别构响应型受体色谱方法建立及评价

别构配体是正在新兴的G蛋白偶联受体（GPCR）潜在高效药物，迫切需要发展新原理、新技术和新方法，实现其高效筛选。本项目以β2-肾上腺素受体（β2-AR）为例，用指数富集配体系统进化技术筛选DNA随机文库，获得受体特异性核酸适配体；用该适体捕获单一活性构象态受体，通过碱基互补配对和Ni2+-组氨酸标签双元作用，将受体固载于大孔微球表面，制备别构响应型受体色谱固定相；扫描电镜等技术和工具药保留行为表征固定化受体形貌及正构配体识别活性；以肾上腺素等内源性正构配体为探针，通过测定特异性反激动剂等工具药对探针与固定化受体相互作用热力学和动力学参数影响规律表征受体别构响应活性，建立别构响应型固定化受体色谱方法；用该法对三子养亲汤进行分析，建立β2-AR靶向别构配体色谱筛选方法，有望填补色谱技术在别构配体筛选领域的空白，对靶向G-蛋白偶联受体药物研发，推动分析化学学科色谱分析领域发展具有重要意义。

Allosteric ligand is the potential emerging highly effective drugs for G protein coupled receptors (GPCRs). It is urgent to develop new principles, new technologies and new methodologies to achieve the efficient screening of the allosteric drug candidates. Using β2-adrenoceptor (β2-AR) as a probe, receptor specific nucleic acid aptamer is obtained by screening a random DNA library using exponential enrichment ligand system evolution technique (SELEX). The single active conformation of the receptor is stabilized by the selected aptamer. To prepare the allosteric responsive receptor stationary phase, the receptor is immobilized on the surface of macroporous microspheres by complementary base pairing and metal chelate interaction. The morphology and orthosteric ligand recognition activity of the immobilized receptor are characterized by Scanning Electronic Microscopy (SEM) and the retention behavior of specific drugs. To establish the allosteric responsive receptor chromatographic method, we will use adrenaline and other orthosteric ligands as probes. The allosteric responsive activity of the receptor can be characterized by determining the thermodynamic and kinetic parameters between the probes and immobilized receptor under the presence of anti-agonists of the receptor. We can establish a chromatographic methodology for screening the targeted beta2-AR allosteric ligands in Sanziyangqin Decoction. This methodology sheds light on filling the gap of chromatographic technology in allosteric ligand screening. It is also of great significance to the research and development of drugs targeting G-protein coupled receptors and to promote the development of chromatographic analysis in analytical chemistry.