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J. Biol. Chem., Vol. 281, Issue 22, 15277-15286, June 2, 2006

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Structural and Functional Characterization of HQL-79, an Orally Selective Inhibitor of Human Hematopoietic Prostaglandin D Synthase^*

Kosuke Aritake¹, Yuji Kado¹, Tsuyoshi Inoue, Masashi Miyano^¶, and Yoshihiro Urade²

From the Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4, Furuedai, Suita, Osaka 565-0874, Japan, the Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan, and the ^¶Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1, Kouto, Sayo, Hyogo 679-5148, Japan

We determined the crystal structure of human hematopoietic prostaglandin (PG) D synthase (H-PGDS) as the quaternary complex with glutathione (GSH), Mg²⁺, and an inhibitor, HQL-79, having anti-inflammatory activities in vivo, at a 1.45-Å resolution. In the quaternary complex, HQL-79 was found to reside within the catalytic cleft between Trp¹⁰⁴ and GSH. HQL-79 was stabilized by interaction of a phenyl ring of its diphenyl group with Trp¹⁰⁴ and by its piperidine group with GSH and Arg¹⁴ through water molecules, which form a network with hydrogen bonding and salt bridges linked to Mg²⁺. HQL-79 inhibited human H-PGDS competitively against the substrate PGH₂ and non-competitively against GSH with K_i of 5 and 3 µM, respectively. Surface plasmon resonance analysis revealed that HQL-79 bound to H-PGDS with an affinity that was 12-fold higher in the presence of GSH and Mg²⁺ (K_d, 0.8 µM) than in their absence. Mutational studies revealed that Arg¹⁴ was important for the Mg²⁺-mediated increase in the binding affinity of H-PGDS for HQL-79, and that Trp¹⁰⁴, Lys¹¹², and Lys¹⁹⁸ were important for maintaining the HQL-binding pocket. HQL-79 selectively inhibited PGD₂ production by H-PGDS-expressing human megakaryocytes and rat mastocytoma cells with an IC₅₀ value of about 100 µM but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between H-PGDS and cyclooxygenase. Orally administered HQL-79 (30 mg/kg body weight) inhibited antigen-induced production of PGD₂, without affecting the production of PGE₂ and PGF₂, and ameliorated airway inflammation in wild-type and human H-PGDS-overexpressing mice. Knowledge about this structure of quaternary complex is useful for understanding the inhibitory mechanism of HQL-79 and should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit PGD₂ production specifically.

Received for publication, June 13, 2005 , and in revised form, March 15, 2006.

^* This work was supported by the Applied Research Pilot Project for the Industrial Use of Space promoted by JAXA and Japan Space Utilization Promotion Center (JSUP), a grant from Japan Foundation for Applied Enzymology (to Y. U.), the 21st Century Center of Excellence (21COE) Program "Creation of Integrated EcoChemistry" of Osaka University (to Y. K.), a Grant-in-aid for Scientific Research of MEXT (17659022) (to K. A.), PRESTO (to T. I.), Japan Science and Technology Agency, and the National Project on Protein Structural and Functional Analyses (to T. I.), and Osaka City. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 2CVD) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4, Furuedai, Suita, Osaka 565-0874, Japan. Tel.: 81-6-6872-4851; Fax: 81-6-6872-2841; E-mail: uradey{at}obi.or.jp.

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