School of Pharmacy, Xi'an Jiaotong University, 76# Yanta West Road, Xi'an 710061, China.
Institute of Pharmaceutical Science and Technology, Western China Science and Technology Innovation Harbour, Xi'an 710115, China.
Anal Chem. 2024 Aug 6;96(31):12927-12935. doi: 10.1021/acs.analchem.4c03451. Epub 2024 Jul 23.
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 (SiO-IB) stepwise. Finally, we immobilized Avi-tagged MrgprX2 cell membranes on SiO-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 from extract via a 2D/CMC platform. and 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.
Mas 相关 G 蛋白偶联受体 X2（MrgprX2）在过敏反应和过敏性疾病中发挥着关键作用。已经报道了一些具有合理效力和选择性的拮抗剂。细胞膜色谱（CMC）是发现配体的有效方法。基于蛋白标签的 CMC 模型（如 SNAP 标签和 HALO 标签）提高了性能，但也增加了小分子的非特异性吸附。Avi 标签是一种短肽序列，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）可以作为 BirA 的底物，从而可以基于 IB 的化学性质（修饰羧基硅胶和特异性标记 Avi 标签）开发新的细胞膜固定相（CMSP）。首先，我们构建了 MrgprX2-Avi 标签 HEK293T 细胞系。然后，我们逐步合成了 IB 修饰的硅胶（SiO-IB）。最后，我们在 BirA 催化下将带有 Avi 标签的 MrgprX2 细胞膜固定在 SiO-IB 上。我们对开发的 CMSP 进行了表征，并使用它建立了 MrgprX2-Avi 标签/CMC-HPLC/MS 二维筛选平台，成功地通过 2D/CMC 平台从提取物中筛选出牡荆素。实验证实牡荆素靶向 MrgprX2 受体，具有抗过敏作用。我们基于 IB-Avi 标签的 CMC 方法有效地减少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药物候选物。
