Designing of inhibitors for prostaglandin D synthases based on crystallographic analyses

前列腺素D_2（Prostaglandin，PG）D_2是中枢神经系统产生的活性物质，具有诱导睡眠、调节疼痛等多种中枢作用。POD 2也由肥大细胞和Th 2细胞产生，并作为炎症和过敏介质释放。POD合酶（PGDS）有两种不同的类型：一种是位于中枢神经系统的脂质运载蛋白型POD合酶（L-PGDS），另一种是在过敏相关细胞中表达的造血POD合酶（H-PODS）。这两种酶催化的是从POH_2到POD_2的相同反应，但它们的氨基酸序列没有任何同源性。重组小鼠L-PGDS和人H-PGDS与几种对每种PODS具有选择性的抑制剂共结晶。我们在KEK和Spring-8同步加速器上获得了这些晶体的X射线衍射数据。测定了L-PODS与其抑制剂AT-056复合物的三维坐标，分辨率为2.5 μ m。我们还测定了H-PODS与 ...更多信息 h Mg^2+或Ca^2+和4种不同的抑制剂，包括口服有效的抑制剂HQL-79，分辨率为1.2至1.8 μ m。L-PGDS为β-barrell结构，具有疏水口袋; H-PGDS为α+β结构，在2个亚结构域中有一个谷胱甘肽结合位点。基于晶体学结构，我们引入定点突变来鉴定L-PODS和H-PODS催化反应所涉及的氨基酸残基。然后，我们调查了在β-actin启动子控制下过表达人L-PODS或H-PODS的转基因（TG）小鼠的功能异常。我们发现L-PGDS-TG小鼠在剪尾刺激后非快速眼动睡眠时间显著增加。TO小鼠的睡眠诱导与脑中POD 2的产生呈正相关。在卵白蛋白诱导的哮喘模型中，L-PODS-或H-PGDS-TG小鼠表现出明显的嗜酸性粒细胞和淋巴细胞浸润到肺中，支气管肺泡灌洗液中Th 2细胞因子上调。这些TO小鼠可用于监测先导化合物的体内功效，以开发用作抗瞌睡药物或抗过敏药物的PGDS抑制剂。少

Prostaglandin (PG) D_2 is actively produced in the central nervous system and exhibits a variety of central actions such as sleep induction and pain modulation. POD2 is also produced by mast cells and Th2 cells and is released as an inflammatory and allergic mediator. There are two distinct types of POD synthase (PGDS) : one is lipocalin-type PODS (L-PGDS) localized in the central nervous system and the other is hematopoietic POD synthase (H-PODS) expressed in those allergy-related cells. These two enzymes catalyze the same reaction from POH_2 to POD2 but possess no homology at all in their amno acid sequences. Recombinant mouse L-PGDS and human H-PGDS were cocrystallized with several inhibitors selective to each PODS. We obtained the X-ray diffraction data of these crystals at KEK and Spring-8 synchrotron facilities. The 3-D coordinates of L-PODS complexed with its inhibitor AT-056 were determined at a resolution of 2.5 Å. We also determined the 3-D coordinates of H-PODS complexed wit … More h Mg^<2+> or Ca^<2+> and 4 different inhibitors, including an orally effective inhibitor HQL-79, at 1.2 to 1.8 Å resolution. As predicted, the tertiary structures of two enzymes are quite different from each other L-PGDS possesses β-barrell structure with hydrophobic pocket and H-PGDS, α+β structure with a catalytic creft containing the glutathione-binding site within 2 subdomains. Based on the crystallographic structures, we introduced the site-directed mutagenesis to identify the amino acid residues involved in the catalytic reactions by L-PODS and H-PODS. We then surveyed the functional abnormality of trarisgenic (TG) mice that over-expressed human L-PODS or H-PODS under the control of the β-actin promoter. We found that L-PGDS-TG mice showed a striking time-dependent increase in non-rapid eye movement sleep afler stimulation by tail clipping. The sleep induction in TO mice was positively correlated with the POD2 production in the brain. In an ovalbumin-induced asthma model, L-PODS- or H-PGDS-TG mice showed a pronounced infiltration of eosinophils and lymphocytes into the lung and upregulation of Th2 cytokines in the bronchoalveolar lavage fluid. These TO mice are useful to monitor the in vivo efficacy of the lead compounds to develop the PGDS inhibitors act as anti-doze drugs or anti-allergy drugs. Less