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

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.

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。

本项目在国家自然科学基金的资助下，针对现有亲和色谱药物活性成分筛选方法准确度较低的问题，以Beta2-肾上腺素受体（β2AR）为例，围绕受体蛋白高活性固定化方法及中药活性成分筛选展开研究。通过将卤代烷烃脱卤素酶（Halo）作为标签重组至β2AR非活性末端，大肠杆菌系统快速表达获得受体蛋白细胞裂解液；利用受体蛋白结构中酶与其底物6-氯己酸修饰的硅胶微球间特异性共价反应，建立了基于一步共价反应的复杂体系中目标蛋白质捕获及固定化方法;创新性的荧光成像等技术引入至固定化受体的形貌及活性表征，建立了固定化受体全面多息表征方法。在此基础上，以特异性G-蛋白相关肽作为内在活性探针，建立了固定化受体内在活性色谱表征方法，并对黄连等中药提取液进行了分析，发展形成了基于亲和色谱的中药高内在活性成分筛选技术.上述研究表明：基于大肠杆菌系统能实现mg/L水平跨膜蛋白质规模化表达；基于Halo标签的跨膜蛋白质固定化方法可在10min内完成复杂样品中目标蛋白质的捕获，实现目标蛋白一步化固定，克服传统蛋白质固定化方法需要分离纯化获得高纯度目标蛋白的不足，解决现有固定化方法蛋白质活性损失大的问题，具有简便、快速、蛋白质稳定性好和活性强的优点；以荧光成像技术为核心的固定化蛋白质表征策略能准确测定固定化蛋白质的取向和构象等活性信息，解决了液固界面蛋白质活性及构象表征缺乏直接证据的问题；以G-蛋白相关肽为核心的固定化蛋白质活性及构象色谱表征策略能全面反应蛋白质的配体识别活性和信号传导活性双重活性，能克服传统筛选方法缺乏受体信号传导活性的不足，解决了现有筛选技术准确度低的问题，实现高内在活性药物成分筛选，具有筛选准确度高，可应用于复杂体系的优点。围绕上述研究，项目组取得的重要成果包括：在Chemical Science，Analytical Chemistry和Journal of Chromatography A等杂志发表论文13篇；申请国家发明专利4项，其中2项获的专利授权并实现转化；获软件著作权1项；培养青年骨干师资2名，博士毕业生4名，硕士毕业生6名；获陕西省科学技术进步奖一等奖1项。

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