CAJ | 학술논문

目的：通过计算机虚拟筛选和细胞活性筛选，获得能靶向抑制肿瘤坏死因子β（TNFβ）的细胞毒活性的小分子抑制剂。方法根据TNFβ与肿瘤坏死因子Ⅰ型受体（TNFR1）结合的复合晶体结构，采用计算机虚拟筛选方法初步选择对接结果较好的105个小分子化合物（C1~C105），并对其进行细胞活性筛选；MTT法检测小分子化合物抑制TNFβ对L929细胞的细胞毒性；流式细胞术测定小分子化合物对TNFβ引起的细胞凋亡的抑制作用；采用碘化丙啶（PI）单染和流式分析技术检测小分子化合物对L929细胞周期的影响；采用Western blot和双转盘激光共聚焦显微镜分析小分子化合物对TNFβ引起的下游Caspase 3活化的抑制作用。结果 C35能有效抑制TNFβ引起的细胞毒作用，且这种抑制作用呈现剂量依赖性（半数抑制浓度=8.19μmol/L)；C35具有较低的细胞毒性，且对L929细胞周期无影响；C35能阻断TNFβ与其受体结合后引起的细胞凋亡通路，显著抑制TNFβ引起的L929细胞凋亡。结论成功筛选到一个TNFβ小分子抑制剂C35，其能阻断TNFβ引起的细胞凋亡通路，高效抑制TNFβ的细胞毒活性。
목적：통과계산궤허의사선화세포활성사선，획득능파향억제종류배사인자β（TNFβ）적세포독활성적소분자억제제。방법근거TNFβ여종류배사인자Ⅰ형수체（TNFR1）결합적복합정체결구，채용계산궤허의사선방법초보선택대접결과교호적105개소분자화합물（C1~C105），병대기진행세포활성사선；MTT법검측소분자화합물억제TNFβ대L929세포적세포독성；류식세포술측정소분자화합물대TNFβ인기적세포조망적억제작용；채용전화병정（PI）단염화류식분석기술검측소분자화합물대L929세포주기적영향；채용Western blot화쌍전반격광공취초현미경분석소분자화합물대TNFβ인기적하유Caspase 3활화적억제작용。결과 C35능유효억제TNFβ인기적세포독작용，차저충억제작용정현제량의뢰성（반수억제농도=8.19μmol/L)；C35구유교저적세포독성，차대L929세포주기무영향；C35능조단TNFβ여기수체결합후인기적세포조망통로，현저억제TNFβ인기적L929세포조망。결론성공사선도일개TNFβ소분자억제제C35，기능조단TNFβ인기적세포조망통로，고효억제TNFβ적세포독활성。
Objective To explore a novel and highly specific small-molecule TNFβinhibitor by using computer-aid?ed virtual screening and cell-based assays in vitro. Methods Computer-aided drug design and virtual screening were used to design and identify chemical compounds that targeted TNFβbased on the crystal structure of the TNFβ-TNFR1 com?plex. The effect of the small-molecule compound against TNFβ-induced cytotoxicity of L929 cells was detected by MTT as?say, and the efficacy of the compound to inhibit TNFβ-induced apoptosis of L929 cells was determined by flow cytometry as?say. The impact of the compound on L929 cell cycle was examined by Propidium Iodide (PI) staining and flow cytometry, and the influence of the compound on TNFβ-triggered signal pathway was analyzed by Western blot assay and Ultra VIEW VOX 3D Live Cell Imaging System. Results No.35 compound (named as C35 thereafter) could effectively inhibit TNFβ-induced cell death in a dose dependent manner, and the half-maximum inhibition concentration (IC50) was 8.19μmol/L. Furthermore, C35 had lower cytotoxicity and minimal effect on L929 proliferation. Here we further revealed that C35 could affect TNFβ-induced apoptotic pathway by blocking the activation of Caspase 3, and markedly reduce L929 cell apoptosis induced by TNFβ. Conclusion A novel TNFβsmall-molecule inhibitor was identified by combining computer-aided virtual screening with functional assays, and which could block TNFβ-triggered apoptotic pathway and efficiently inhibit the cell death in?duced by TNFβ.