转化生长因子βⅠ型受体抑制剂的体外筛选及构效关系分析

发布时间：2018-10-23 08:09

【摘要】：目的筛选转化生长因子(TGF-β)信号通路Ⅰ型受体抑制剂,并探讨活性化合物的结构特征。方法利用SMAD3荧光素酶报告系统对靶向TGF-β信号通路Ⅰ型受体激活素受体样激酶(ALK)设计合成的170个化合物进行活性初筛;通过分子水平激酶活性分析评价初筛活性化合物对ALK4、ALK5或ALK7激酶的选择性抑制活性;采用EGFP-SMAD2荧光蛋白转核模型,对筛选结果进行确证;参照SB431542与ALK5分子对接情况,对活性化合物进行构效关系的分析。结果初筛发现15个化合物对TGF-β引起的SMAD3荧光素酶报告基因表达达到≥25%的抑制程度;在分子水平对ALK4、ALK5或ALK7也具有抑制活性,并表现出一定的选择性。其中,化合物63对ALK4和ALK7的IC50值分别为0.234和0.370μmol/L,对ALK5选择性较差,化合物64对ALK4、ALK5和ALK7的IC50值分别为10、6和85 nmol/L。这两个化合物在TGF-β1诱导EGFP-SMAD2核转位中同样表现出浓度依赖性抑制活性,IC50值分别为0.45和6.30μmol/L。MTT细胞增殖抑制实验表明,63和64在有效浓度下均无细胞毒性。构效关系分析表明,1,2,4-三芳基-1H-咪唑母核、1,3,5-三芳基-1H-吡唑母核、3,4-亚甲二氧基苯基、6-甲基-吡啶-2基和4-氨基羧基取代基团的化合物预计具有更好的活性。结论筛选得到2个与阳性化合物SB431542活性相当的TGF-β信号通路Ⅰ型受体抑制剂,其中63对ALK4和ALK7具有选择性,64对ALK4、ALK5和ALK7均有抑制活性。
[Abstract]:Objective to screen type I receptor inhibitors of transforming growth factor (TGF- 尾) signaling pathway and to investigate the structural characteristics of active compounds. Methods using SMAD3 luciferase reporting system, 170 compounds designed and synthesized by type I receptor activator receptor-like kinase (ALK) targeting TGF- 尾 signaling pathway were screened. The selective inhibitory activity of active compounds against ALK4,ALK5 or ALK7 kinase was evaluated by molecular level kinase activity analysis. The screening results were confirmed by EGFP-SMAD2 fluorescent protein transnucleation model. The structure-activity relationship of active compounds was analyzed. Results it was found that 15 compounds could inhibit the expression of SMAD3 luciferase reporter gene induced by TGF- 尾 by more than 25%, and also had inhibitory activity on ALK4,ALK5 or ALK7 at molecular level, and showed certain selectivity. The IC50 values of compound 63 to ALK4 and ALK7 were 0.234 and 0.370 渭 mol/L, respectively, and the IC50 values of compound 64 to ALK4,ALK5 and ALK7 to ALK5 were 10 6 and 85 nmol/L., respectively. The two compounds also showed concentration-dependent inhibitory activity in TGF- 尾 1-induced EGFP-SMAD2 nuclear translocation. The IC50 values were 0.45 and 6.30 渭 mol/L.MTT, respectively. The results showed that 63 and 64 had no cytotoxicity at the effective concentration. The results of structure-activity relationship analysis showed that the compounds of 1o 2n 4- triaryl-1H imidazole mother nucleus, 1o 3H aryl 1 H pyrazole parent nucleus, 3o 4 methylene dioxy phenyl, 6 methyl pyridyl 2 group and 4 amino carboxyl substituent group were expected to have better activity. Conclusion two type I receptor inhibitors of TGF- 尾 signaling pathway were obtained which were comparable to the SB431542 activity of the positive compounds. 63 of them were selective to ALK4 and ALK7, and 64 to ALK4,ALK5 and ALK7.
【作者单位】：军事医学科学院毒物药物研究所抗毒药物与毒理学国家重点实验室;北京师范大学生命科学学院;
【基金】：国家自然科学基金重点项目资助(81430090) 国家“重大新药创制”科技重大专项资助项目(2012ZX09301003-003;2009ZX09501031)
【分类号】：R91

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