PO-322抑制Th1/Th17应答及抗银屑病的作用机制

Seeking novel therapeutics for psoriasis has never been stopped because of the limitations of current therapies. Recently, a novel compound PO-322 has been synthesized. We found that PO-322 inhibited T cell proliferation in vitro and attenuated T cell-mediated inflammation in delayed-type hypersensitivity reaction mouse model in vivo. Furthermore, PO-322 had no effect on IL-2, IL-4 and IL-10 production and CD25 expression, but inhibited IFN-γ, IL-6 and IL-17 in anti-CD3/CD28 mAbs induced PBMC. The structure and effect on cytokine production of PO-322 were different from current immunosuppresants, indicating PO-322 may have new target. Our findings suggest that PO-322 may be developed as a novel lead compound for psoriasis treatment by inhibiting Th1/Th2 polarization and regulating Th17/Treg balance. In this project, we will explore the mechanism of PO-322 in treating psoriasis on target cells, signalling pathway and animal model. First, we will confirm PO-322 inhibits Th1/Th2 polarization and regulates Th17/Treg balance by observing specific transcription factor expression and cytokine production using Flow cytometry and ELISA. Next, we will explore the signalling pathway of PO-322 by observing the signalling pathway regulating Th1/Th17 responses, such as MAPK/ERK, PI3K/Akt and JAK/STAT signalling pathway, using western blotting and finding the target protein kinase of PO-322 using kinase screen. Finally, we will provide evidence that PO-322 can attenuate the psoriasis skin inflammation in imiquimod induced psoriasis mouse model. This project will help us understand the mode of action of PO-322 in treating psoriasis, explore the new pathogenesis of psoriasis and develop new drug for the treatment of psoriasis.

我们前期合成了新型小分子化合物PO-322，它可完全抑制T细胞增殖，显著缓解迟发型超敏反应。PO-322的结构与细胞因子作用特点有别于现有免疫抑制剂，提示PO-322可能存在新的作用靶点，并有望通过调控Th1/Th2极化，调节Th17/Treg平衡，缓解银屑病皮肤炎症。本项目拟从靶细胞、信号通路与动物模型三个方面探索PO-322抗银屑病的作用机制：①流式与ELISA观察特异性的转录因子表达与细胞因子产生，证实PO-322调控Th1/Th2极化并调节Th17/Treg平衡；②免疫印迹检测MAPK/ERK、PI3K/Akt与JAK/STAT等参与Th1/Th17应答的信号通路，激酶筛选寻找可能作用的蛋白激酶，探索PO-322作用的信号通路；③在小鼠银屑病模型中，证实PO-322可有效缓解皮肤炎症。本项目的实施将为揭示银屑病发病的新机制，发现更多的治疗靶点与候选药物奠定坚实基础。

银屑病是一种以慢性皮肤炎症为特征的自身免疫性疾病。机体免疫系统在银屑病的发病过程中发挥着关键性的作用。银屑病当前的治疗药物也多以免疫细胞或免疫系统活化相关的蛋白为靶细胞或靶点。近年，免疫抑制药物在银屑病的治疗中取得了极好的疗效。但是，不同患者的药物敏感性存在显著个体差异，治疗过程中发现单一疗法效果不明显时，还需给予联合、轮换或序贯治疗。继续探索银屑病的发病机制，发现更多的治疗靶点和全新结构的先导化合物，将为患者提供更多的选择与希望。噁唑类化合物是一类特殊结构的化合物，具有多种生物学功能。但目前尚未有文献报道噁唑类化合物具有对T细胞的免疫抑制活性以及对银屑病的治疗作用。本课题合成了噁唑类化合物PO-322 [1H-indole-2,3-dione 3-(1,3-benzoxazol-2-ylhydrazone)]，并对一系列噁唑类化合物进行了免疫抑制活性的检测。我们发现PO-322的活性最强，它抑制抗CD3/CD28抗体刺激的人T细胞增殖和混合淋巴细胞反应且无显著细胞毒性。PO-322不影响活化T细胞表达CD25和CD69以及产生IL-2，但阻滞活化T细胞的细胞周期于G0/G1期。PO-322抑制活化T细胞产生炎性细胞因子IFN-γ，IL-6和IL-17。随后，我们观察了PO-322作用的信号通路。结果显示PO-322抑制IL-2刺激的T细胞增殖，提示其作用的信号通路位于IL-2受体下游。进一步发现，PO-322不抑制AKT和ERK 1/2分子的表达和磷酸化，不抑制p70S6K分子的表达，但显著降低p70S6K分子的磷酸化，提示PO-322作用于mTOR/p70S6K信号通路。同时，我们发现PO-322不抑制STAT5分子的表达，但促进了STAT5分子的磷酸化。mTOR/p70S6K信号通路在银屑病的发病过程中起重要作用。我们建立了咪喹莫特诱导小鼠发生银屑病样皮肤炎症的模型，并发现PO-322能够有效地减缓咪喹莫特诱导的皮肤炎症。体内实验过程中，PO-322不会引起小鼠死亡、明显饮食行为异常与疾病表现。因此，PO-322具有成为先导化合物设计和开发治疗银屑病的新型药物的潜力。