Abstract
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-A 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 Ki of 5 and 3 microm, 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+ (Kd, 0.8 microm) 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 microm 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 PGF2alpha, 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.
Highlights
PGD2 is formed from arachidonic acid by successive enzyme reactions mediated by PG endoperoxide synthase and PGD synthase (PGDS)
Human hematopoietic prostaglandin (PG) D synthase (H-PGDS) Inhibitor HQL-79 was originally developed as an antagonist for histamine H1 receptors (H1R), a part of the anti-allergic and anti-asthmatic effects of HQL-79 was proposed to be mediated by the inhibition of PGD2 production, because HQL-79 inhibited the conversion of PGH2 to PGD2 in crude extracts of mouse spleen [18]
Inhibition of Allergic Lung Inflammation and PGD2 Production by HQL-79 in Vivo—We applied HQL-79 (Fig. 1) to the allergic-airway inflammation model of human H-PGDS overexpressing TG mice and WT mice (FVB strain) sensitized to OVA, and determined the contents of PGD2, PGE2, and PGF2␣ in the BALF obtained from these mice (Fig. 2A)
Summary
PGD2 is formed from arachidonic acid by successive enzyme reactions mediated by PG endoperoxide synthase (cyclooxygenase, COX) and PGD synthase (PGDS). In the absence of Mg2ϩ, HQL-79 showed the same kinetic profile of the H-PGDS inhibition as that in the presence of Mg2ϩ; the Ki value was increased to 55 M for PGH2 and to 40 M for GSH (data not shown).
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