Abstract

We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3–250 μm)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a competitive manner against the substrate PGH2 (Km = 14 μm) with a Ki value of 75 μm but did not inhibit the binding of 13-cis-retinoic acid, a nonsubstrate lipophilic ligand, to L-PGDS. NMR titration analysis revealed that AT-56 occupied the catalytic pocket, but not the retinoid-binding pocket, of L-PGDS. AT-56 inhibited the production of PGD2 by L-PGDS-expressing human TE-671 cells after stimulation with Ca2+ ionophore (5 μm A23187) with an IC50 value of about 3 μm without affecting their production of PGE2 and PGF2α but had no effect on the PGD2 production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (<30 mg/kg body weight) decreased the PGD2 production to 40% in the brain of H-PGDS-deficient mice after a stab wound injury in a dose-dependent manner without affecting the production of PGE2 and PGF2α and also suppressed the accumulation of eosinophils and monocytes in the bronco-alveolar lavage fluid from the antigen-induced lung inflammation model of human L-PGDS-transgenic mice.

Highlights

  • PGD24 is a lipid mediator involved in sleep [1, 2] and inflammatory responses [3]

  • PGD2 is formed by the following sequence of enzyme reactions after cell activation: 1) cytosolic phospholipase A2 is translocated to the endoplasmic reticulum and perinuclear membranes in a Ca2ϩ-dependent manner, where it cleaves arachidonic acid from the membrane phospholipids; 2) arachidonic acid is converted to PGH2, a common precursor of various prostanoids, by the membrane-bound cyclooxygenases (COXs); and 3) PGH2 is further isomerized to PGD2 by PGD synthase (PGDS)

  • In addition we found that AT-56 inhibited the production of PGD2 by lipocalin-type prostaglandin (PG) D synthase (L-PGDS)-expressing cultured cells, hematopoietic PGD synthase (H-PGDS) gene knock-out (KO) mice, and human L-PGDS overexpressing transgenic (TG) mice

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Summary

EXPERIMENTAL PROCEDURES

Chemicals—AT-56 was a generous gift from TAIHO Pharmaceutical Company (Saitama, Japan). The culture media were harvested and centrifuged at 10,000 ϫ g for 5 min to remove the cells, and the supernatant was removed and stored at Ϫ80 °C until the measurements of PGs could be made In some experiments, these cells were prelabeled with [1-14C]arachidonic acid (3.7 kBq/ well) for 12 h before the assay. Measurement of PG Contents—The amounts of PGs in the cell culture media and brain tissues were determined as described previously [22]. The cell culture media and the frozen brain tissues were homogenized in ethanol containing 0.02% HCl at pH 2.0 and [3H]PGD2, [3H]PGE2, and [3H]PGF2␣ (PerkinElmer Life Sciences) as tracers to estimate the recovery during the purification procedure. Statistics—Comparisons were analyzed for statistical significance by Dunnett’s multicomparison test using SigmaPlot (Systat Software, CA). p Ͻ 0.05 was considered significant

RESULTS
Amino acid residues
DISCUSSION
Per os
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