were observed by flow cytometry when cells treated with 6-azidofucose were labeled with the click-activated fluorogenic probe or biotinylated alkyne. The intracellular localization of fucosylated glycoconjugates was visualized by using fluorescence microscopy. This technique will allow dynamic imaging of cellular fucosylation and facilitate studies of fucosylated glycoproteins and glycolipids.

The beta3-adrenergic receptor (beta3-AR) has been shown to mediate various pharmacological and physiological effects such as lipolysis, thermogenesis, and relaxation of the urinary bladder. Activation of the beta3-AR is thought to be a possible approach for the treatment of obesity, type 2 diabetes mellitus, and frequent urination. Therefore, the beta3-AR is recognized as an attractive target for drug discovery. On the other hand, activation of the beta1- or beta2-AR can cause undesirable side effects such as increased heart rate or muscle tremors. Consequently, a number of recent efforts in this field have been directed toward the design of selective agonists for the beta3-AR. This review summarizes recent advances in beta3-AR agonists with an emphasis on recent attempts to create potent, selective and orally bioavailable small-molecule agonists.

The synthesis and evaluation of a novel series of 1,7-cyclized indole-based human adrenergic receptor (β 3 -AR) agonists are reported. The synthesis of a variety of 1,7-cyclized indole part was accomplished by the Mitsunobu reaction or a ring closing metathesis (RCM) reaction. SAR studies revealed that expansion of the ring size resulted in considerable selectivity against the β 1 - and β 2 -ARs. Compound 26 , an eight-membered ring analogue with a double bond on its 1,7-linker portion, was found to be a potent β 3 -AR agonist (EC 50 = 0.75 nM, IA = 90%) with extremely high selectivity for the β 3 -AR over the β 1 - and β 2 -ARs.