Hitoshi Sakashita

Structural basis for telmisartan-mediated partial activation of PPAR gamma

Telmisartan, a selective angiotensin II type 1 receptor blocker, has recently been shown to act as a partial agonist for peroxisome proliferator-activated receptor gamma (PPARγ). To understand how telmisartan partially activates PPARγ, we determined the ternary complex structure of PPARγ, telmisartan, and a coactivator peptide from steroid receptor coactivator-1 at a resolution of 2.18 Å. Crystallographic analysis revealed that telmisartan exhibits an unexpected binding mode in which the central benzimidazole ring is engaged in a non-canonical—and suboptimal—hydrogen-bonding network around helix 12 (H12). This network differs greatly from that observed when full-agonists bind with PPARγ and prompt high-coactivator recruitment through H12 stabilized by multiple hydrogen bonds. Binding with telmisartan results in a less stable H12 that in turn leads to attenuated coactivator binding, thus explaining the mechanism of partial activation.

4-Hydroxypyridazin-3(2H)-one Derivatives as Novel d-Amino Acid Oxidase Inhibitors

D-Amino acid oxidase (DAAO) catalyzes the oxidation of d-amino acids including d-serine, a coagonist of the N-methyl-d-aspartate receptor. We identified a series of 4-hydroxypyridazin-3(2H)-one derivatives as novel DAAO inhibitors with high potency and substantial cell permeability using fragment-based drug design. Comparisons of complex structures deposited in the Protein Data Bank as well as those determined with in-house fragment hits revealed that a hydrophobic subpocket was formed perpendicular to the flavin ring by flipping Tyr224 in a ligand-dependent manner. We investigated the ability of the initial fragment hit, 3-hydroxy-pyridine-2(1H)-one, to fill this subpocket with the aid of complex structure information. 3-Hydroxy-5-(2-phenylethyl)pyridine-2(1H)-one exhibited the predicted binding mode and demonstrated high inhibitory activity for human DAAO in enzyme- and cell-based assays. We further designed and synthesized 4-hydroxypyridazin-3(2H)-one derivatives, which are equivalent to the 3-hydroxy-pyridine-2(1H)-one series but lack cell toxicity. 6-[2-(3,5-Difluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-one was found to be effective against MK-801-induced cognitive deficit in the Y-maze.

Identification of a key element for hydrogen‐bonding patterns between protein kinases and their inhibitors

In this article, we report crystal structures for inhibitor‐kinase complexes in which the inhibitor has different binding orientations and hydrogen‐bonding patterns with extracellular‐signal regulated kinase 2 and insulin receptor tyrosine kinase. Our crystallographic studies, and sequence and structural analyses of 532 coordinates of kinases held in the Protein Data Bank, suggest that the length of the “specificity linker” described here is a key structural element of the hydrogen‐bonding patterns between protein kinases and their inhibitors. Proteins 2008.

Structures of complexes of type 5 17β‐hydroxysteroid dehydrogenase with structurally diverse inhibitors: insights into the conformational changes upon inhibitor binding

Type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5) is an aldo-keto reductase expressed in the human prostate which catalyzes the conversion of androstenedione to testosterone. Testosterone is converted to 5α-dihydrotestosterone, which is present at high concentrations in patients with castration-resistant prostate cancer (CRPC). Inhibition of 17β-HSD5 is therefore considered to be a promising therapy for treating CRPC. In the present study, crystal structures of complexes of 17β-HSD5 with structurally diverse inhibitors derived from high-throughput screening were determined. In the structures of the complexes, various functional groups, including amide, nitro, pyrazole and hydroxyl groups, form hydrogen bonds to the catalytic residues His117 and Tyr55. In addition, major conformational changes of 17β-HSD5 were observed following the binding of the structurally diverse inhibitors. These results demonstrate interactions between 17β-HSD5 and inhibitors at the atomic level and enable structure-based drug design for anti-CRPC therapy.

AR‐NE3A, a New Macromolecular Crystallography Beamline for Pharmaceutical Applications at the Photon Factory

Recent advances in high‐throughput techniques for macromolecular crystallography have highlighted the importance of structure‐based drug design (SBDD), and the demand for synchrotron use by pharmaceutical researchers has increased. Thus, in collaboration with Astellas Pharma Inc., we have constructed a new high‐throughput macromolecular crystallography beamline, AR‐NE3A, which is dedicated to SBDD. At AR‐NE3A, a photon flux up to three times higher than those at existing high‐throughput beams at the Photon Factory, AR‐NW12A and BL‐5A, can be realized at the same sample positions. Installed in the experimental hutch are a high‐precision diffractometer, fast‐readout, high‐gain CCD detector, and sample exchange robot capable of handling more than two hundred cryo‐cooled samples stored in a Dewar. To facilitate high‐throughput data collection required for pharmaceutical research, fully automated data collection and processing systems have been developed. Thus, sample exchange, centering, data collection, and ...

An NMR Biochemical Assay for Fragment-Based Drug Discovery: Evaluation of an Inhibitor Activity on Spermidine Synthase of Trypanosoma cruzi

Although NMR in fragment-based drug discovery is utilized almost exclusively to evaluate physical binding between molecules, it should be also a powerful tool for biochemical assay, evaluating inhibitory effect of compounds on enzymatic activity. Time-dependent spectral change in real-time monitoring or inhibitor concentration-dependent spectral change after constant-time reaction was processed by factor analysis, by which reaction rate or IC50 value was obtained. Applications to spermidine synthase of Trypanosoma cruzi, which causes Chagas disease, are described.