基于结构的GIIE分泌型磷脂酶A2的新型小分子抑制剂选择性及与受体结合的机制研究

Secreted phospholipases A2s (sPLA2s) comprise 11 isoforms, which play a complementary or antagonistic role in diseases associated with inflammation and lipid metabolism. Inhibitors of sPLA2s have limited use in basic and clinical research because of its poor sub-type selectivity. GIIE, One of the sPLA2s members, has been proved to have anti-atherosclerotic function. Through the structure study of human GIIE, we understood the binding mechanism between GIIE and its non-specific inhibitors, and discussed the relationship between its enzymatic activity and physiological function. However it is unclear how GIIE play the role in biological function by binding to its receptors. Recently, we obtained new kinds of subgroup-selective inhibitors of GIIE by computer-aided high throughout virtual screening. Based on this work, we will verify the sub-type selectivity of these inhibitors using enzymatic method, and explore their structural-inhibitor relationship using structural biology approaches. Using the inhibitors obtained as the probe, we plan to study the colocalization of GIIE to its receptor PLA2R1 and its regulation of the lipid transport in cellular experiment. This will give us insight into the receptor binding mechanism of GIIE in its anti-atherosclerotic function. This project will provide scientific basis for the development of sPLA2 inhibitors and the treatment of atherosclerosis.

分泌型磷脂酶A2（sPLA2）家族成员众多，在一些炎症及脂代谢疾病中起互补或相反作用。目前已报道的sPLA2抑制剂亚型选择性较差，限制了其在基础研究和临床中的应用。sPLA2成员GIIE能拮抗动脉粥样硬化的发生。申请人前期通过人源GIIE的结构生物学研究，探讨了GIIE与已知小分子抑制剂的结合机制、GIIE酶活性与功能的关系，但是尚不清楚GIIE如何通过受体结合发挥生理功能。我们近来采用计算机虚拟筛选初步获得了GIIE的选择性小分子。本项目将采用酶学等方法进一步筛选GIIE的选择性小分子，同时开展小分子与GIIE的复合物晶体结构研究，阐明小分子的选择机制；并以新小分子为工具，在细胞水平研究GIIE与受体PLA2R1的共定位和GIIE对脂质转运的调节作用，探究GIIE通过受体途径拮抗动脉粥样硬化的结构基础和分子机制。本项目将为sPLA2亚型选择性抑制剂的开发和动脉粥样硬化的防治提供理论基础。

分泌型磷脂酶A2（sPLA2）家族成员众多，不同亚型sPLA2结构类似，功能迥异。目前已报道的sPLA2抑制剂亚型选择性较差，限制了其在基础研究和临床中的应用。我们前期获得了GIIE的晶体结构，基于GIIE的结构，本研究利用计算机虚拟筛选手段获得了22种sPLA2亚型选择性小分子抑制剂。采用原核表达系统和真核表达系统对不同亚型的sPLA2（GIIE、GIB、GIIA和GX）进行了表达，并采用包涵体变复性、亲和层析、离子交换层析和分子排阻法对蛋白进行了纯化。采用sPLA2酶活性测定试剂盒，验证了虚拟筛选的抑制剂对sPLA2的选择性抑制效果。通过对GIIE突变体N21G的结构研究，以及N21G与抑制剂的复合物结构研究，分析了GIIE独特的钙离子结合机制和催化机制；通过对GIIE的E54进行突变研究，分析了GIIE的底物选择性机制。GIIE敲除小鼠实验表明GIIE能拮抗动脉粥样硬化的发生。本研究通过泡沫细胞模型实验发现，外源添加GIIE蛋白抑制泡沫细胞的形成。通过分子模拟构建了GIIE与受体的复合物结构，为探究GIIE通过受体途径拮抗动脉粥样硬化提供结构基础。

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