鸡球虫药物靶标丙二酰单酰辅酶A：ACP转酰基酶的结构及作用机制初步研究

Fatty acids play a critical role in cells as metabolic precursors for biological membranes, they required to complete successive cycles are catalysed by distinct enzymes encoded by unique genes involved in type II fatty acid synthesis pathway in Eimeria tenella de novo. This is in contrast with the Type I pathway in its host, where a multifunctional enzyme catalyses all the steps of the pathway. Malonyl-CoA:acyl-carry protein transacylase (MCAT) encoded by the fabD gene is one of the essential enzymes in the FAS II system, it has been considered as attractive drug targets for chemotherapeutic intervention. Structure-based drug design refers specifically to finding and complementing the 3D structure (binding and/or active site) of a drug target molecule. MCAT from E.tenella (EtMCAT) has been conformed as a drug target against coccidian in our previous study. In order to find structure and mechanism of EtMCAT for further drug design, EtMCAT and EtMCAT/ACP complex will be crystallized in hanging droplets, Protein and complex crystals will be diffracted X-rays with a Bruker Proteum CCD Detector system coupled with a Rigaku generator. The data were integrated and reduced using Proteum software. The putative active site residues in EtMCAT based on 3D structure of EtMCAT and EtMCAT/ACP complex were site-directed mutated. The active center and mechanism of the drug target will be obtained from the differences among wild-type and mutant enzymes,the 3D data of enzyme and enzyme/substrate. The complementing of 3D structure and mechanism of EtMCAT will provide essential information for structure-based drug design against coccidian.

长链脂肪酸是所有生物生命活动不可或缺的物质之一，大多数寄生性原虫，只能通过不同于其宿主的Ⅱ型脂肪酸代谢途径(FASⅡ)才能最终合成得到；丙二酰单酰辅酶A：ACP转酰基酶（Malonyl-CoA:ACP trabsacylase，MCAT）作为FASⅡ中的关键酶，被认为是研制新型抗原虫药物的靶标，药靶分子结构和作用机制解析是当前药物研制的技术瓶颈；本课题组前期研究中证实对养鸡业危害最为严重的柔嫩艾美耳球虫（Eimeria tenella）MCAT可以作为研制抗E.tenella药物的靶标。该申请项目拟在前期研究基础上，利用稀疏矩阵法和坐滴法蒸发扩散技术对EtMCAT和EtMCAT/ACP复合物进行蛋白结晶，通过X-射线衍射获得其空间构象，利用定点突变技术验证其活性中心，初步解析其作用机制，所获结果可望为新型抗球虫药物的研制提供理论和实验基础。

长链脂肪酸是所有生命活动不可或缺的物质之一，大多数寄生性原虫，只能通过不同于其宿主的II型脂肪酸代谢途径（FASII）才能最终合成：而丙二酰单酰辅酶A：ACP转酰基酶（Malony-CoA:ACP trabsacylase, MACT）作为FASII中的关键酶，被认为是研制新型抗原虫药物的靶标，药靶分子结构和作用机制解析是当前药物研制的技术瓶颈：本课题组前期研究中心证实对养鸡业最为严重的柔嫩艾美耳球虫（Eimeria tenella） MCAT可以作为研制抗E. tenella药物的靶标。该申请项目拟在前期研究基础上，利用稀疏矩阵法和坐滴法蒸发扩散技术对EtMACT和EtMACT/ACP复合物进行蛋白结晶，通过X-射线衍射分析解析其空间构想和活性中心及其表面结合口袋的小分子化合物，借助分子对接原理和分析技术，利用商业和公共数据库筛选获得具有效应的小分子化合物104种；分析显示S92、R117与H214三个氨基酸残疾构成了EtMCAT的活性中心，利用定点突变技术制备EtMCAT:S92P、EtMCAT: R117K和EtMCAT: H214N，酶活性实验发现3个突变体的酶活性分别为EtMCAT（100%）酶活性的59%、35%和5%。通过体外抑制动力学分析小分子化合物的抑制效果；并在细胞培养平台上，建立了以Real time RT-PCR技术为基础的药物筛选技术，利用该平台筛选到特异性抑制剂2种corytuberine和hexachlorophene，该研究结果将为新型抗球虫药物的研制提供理论和实验基础。