Effects And Potential Mechanism Of The Novel Benzothiazole Derivative Inhibiting T Cell Proliferation

T cells play a pivotal role in immune responses against pathogens and aberrant. T cell responses can also mediate organ transplantation rejection and a variety of autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis and psoriasis. Currently, there are several immunosuppressants available in the clinic for promoting the therapeutic effects of graft rejection and autoimmune diseases through inhibiting T cell proliferation by different mechanisms.However, these diseases are triggered by kinds of factors and involve many types of cell and signalling pathways. The inducement, pathogenesis and drug sensitivity of different patients exhibited remarkable difference. If the clinical effect of monotherapy is not obvious in process of treatment, it is necessary to use combined, rotation and sequential method. Therefore, continue investigating the pathogenesis and seeking more new targets for therapy and novel leading compounds have never been stopped, because they can provide more choices and hopes to the patients.In our laboratory, we screened small molecule compounds from traditional Chinese drug, plant, metabolite of pathogenic microorganism and synthesized monomer small molecule compound laboratory with the goal of developing novel and potent immunosuppressive agents by using flow cytometry on cellular level. Then, we investigated the mechanism of the novel small molecule compounds on purified T cells with influence on cytotoxicity, the production of IL-2and expression of CD25, cell cycle, and the signalling pathway. Finally, we confirmed the activity of the novel small molecule compounds on T cell-mediated delayed-type hypersensitivity mouse model.Part I Seeking the novel small molecule compounds with immunosuppressive activityWe not only screened small molecule compounds with immunosuppressive activity from traditional Chinese drug, plant, metabolite of pathogenic microorganism, but also synthesized and tested small molecule compounds with new structure with the goal of developing novel and potent immunosuppressive agents by using flow cytometry on cellular level.Benzothiazole is an aromatic heterocyclic compound and many of its derivatives have variable bioactivities, such as antitumor, antimicrobial, anticonvulsant activities and inhibiting protoporphyrinongen oxidase. However, the immunosuppressive activity of benzothiazole derivatives is still unseen. To discover new immunosuppressants, a series of novel benzothiazole derivatives were synthesized and screened for the immunosuppressive activity. We found that BD711, BD713, BD750, BD751, BD753, BD755, BD756, BD757, BD759, BD760, BD761, BD763and BD764remarkably inhibited the proliferation of mouse splenocyte stimulated by ConA and human PBMC activated by PHA. BD750[2-(2-benzothiazoleyl)-4,5,6,7-tetrahydro-2H-indazol-3-ol, C14H13N3OS, MW:271.3], had the most effective activity among these compounds.The structure of these novel benzothiazole derivatives was different from current immunosuppresants, such as CsA, RAPA, FK506, MPA, etc, and new compound drugs in clinical trials, such as CP-690,550, ASP-015K, INCBO18424, etc. The target and function of compound depend on its structure indicating that these novel benzothiazole derivatives may have new target, according to the principle of structural biology. Therefore, the finding of these novel benzothiazole derivatives not only used as a lead compound for the design and development of new immunosuppressants for the intervention of graft rejection and autoimmune diseases, but also acted as molecular probe for investigating the pathogenesis of autoimmune diseases, according chemical biology.Part Ⅱ BD750, a benzothiazole derivative, inhibits T cell proliferation by affecting the JAK3/STAT5signalling pathwayWe found that BD750inhibited human T cell proliferation stimulated either by anti-CD3/anti-CD28mAbs or by alloantigen in a dose-dependent manner with IC50values of1.1±0.2μM and1.3±0.2μM, respectively, by flow cytometry with CFSE labeling. In addition, ConA, PMA/ionomycine or alloantigen-induced mouse T cell proliferation and PHA or PMA/ionomycine-induced human T cell proliferation were inhibited by BD750.Many compounds inhibit T cells proliferation by cytotoxicity, but not immunosuppressive activity. To test the cytotoxicity of BD750, the cell viability of BD750-treated human resting naive T cells, IL-4treated-activated T cells and FLS was determined by the CCK-8assay. Resting naive T cells were not activated and did not proliferate. IL-4treated-activated T cells were derived from naive T cells stimulated by anti-CD3/anti-CD28mAbs for72h, then washed and incubated with IL-4. IL-4could prevent the deaths of activated T cells and IL-4treated-activated T cells did not proliferate in vitro. FLS were isolated from RA patients’ synovial tissues as the non-specific cellular control. FLS can remain proliferation at least10generations in vitro in DMEM supplemented with10%FCS. We found that there was no significant difference in the relative viability of BD750-treated human resting naive T cells, IL-4treated-activated T cells and FLS to control cells among different groups by CCK-8assay. These results suggested that BD750had no obvious cytotoxicity against these cells in our experimental condition, indicating that BD750selectively inhibited activated T cell proliferation.Engagement of TCR on naive T cells by anti-CD3in the presence of anti-CD28 for co-stimulation can induce IL-2production and CD25/CD69expression at the early stage of T cell activation. We found that BD750did not inhibit CD25and CD69expression as well as IL-2secretion by ELISA and flow cytometry, indicating that BD750did not affect naive T cell activation.During the proliferation, activated T cells undergo a cell cycling process. To understand the mechanisms underlying the action of