天然产物中FFA1受体新激动剂的发现与研究

Discovery and study of new FFA1 receptor agonists from natural products

目的:发现游离脂肪酸受体1(FFA1)的新型激动剂,为该受体的生物学和功能研究提供工具分子。方法:(1)筛选实验:构建FFA1受体高表达的CHO-K1细胞(CHO-FFA1细胞)。采用无标记细胞整合药理学(CLIP)技术在CHO-FFA1细胞上筛选熊果酸、白藜芦醇、黄芪甲苷、甘草酸、黄柏碱、苍术素、苍术酮等80个天然化合物对FFA1受体的激动活性。(2)验证实验:采用FFA1受体选择性拮抗剂GW1100,对筛选得到的潜在活性化合物进行FFA1受体激动活性验证。(3)量效实验:以不同浓度的活性化合物刺激CHO-FFA1细胞,观察其作用的量效曲线,测定化合物对FFA1受体激动效应的半数效应浓度(EC50),同时在CHO-K1细胞上评估化合物的响应信号。(4)分子对接:利用autodock分子模拟软件研究活性化合物与FFA1受体的结合模式。结果:(1)活性筛选得到4个潜在的FFA1受体激动剂,分别为苍术素、苍术酮、五味子醇甲和原花青素。(2)验证实验发现,GW1100能剂量依赖性抑制苍术素和苍术酮产生的DMR信号,而对五味子醇甲和原花青素引发的DMR信号无明显影响,提示苍术素和苍术酮为FFA1受体激动剂。(3)量效实验测得苍术素和苍术酮的EC50值分别为87.2、58.4μmol/L,且两者均为部分激动剂。苍术酮在CHO-K1细胞上有微弱的DMR信号,也可能作用于CHO-K1细胞上的其他靶点。(4)分子对接显示,苍术素和苍术酮在FFA1受体部分激动剂对应的变构位点有较好的结合;两者作为疏水性分子,主要与FFA1的疏水残基相互作用,且均可与Trp174形成p-π相互作用。结论:从天然产物中发现2个新的FFA1受体激动剂苍术素和苍术酮,并分析其与受体的结合方式。

Objective: To discover new agonist of free fatty acid receptor 1(FFA1), and provide pharmacological tools for the study on biology and function of this receptor. Methods:(1)Screening assay: CHO-K1 cells with high expression of FFA1 receptor(as CHO-FFA1 cells) were constructed. In CHO-FFA1 cells, cellular label-free integrative pharmacology(CLIP) technology was used to screen the agonist of FFA1 receptor from 80 natural compounds, including ursolic acid, resveratrol, astragaloside A, glycyrrhizic acid, phellodendrine, atractylodin, atractylon and so on.(2)Verification assay: The FFA1 receptor selective antagonist GW1100 was used to verify the activity of the potential compounds.(3)Dose-effect assay:CHO-FFA1 cells were stimulated with different concentrations of active compounds. The dose-effect curve was observed and the half effective concentration(EC50) value of the compound on FFA1 receptor was determined. Meanwhile, the response signals of the compounds were evaluated in CHO-K1 cells.(4)Molecular docking: Autodock software was employed to dock the binding mode of active compounds with FFA1 receptor. Results:(1)Four potential FFA1 receptor agonists,including atractylodin,atractylon,schisandrin and procyanidin,were obtained through activity screening assay.(2)The verification assay showed that GW1100 could inhibit the DMR signal induced by atractylodin and atractylon in a dose-dependent manner,but not affect that of schisandrin and procyanidin,indicating that atractylodin and atractylon were FFA1 receptor agonists.(3)In dose-effect assay,the EC50 values of atractylodin and atractylon were 87. 2 and 58. 4 μmol/L,respectively. Both of them were partial agonists. Atractylon showed a weak DMR signal in CHO-K1 cells,suggesting that it might also act on other targets.(4) Molecular docking revealed that atractylodin and atractylon bound well at the allosteric site corresponding to the partial agonist of FFA1 receptor. As hydrophobic molecule,atractylodin and atractylon mainly interacted with the hydrophobic residues of FFA1,and formed p-π interaction with Trp174.Conclusion: Two new agonists of FFA1 receptor,atractylodin and atractylon,are found from natural products,and their binding modes with receptor are analyzed.