Abstract：
Mas-related G protein-coupled receptor X2 (MrgprX2) plays a crucial role in anaphylactoid reactions and allergic diseases. Some antagonists with reasonable potency and selectivity have been reported. Cell membrane chromatography (CMC) is effective for discovering ligands. Protein-tag-based CMC models (e.g., SNAP tags and HALO tags) have enhanced performance but also increased nonspecific adsorption of small molecules. The Avi tag, a short peptide sequence, binds biotin specifically via BirA catalysis. Our study showed that 2-iminobiotin (IB) can be a BirA substrate, enabling the development of a new cell membrane stationary phase (CMSP) based on the chemical properties (modifying carboxyl silica gel and specifically labeling the Avi tag) of IB. First, we constructed the MrgprX2-Avi-tag HEK293T cell line. Next, we synthesized IB-modified silica gel (SiO2-IB) stepwise. Finally, we immobilized Avi-tagged MrgprX2 cell membranes on SiO2-IB under BirA catalysis. We characterized the developed CMSP and used it to establish a MrgprX2-Avi-tag/CMC-HPLC/MS two-dimensional screening platform, successfully screening vitexicarpin fromViticis Fructus extract via a 2D/CMC platform. In vitro and in vivo experiments confirmed that vitexicarpin targets the MrgprX2 receptor, demonstrating antiallergic effects. Our IB-Avi tag-based CMC approach effectively decreased nonspecific adsorption of the screening materials. The Avi-tag-based 2D/CMC platform is suitable for screening potential drug candidates.
摘要：
Mas相关G蛋白偶联受体X2(MrgprX2)在类过敏反应和过敏性疾病中起着至关重要的作用。已经报道了一些具有合理效力和选择性的拮抗剂。细胞膜层析(CMC)对于发现配体是有效的。基于蛋白质标签的CMC模型(例如，SNAP标签和HALO标签）具有增强的性能，但也增加了小分子的非特异性吸附。Avi标签,一个短的肽序列，通过BirA催化特异性结合生物素。我们的研究表明，2-亚氨基生物素（IB）可以是BirA底物，能够基于IB的化学性质（修饰羧基硅胶并专门标记Avi标签）开发新的细胞膜固定相（CMSP）。首先,我们构建了MrgprX2-Avi-tagHEK293T细胞系。接下来,我们逐步合成了IB改性硅胶（SiO2-IB）。最后,我们在BirA催化下将Avi标记的MrgprX2细胞膜固定在SiO2-IB上。我们对开发的CMSP进行了表征，并将其用于建立MrgprX2-Avi-tag/CMC-HPLC/MS二维筛选平台，通过2D/CMC平台成功筛选出维甲酸提取物中的维甲酸。体外和体内实验证实，viteticarpin靶向MrgprX2受体，展示抗过敏作用。我们基于IB-Avi标签的CMC方法有效地降低了筛选材料的非特异性吸附。基于Avi标签的2D/CMC平台适用于筛选潜在的候选药物。