BD750, we characterized T cell cycling by flow cytometry with PI staining and found that following stimulation with anti-CD3/anti-CD28, significantly higher frequency of BD750-treated T cells was at G0/G1phase, as compared with that in vehicle-treated T cells. Furthermore, we found treatment with BD750significantly reduced the levels of cyclin D3and CDK6expression in anti-CD3/anti-CD28stimulated T cells by western blotting. These data clearly indicated that BD750induced activated T cell cycling arrest at G0/G1phase, which may contribute to the inhibition of T cell proliferation.IL-2can promote cell cycle progression and proliferation of activated T cell through the JAK3/STAT5, phosphoinositide3kinase (PI3K)/AKT and mTOR/p70S6K pathways. To further understand the mechanisms underlying the action of BD750, we found BD750inhibited IL-2induced CTLL-2cell and primary activated T cell proliferation. Although treatment with BD750did not modulate the AKT and p70S6K expression and phosphorylation, the STAT5phosphorylation was significantly reduced by BD750in both IL-2induced CTLL-2cells and primary activated T cells, implying BD750inhibited the JAK3/STAT5signal pathway.The in vivo properties of BD750were illustrated in a T-cell mediated DTH response. T cells, particularly for Thl cells, are crucial players in the pathogenesis of DTH. Administration of BD750mitigated T cell-mediated DTH in mice in a dose-dependent manner in vivo.JAK3/STAT5signalling pathway plays a crucial role in signals transduction of cytokine in immune response. SCID was identified in JAK3or STAT5knockout mice and patients lacking JAK3or STAT5expression. On the other hand, the expression of JAK3is restricted to hematopoietic cells, including immune cells, but not in other tissue cells. In contrast to ubiquitous expressed molecular targets of currently used immunosuppressive drugs, the restricted distribution of JAK3is appealing. The dramatic inhibitory effect of BD750on JAK3/STAT5signalling pathway may underlie its effect on inhibiting T cell proliferation. Therefore, BD750could be used as lead compound to develop a new class of immunosuppressive drugs selectively targeting JAK3and might have therapeutic potential for organ transplant and autoimmune disease.Part III Immunosuppressive activity of a novel water-soluble benzothiazole derivative, BD926, on T cell proliferation in vitro and in vivoHowever, the activity of BD750was limited significance and unstable in T cell-mediated delayed-type hypersensitivity mouse model. This character will limit the development and application of BD750. The main reason may be the poor water solubility of BD750. Its water solubility was not beyond0.05mg/ml in saline. It has been well recognized that drugs with poor water solubility have performance limitations, such as incomplete or erratic absorption, poor bioavailability, and slow onset of action. So, it is necessary to synthesize the derivative of BD750with good water solubility. This is the key to investigate the effect of these novel benzothiazole derivatives in vivo. We had synthesized a novel water-soluble derivative, BD926, based on the structure of BD750by chemical synthetic experiments. The water solubility of BD926exceeds15mg/ml which was300times more than BD750.BD926had the powerful immusuppressive activity in vitro and in vivo without obvious cytotoxicity. T cell proliferation stimulated by anti-CD3/anti-CD28, alloantigen and other T cell stimulators was inhibited significantly by BD926in vitro. And, we did not observe obvious cytotoxicity of BD926against human resting naive T cells, IL-4treated-activated T cells and fibroblast-like synoviocyte in our experimental condition, indicating that BD926selectively inhibited activated T cell proliferation. More importantly, BD926had the significant immusuppressive activity in vivo. In T cell-mediated delayed-type hypersensitivity mouse model, BD926can remarkably reduced ear swelling with5mg/kg/d. We did not observe any abnormality in food and water intake and other obvious signs of sickness and all of mice survived in the experimental period. Our findings indicate that BD926is safe and effective in inhibiting T cell-mediated inflammation in vivo.BD926also can inhibit cell proliferation by affecting the JAK3/STAT5signalling pathway in IL-2-induced primary activated T cells. The effects of BD926on cytokines production showed that BD926had no influence on IL-2, IL-4and IL-10production, but inhibited the secretion of IFN-y, IL-6and IL-17. This result suggested that BD926may inhibit Thl/Th2polarization and regulate Th17/Treg balance.In summary, we synthesized a series of novel benzothiazole derivatives and found that BD750had the most effective immunosuppressive activity among these compounds. BD750significantly inhibited T cell proliferation by affecting the JAK3/STAT5signalling pathway. However, the activity of BD750was limited significance and unstable in vivo, because of its poor water solubility. This character will limit the development and application of BD750. Therefore, we synthesized a novel water-soluble derivative, BD926, based on the structure of BD750by chemical synthetic experiments. BD926not only had the immunosuppressive activity same as BD750in vitro, but also had more significant activity in vivo. The structure of these novel benzothiazole derivatives was different from current immunosuppresants and new compound drugs in clinical trials. The target and function of compound depend on its structure indicating that these novel benzothiazole derivatives may have new target, according to the principle of structural biology. Therefore, the finding of these novel benzothiazole derivatives not only could act as molecular probe for investigating the pathogenesis, according chemical biology, but also used as a lead compound for the design and development of new immunosuppressants for the intervention of graft rejection and autoimmune diseases.