The Biological Evaluation Of Small Non-peptidic CD4 Inhibitors

Immunosuppressants have been studied for more than 80 years. However, the effectof their clinical applications is not ideal because of nonspecificity and undesired side effects. Prof. Li applied the computer-based strategy to design and synthesize some small non-peptidic compounds, which can specifically block stable CD4-MHC classII 2 domain interaction, but the biological effects in vivo and in vitro is not clear.In this study, we primarily screened the 51 organic compounds, and then selected 3 compounds of them that have obvious immunosuppression to study the mechanism at celluar level, molecular level and gene level. It is hoped that these organic compounds can be used to treat transplant rejection and autoimmune diseases.1. The definition of organic compounds with immunosuppressionUsing MTT assay and incorporation of 3H-thymidine in cell lines and human peripheral blood respectively, immunosuppression of these compounds have been observed and evaluated on mixed lymphocyte culture. It was proved that 70% of them have immunosuppressive effect, we selected D2, J2 and N2 to investigate the mechanisms.2. Study on the mechanisms of organic compounds(1) The effect on cell adhesion between CD4-MHC class n molecules Utilizing the HEK-293/CD4 cell that expressed CD4 and green fluorescenceprotein, and Daudi cell that expressed MHC class II highly, which mimic the cell adhesion between CD4 and class II MHC molecules in vitro. We found that the 3 organic compounds can inhibit this kind of cell adhesion distinctly.(2) The effect on cytokines productionCD4+ T cell is activated upon stimulation, then secrete cytokines such as IL-2 and IFN- v . So we use PMA and ionomycin to activate human T cells in order to study secretion of cytokines by Flow Cytometry. The result has indicated that they all inhibited the production of IL-2, but have little effect on the production of IFN-. (3) The effect on nuclear factor of activated T cells (NF-AT)Once CD4+ T cell has been activated, they produce IL-2, whereas NF-AT binds to the sequences within the IL-2 enhancer is necessary for T cell-specific activation. For this reason, human PBMC was activated with PMA and ionomycin, and then extract nuclear proteins binding to NF-AT probe for electrophoresis mobility shift assay (EMSA). The result showed that organic compounds have immunosuppression at the different level.To conclusion, small non-peptidic compounds-D2, J2, N2 can act as new leading compounds for preventing and treating transplantation rejection and autoimmune diseases.