【摘要】：目的篩選轉(zhuǎn)化生長因子(TGF-β)信號通路Ⅰ型受體抑制劑,并探討活性化合物的結(jié)構(gòu)特征。方法利用SMAD3熒光素酶報告系統(tǒng)對靶向TGF-β信號通路Ⅰ型受體激活素受體樣激酶(ALK)設(shè)計合成的170個化合物進(jìn)行活性初篩;通過分子水平激酶活性分析評價初篩活性化合物對ALK4、ALK5或ALK7激酶的選擇性抑制活性;采用EGFP-SMAD2熒光蛋白轉(zhuǎn)核模型,對篩選結(jié)果進(jìn)行確證;參照SB431542與ALK5分子對接情況,對活性化合物進(jìn)行構(gòu)效關(guān)系的分析。結(jié)果初篩發(fā)現(xiàn)15個化合物對TGF-β引起的SMAD3熒光素酶報告基因表達(dá)達(dá)到≥25%的抑制程度;在分子水平對ALK4、ALK5或ALK7也具有抑制活性,并表現(xiàn)出一定的選擇性。其中,化合物63對ALK4和ALK7的IC50值分別為0.234和0.370μmol/L,對ALK5選擇性較差,化合物64對ALK4、ALK5和ALK7的IC50值分別為10、6和85 nmol/L。這兩個化合物在TGF-β1誘導(dǎo)EGFP-SMAD2核轉(zhuǎn)位中同樣表現(xiàn)出濃度依賴性抑制活性,IC50值分別為0.45和6.30μmol/L。MTT細(xì)胞增殖抑制實(shí)驗(yàn)表明,63和64在有效濃度下均無細(xì)胞毒性。構(gòu)效關(guān)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-亞甲二氧基苯基、6-甲基-吡啶-2基和4-氨基羧基取代基團(tuán)的化合物預(yù)計具有更好的活性。結(jié)論篩選得到2個與陽性化合物SB431542活性相當(dāng)?shù)腡GF-β信號通路Ⅰ型受體抑制劑,其中63對ALK4和ALK7具有選擇性,64對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.
【作者單位】： 軍事醫(yī)學(xué)科學(xué)院毒物藥物研究所 抗毒藥物與毒理學(xué)國家重點(diǎn)實(shí)驗(yàn)室;北京師范大學(xué)生命科學(xué)學(xué)院;
【基金】：國家自然科學(xué)基金重點(diǎn)項(xiàng)目資助(81430090) 國家“重大新藥創(chuàng)制”科技重大專項(xiàng)資助項(xiàng)目(2012ZX09301003-003;2009ZX09501031)
【分類號】：R91

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