Toyomichi Nanayama

Cloning and Crystal Structure of Hematopoietic Prostaglandin D Synthase

Hematopoietic prostaglandin (PG) D synthase is the key enzyme for production of the D and J series of prostanoids in the immune system and mast cells. We isolated a cDNA for the rat enzyme, crystallized the recombinant enzyme, and determined the three-dimensional structure of the enzyme complexed with glutathione at 2.3 A resolution. The enzyme is the first member of the sigma class glutathione S-transferase (GST) from vertebrates and possesses a prominent cleft as the active site, which is never seen among other members of the GST family. The unique 3-D architecture of the cleft leads to the putative substrate binding mode and its catalytic mechanism, responsible for the specific isomerization from PGH2 to PGD2.

Discovery of a Highly Potent and Selective MEK Inhibitor: GSK1120212 (JTP-74057 DMSO Solvate).

Inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) represents a promising strategy for the discovery of a new generation of anticancer chemotherapeutics. Our synthetic efforts, beginning from the lead compound 2, were directed at improving antiproliferative activity against cancer cells as well as various drug properties. These efforts led to the discovery of N-{3-[3-cyclopropyl-5-(2-fluoro-4-iodophenylamino)-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamide dimethylsulfoxide solvate (GSK1120212, JTP-74057 DMSO solvate; 1), a selective and highly potent MEK inhibitor with improved drug properties. We further confirmed that the antiproliferative activity correlates with cellular MEK inhibition and observed significant antitumor activity with daily oral dosing of 1 in a tumor xenograft model. These qualities led to the selection of 1 for clinical development.

Identification of JTP‐70902, a p15INK4b‐inductive compound, as a novel MEK1/2 inhibitor

The INK4 family members p16INK4a and p15INK4b negatively regulate cell cycle progression by inhibition of cyclin‐dependent kinase (CDK) 4/6. Loss of p16INK4a functional activity is frequently observed in tumor cells, and is thought to be one of the primary causes of carcinogenesis. In contrast, despite the biochemical similarity to p16INK4a, the frequency of defects in p15INK4b was found to be lower than in p16INK4a, suggesting that p15INK4b‐inductive agents may be useful for tumor suppression. Here we report the discovery of a novel pyrido‐pyrimidine derivative, JTP‐70902, which exhibits p15INK4b‐inducing activity in p16INK4a‐inactivated human colon cancer HT‐29 cells. JTP‐70902 also induced another CDK‐inhibitor, p27KIP1, and downregulated the expression of c‐Myc and cyclin D1, resulting in G1 cell cycle arrest. MEK1/2 was identified by compound‐immobilized affinity chromatography as the molecular target of JTP‐70902, and this was further confirmed by the inhibitory activity of JTP‐70902 against MEK1/2 in kinase assays. JTP‐70902 suppressed the growth of most colorectal and some other cancer cell lines in vitro, and showed antitumor activity in an HT‐29 xenograft model. However, JTP‐70902 did not inhibit the growth of COLO320 DM cells; in these, constitutive extracellular signal‐regulated kinase phosphorylation was not detected, and neither p15INK4b nor p27KIP1 induction was observed. Moreover, p15INK4b‐deficient mouse embryonic fibroblasts were found to be more resistant to the growth‐inhibitory effect of JTP‐70902 than wild‐type mouse embryonic fibroblasts. These findings suggest that JTP‐70902 restores CDK inhibitor‐mediated cell cycle control by inhibiting MEK1/2 and exerts a potent antitumor effect. (Cancer Sci 2007; 98: 1809–1916)

Pharmacokinetics and disposition of dalcetrapib in rats and monkeys

Abstract 1. The pharmacokinetics and metabolism of dalcetrapib (JTT-705/RO4607381), a novel cholesteryl ester transfer protein inhibitor, were investigated in rats and monkeys. 2. In in vitro stability studies, dalcetrapib was extremely unstable in plasma, liver S9 and small intestinal mucosa, and the pharmacologically active form (dalcetrapib thiol) was detected as major component. Most of the active form in plasma was covalently bound to plasma proteins via mixed disulfide bond formation. 3. Following oral administration of 14C-dalcetrapib to rats and monkeys, active form was detected in plasma. The active form was mainly metabolized to the glucuronide conjugate and the methyl conjugate at the thiol group. Several minor metabolites including mono- and di-oxidized forms of the glucuronide are also detected in the plasma and urine. 4. The administered radioactivity was widely distributed to all tissues and mainly excreted into the feces (85.7 and 62.7% of the dose in rats and monkeys, respectively). Most of the radioactivity was recovered by 168 h. Although the absorbed dalcetrapib was hydrolyzed to the active form and was bound to endogenous thiol via formation of disulfide bond, it was relatively rapidly eliminated from the body and was not retained.

Structure and tumor necrosis factor-inducing activity of dispersed particles of a lipid A analogue, GLA-60, and phosphatidylcholine mixture

Abstract A synthetic monosaccharide-analogue of lipid A, GLA-60, was co-dispersed with egg PC in aqueous solution. The size distribution and structure of the dispersed particles were investigated by the dynamic light scattering (DLS) and transmission electron microscopic (TEM) methods. The particles were of liposomal structure with homogeneous size. The leakage of CF out of the liposomes in rat plasma was suppressed by augmenting GLA-60. Fluorescence anisotropy measurements showed that GLA-60 in lipid bilayers led to a rigid structure of the membranes. The activity of tumor necrosis factor (TNF) induction in mice increased with the content of GLA-60 in sonicated liposomes. The results showed that these liposomes were useful as GLA-60 formulations.