A study for drug-discovery based on physiological actions of prostanoid receptors

我们建立了每种类型前列腺素受体缺陷的小鼠，并通过检测每种基因敲除小鼠的表型来阐明前列腺素的生理作用：FP缺陷小鼠由于卵巢中孕酮的持续产生而无法分娩，表明PGF2α在正常分娩所需的黄体溶解中发挥作用。IP基因缺陷小鼠血栓形成倾向增加，炎症反应减弱，未见醋酸扭体反应，提示PG12参与了炎症和痛觉的促进作用。EP3缺陷小鼠对内源性和外源性热原均不表现出发热反应；PGE2通过EP3介导热原诱导的发热产生。EP4基因缺陷小鼠因动脉导管通畅而在出生后3天内死亡。PGE2通过EP4在新生儿动脉导管闭合中起重要作用。PGE2…基因缺陷的雌性小鼠由于排卵和受精障碍而表现出生育力下降更多的是通过EP2刺激卵丘扩张和cAMP水平的增加而促进排卵和受精。通过这些研究，我们证明了受体亚型特异性活性的重要性。此外，通过对通过定点突变方法在CHO细胞中表达的突变型EP3受体的分析，我们发现，精氨酸残基(小鼠EP3受体中的Arg-306)的正电荷与配体上游离羧基的负电荷之间的离子相互作用是Gs和Gq蛋白激活所必需的，但这些残基中的氢相互作用足以激活Gi蛋白。基于这些发现，我们开发了四种EP受体亚型激动剂(EP1：ONO-DI-004，EP2：ONO-AE1-259，EP3：ONO-EA-248，EP4：ONO-AE1-329)和一种EP1特异性拮抗剂(ONO-8711)，它们具有良好的受体特异性。这些化合物有可能被开发为疗效更特异、副作用更少的强效药物。较少

We established mice deficient in each type of prostanoid receptors, and clarified physiological roles of prostanoids by examining phenotypes in each knockout mice : FP-deficient mice fail to deliver their pups due to persistent production of progesterone in ovaries, indicating that PGF2α plays a role in luteolysis required for normal parturition. IP-deficient mice showed increased tendency of thrombosis, reduced responses of inflammation, and did not show acetic acid-induced writhing responses, indicating that PG12 participates in promotion of inflammation and pain sensation. EP3-deficent mice did not show febrile responses to both endogenous and exogenous pyrogen; PGE2 mediates pyrogen-induced fever generation via EP3. EP4-deficient mice died within three days after birth due to patency of ductus arteriosus. PGE2 plays an essential role in neonatal closure of ductus arteriosus via EP4. EP2-deficent female mice showed reduced fertility due to impaired ovulation and fertilization; PGE2 … More contributes to ovulation and fertilization through stimulation of cumulus expansion via EP2 and the resultant increase in cAMP level. Through these studies, we demonstrated importance of receptor subtype-specific activities.Furthermore, by the analysis of mutant EP3 receptor expressed in CHO cells generated by the method of site-directed mutagenesis, we found that ionic interaction between plus charge of arginine-residue (Arg-306 in mouse EP3 receptor) and minus charge of free carboxyl-residue in ligands is essential for the activation of Gs and Gq proteins, but hydrogen interaction in these residues is enough for the activation of Gi protein. Based on these findings, we developed four agonists for EP receptor subtype (EP1 : ONO-DI-004, EP2 : ONO-AE1-259, EP3 : ONO-EA-248, EP4 : ONO-AE1-329) and an EP1-specific antagonist (ONO-8711) which are excellent in their receptor-specificity. These compounds have potential to be developed as powerful drugs with more specific therapeutic effects and less side effects. Less