CCR6小分子拮抗剂的发现及该受体作为治疗自身免疫疾病新靶点的研究

Interleukin 17-producing T helper cells (Th-17 cells) play critical roles in the pathogenesis of autoimmune diseases, including multiple sclerosis, rheumatoid arthritis and Crohn's disease. CCR6, a chemokine receptor highly expressed on the surface of Th17 cells, is required for the migration of Th17 cells into inflammatory tissues. So blockade of CCR6 has been speculated to hold promise for the treatment of Th17-related inflammatory conditions. Experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis, is best associated with Th17 cells. The susceptibility of CCR6-/- mice to EAE has been analyzed by several groups, but the results are quite contradictory. In two studies, CCR6?/? mice developed milder EAE when compared to WT mice, while three other groups described more severe EAE in CCR6?/? mice, with or without delayed disease onset. The reasons for the contradictions among these studies remain unclear, the use of different CCR6?/? mouse strains and different experimental protocols might contribute to the problem. And the redundancy of chemokine receptors might also complicate the problem. Once CCR6 was genetically deleted from birth, other chemokine receptor might replace its function during development. So we would like to study the function of CCR6 in EAE with small molecule inhibitors, which are more easy to manipulate in dose and treatment duration compared to genetic methods. CCR6 has been proven difficult in small molecule screening, and until now, there is no report about small molecule antagonists of CCR6. Fortunatelly, via high-throughtput screening, we have identified a small molecule inhibitor of CCR6. In vitro, it blocks CCR6 mediated calcium mobilization and chemotaxis with IC50 value at 5 uM. Our preliminary in vivo data also indicated that this compound can reduce the severity of EAE. In this project, we will study the role of CCR6 in the pathogenesis of EAE with this small molecule inhibitor and explore the possibility that CCR6 can be used as a new drug target for autoimmune diseases.

Th17在自身免疫性病中发挥重要致炎作用。趋化因子受体CCR6在Th17表面特异性高表达，是其向炎症部位迁移所必须的。因此，CCR6可以作为治疗Th17相关自身免疫疾病的新靶点。人类多发性硬化症的动物模型实验性自身免疫性脑脊髓炎（EAE）是与Th17非常相关的一种自身免疫疾病。但CCR6敲除小鼠诱导EAE却得出两个完全相反的结论，可能由于基因敲除小鼠本身存在缺陷，并不能很好的反应疾病的真实情况。与基因敲除方法相比，小分子拮抗剂具有时间、剂量的可控性及可逆性，更利于研究CCR6在EAE发病过程中的作用。目前，还没有CCR6抑制剂的报道，而我们通过高通量筛选的方法，已经得到一个CCR6的拮抗剂，它能抑制CCR6的配体所引起的钙流及趋化，半数抑制浓度在5uM左右。并且动物实验证明该化合物也能缓解EAE的病情。接下来，我们将要进一步探索CCR6拮抗剂治疗EAE疾病的详细机制及其作为新靶点的可能性。

多发性硬化症（Multiple Sclerosis，MS）是一种以CD4+ T细胞浸润中枢神经系统引起神经元轴突髓鞘脱落为特点的自身免疫性疾病。CD4+ T细胞的过度激活，尤其是Th1和Th17的激活被认为是MS的直接致病原因。趋化因子受体作为介导免疫细胞迁移的重要G蛋白偶联受体（G-protein coupled receptors, GPCRs），在MS病情发展过程中也发挥了重要作用。不同的免疫细胞特异性高表达不同的趋化因子受体，比如Th1细胞优先表达CCR5和CXCR3，而Th17细胞特异性高表达CCR6。.在本项研究中，我们期望找到CCR6的拮抗剂，通过抑制CCR6的功能，阻滞Th17细胞向中枢神经系统（Central nervous system, CNS）的迁移，从而达到减轻EAE病情的目的。首先，我们用钙流实验高通量筛选平台得到金精三羧酸（Aurintricarboxylic Acid，ATA），ATA为CCR6的非特异性拮抗剂，可抑制CCR4、CCR5、CCR6、CCR7、CCR9、CXCR4、CXCR5及CXCR6等趋化因子受体的功能。但接下来的结果表明，ATA则能通过抑制DC由血液向脾脏的迁移，使Th1和Th17的活化增殖分化比例减少，同时阻滞炎症性淋巴细胞透过血脑屏障浸润CNS的过程，显著改善了EAE的临床症状。CCR6基因敲除会减轻被动EAE模型小鼠的临床评分。因此，CCR6具有作为自身免疫性病新靶点的可能性。