苯并噻唑衍生物BD750的JAK/STAT信号通路抑制活性及其抗类风湿性关节炎的作用机制

Seeking novel therapeutics for rheumatoid arthritis (RA) has never been stopped because of the limitations of current therapies. We recently found a novel benzothiazole derivative, BD750, significantly inhibited IL-2 induced-STAT5 phosphorylation. Furthermore, BD750 completely blocked mouse and human T cell proliferation in a dose-dependent manner in vitro, which was stimulated by either anti-CD3/anti-CD28 mAbs or alloantigen. Our novel findings suggest that BD750 may be developed as a lead compound for a new category of immunosuppressants in treating RA. Therefore, we hypothesized that BD750 regulates T cell differentiation and RA fibroblast-like synoviocyte (FLS) inflammatory response via JAK/STAT signaling pathway, providing a signal mechanism in attenuating collagen-induced mouse arthrities (CIA). The specific aims designed to test the hypothesis are: 1)mechanistically, to confirm whether BD750 effects on JAK/STAT signaling pathway is through common γ chain or non γ chain; 2)on cellular basis, to determine the functional effects of BD750 on immuno cells, including inhibition of Th1 and Th17 cell differentiation in na?ve T cell, promotion of the regulatory T cell (Treg) production in vitro, inhibition of the FLS proliferation as well as the release of the chemokine and inflammatory mediators induced by cytokines such as IL-6,TNF-α,IL-1β and IL-21; 3) on animal level, to investigate the therapeutic effects of BD750 on disease model of CIA and its relationship with JAK/STAT signaling pathways and inflammatory mediators inhibition. Studies from this project will broaden our understanding in the mode of action of benzothiazole derivative on JAK/STAT signaling pathways and the signal mechanisms on how BD750 improves inflammatory immune responses in RA.

我们前期实验发现，苯并噻唑类衍生物BD750可抑制小鼠和人T淋巴细胞的STAT5磷酸化，完全阻断T细胞增殖，推测BD750有望作为全新结构的JAK/STAT信号通路抑制剂，通过抑制T细胞增殖分化、调控FLS细胞炎性应答等机制，应用于类风湿性关节炎（RA）的治疗。本项目拟进一步验证上述推测：1.探讨BD750作用的信号通路，证实BD750抑制JAK家族成员介导的共用γ链或非共用γ链信号通路；2. 探索BD750作用的靶细胞，验证BD750抑制T细胞的Th1分化和Th17/Treg失衡; 抑制炎性细胞因子诱导RA患者FLS的活化增殖和释放趋化因子、炎症介质；3. 建立CIA小鼠模型，证实BD750通过阻断诱导期自身免疫反应、抑制炎症因子网络的正向反馈激活等机制，缓解CIA小鼠关节病理学损伤。为深入了解苯并噻唑类衍生物的JAK/STAT通路抑制活性，及其抗RA的体内外作用机制奠定坚实基础。

本项目组在前期实验中发现，苯并噻唑类衍生物BD750 可抑制小鼠和人T 淋巴细胞的STAT5 磷酸化，阻断T 细胞增殖。本项目围绕BD750及其衍生物的体内外活性和作用机制开展了系列研究。我们首先观察了BD750的体外活性及其作用的信号通路，结果发现BD750可特异性抑制T细胞增殖，而不能抑制T细胞活化；可在细胞实验中抑制STAT5磷酸化，但不能在体外蛋白激酶实验中直接抑制JAK2、JAK3等132种常见蛋白激酶的活性，提示BD750可能作用于某种未知靶蛋白，间接影响JAK3/STAT5信号通路。对BD750作用的靶细胞进行研究发现，BD750不能抑制多种肿瘤细胞增殖，也不能抑制多种炎症因子诱导的人滑膜成纤维细胞增殖，提示BD750的细胞增殖抑制活性具有T淋巴细胞特异性，而非一种广泛的细胞增殖抑制剂。在小鼠CIA关节炎模型中，未能证实BD750可显著缓解小鼠关节肿胀情况。推测可能存在两方面的原因：一是BD750水溶性较差，导致血药浓度太低；二是在小鼠CIA模型中，BD750单纯抑制T细胞增殖，而不能抑制T细胞活化，也不能抑制滑膜成纤维细胞增殖， 因而尚不足以扭转病理改变。为解决BD750的水溶性问题，同时争取发现具有更高活性的BD750衍生物。本项目另行合成和筛选了30余种BD750的衍生物和类似物，其中苯并噻唑衍生物BD691、BD759、BD960, 苯并咪唑衍生物BM935显示了良好的T细胞增殖抑制活性； BD926的水溶性较BD750提高了300倍，可显著抑制T细胞增殖，抑制活化T细胞分泌IFN-γ, IL-6和IL-17；BD750的苯并咪唑类似物BMT-1可通过抑制H+/K+-ATP酶的活性，促进活化T细胞酸化，抑制T细胞增殖。上述研究为评估BD750及其系列衍生物的应用前景奠定了实验基础，提示BD750作用的靶蛋白明显不同于目前已知的各种免疫抑制剂，因而值得作为一种分子探针，用于探索T细胞增殖环节的关键分子和信号通路。此外，本项目在建立细胞模型的过程中，意外发现TLR3配体Poly（I:C）可刺激人滑膜成纤维细胞分泌抗炎细胞因子，既往未见任何文献报道，也值得做进一步的探索和研究。