Masaaki Sawa

Recent developments in the design of orally bioavailable β3-adrenergic receptor agonists

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.

Therapeutic targets in the Wnt signaling pathway: Feasibility of targeting TNIK in colorectal cancer

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.

Targeting the Wnt signaling pathway in colorectal cancer

Introduction: 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. Areas covered: 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. Expert opinion: 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.

Compound Selectivity and Target Residence Time of Kinase Inhibitors Studied with Surface Plasmon Resonance

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.

Toward the antibody-catalyzed chemiluminescence. Design and synthesis of hapten

Hapten 4 was synthesized to generate catalytic antibodies triggering chemiluminescence by catalyzing the decomposition of the 1,2-dioxetane 3. The hapten 4 was so designed as to elicit a negatively charged functional group in the antibody combining site to catalyze the beta-elimination of the protecting group in 3 as well as to lock the protecting group into an energetically favorable anti-periplanar conformation.

5Z-7-Oxozeaenol covalently binds to MAP2K7 at Cys218 in an unprecedented manner.

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.

Design and combinatorial synthesis of a novel kinase-focused library using click chemistry-based fragment assembly.

Fragment-based lead discovery is a new approach for lead generation that has emerged in the past decade. Because the initial fragments identified in the fragment screening typically show weak binding affinity, an intensive medicinal chemistry effort would be required to grow initial fragments into a potential lead compound. Here we demonstrate a kinase focused evolved fragment (KFEF) library, constructed by click chemistry-based fragment assembly, that is a valuable source of kinase inhibitors. This combinatorial assembly of two fragments, kinase-privileged alkyne fragments and diversified azide fragments, by two cycloaddition reactions shows a unique potential for the one-step synthesis of structurally diverse evolved fragments. The screening of this triazole-based KFEF library allowed the rapid identification of potent lead candidates for FLT3 and GSK3β kinase.

Cell panel profiling reveals conserved therapeutic clusters and differentiates the mechanism of action of different PI3K/mTOR, Aurora kinase and EZH2 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.

A crucial role of Cys218 in configuring an unprecedented auto-inhibition form of MAP2K7

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.

Abstract B265: Targeting Wnt signaling: Discovery and characterization of novel thiazole-based Traf2- and NCK-interacting kinase (TNIK) inhibitors.

Introduction: Aberrant activation of Wnt pathway is a major force driving colorectal carcinogenesis, and therefore the Wnt pathway has been thought of as an attractive anticancer drug target. However, its complexity has made it difficult to find druggable targets in the Wnt pathway. Recently, we reported Traf2- and NCK-interacting kinase (TNIK) plays important roles in the Wnt signaling pathway by making a complex with beta-catenin and T-cell factor-4 (TCF4) in a nucleus. We also found that TNIK is activated in human colorectal cancer cell lines, and proliferations of those cells are highly dependent upon the expression and catalytic activity of TNIK. Therefore inhibitors of TNIK may represent a promising therapeutic approach for treating colorectal cancer. Here we describe a discovery and characterization of a novel TNIK inhibitor. Methods: To identify compounds that inhibit TNIK acitivity, we have produced a recombinant catalytic domain of human TNIK, and developed a high throughput screening (HTS) assay system using a peptide substrate. TCF/LEF reporter gene assay using HEK293 was established to validate hit compounds. To study the effects of TNIK inhbitors on the Wnt signal pathway, Wnt target genes were analyzed by RT-PCR and Western blotting. The selected compounds were subjected to a kinase selectivity panel assay. Results: By performing a high throughput screening campaigns of our 10K kinase-focused compound library against a recombinant human TNIK, we identified a series of novel thiazole compounds as a potent inhibitor of TNIK. Hit compound, NCB-0001 exhibited potent inhibitory activity for TNIK with an IC50 value of 8.6 nM. After subsequent optimization, N1662 was identified as a lead compound having a strong anti-proliferative activity in colorectal cancer cell lines. N1662 inhibited tumor growth in a mouse xenograft model of colorectal cancer. Further optimization led to compound N5355 that showed good TNIK enzyme inhibition and potent Wnt signal inhibition in the TCF reporter gene assay, as well as down-regulation of Wnt target genes. N5355 showed excellent kinase selectivity against a panel of 311 kinases. Detailed results will be presented in the conference. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B265. Citation Format: Yuko Uno, Hideki Moriyama, Shigeki Kashimoto, Yusuke Kawase, Miki Shitashige, Masaaki Sawa, Tesshi Yamada. Targeting Wnt signaling: Discovery and characterization of novel thiazole-based Traf2- and NCK-interacting kinase (TNIK) inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B265.