基于G-蛋白偶联受体信号转导通路的复杂体系药物活性成分色谱筛选方法

针对亲和色谱法在药物活性成分筛选方面缺乏药物内在活性等功能信息的问题，将脱卤素酶重组至β2-肾上腺素受体（β2-AR）碳末端作为蛋白标签，通过标签与卤代烷烃衍生物特异性生物正交反应，将受体定向固定在氨基微球表面制备色谱固定相。前沿分析法研究β2-AR特异性激动剂、部分激动剂和拮抗剂等工具药与固定化受体相互作用热力学和动力学规律，表征受体的配体识别活性；G-蛋白及其α单位相关肽与β2-AR相互作用过程的熵变，表征受体信号转导活性。竞争置换法研究工具药对信号转导活性的影响规律，建立固定化受体配体识别活性和信号转导活性的同步检测色谱方法。改进前沿色谱公式，以G-蛋白及其相关活性肽突破时间变化率为活性指标，将方法应用于黄连等中药活性成分筛选，建立基于受体信号转导通路的复杂样品高内在活性药物成分筛选方法。该研究有望解决药物活性成分筛选准确率低的难题，对G-蛋白偶联受体靶向药物研发具有重要意义。

As widely-used assays for screening bioactive compounds, classical affinity chromatographic assays are limited due to the lack of functional information such as drug intrinsic activity. To address this issue, this project will fuse dehalogenase at the C-terminus of beta2-adrenoceptor to construct a tag of the receptor. The obtained receptor is orientally immobilized onto amino-microspheres to prepare new stationary phase by the specific bioorthogonal reaction between the tag and haloalkanes. Using the specific agonist, partial agonist and antagonist as probes, the ligand-recognizing activity is characterized by frontal analysis of the thermodynamic and dynamic behaviors of these control drugs and the immobilized beta2-AR; the receptor activity of signal transportation is characterized by the entropy change of G-protein and its peptides of α domain binding to immobilized β2-AR. Through exploration of the regulating law of the abovementioned control drugs on the receptor activity of signal transportation by zonal elution, we established a new chromatographic method that enables simultaneous detection ligand-recognizing and signal-transporting activities of the receptor. With the improvement of frontal analysis equations and the use of breakthrough time changing rate as bioactivity index, we applied the proposed method in real samples such as Coptis chinensis. This results in the desired methodology for screening bioactive compounds with high intrinsic activity from complex matrice based on the signaling pathway of the immobilized receptor. This study has the potential to address the issue of low accuracy in the case of bioactive compound screening. It plays pivotal role in G-protein coupled receptor targeted drug discovery.