Development and characterization of a selective chromatographic approach to the rapid discovery of ligands binding to muscarinic-3 acetylcholine receptor
Development and characterization of a selective chromatographic approach to the rapid discovery of ligands binding to muscarinic-3 acetylcholine receptor
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DOI：
10.1016/j.chroma.2021.462443
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发表时间：
2021-08-05
影响因子：
4.1
通讯作者：
Zhao, Xinfeng
中科院分区：
文献类型：
--
作者：
Zhao, Xue;Fu, Xiaoying;Zhao, Xinfeng
由于缺乏高效的筛选方法，寻找与毒蕈碱-3乙酰胆碱受体（M3R）结合的新配体被认为是具有挑战性的。为了解决这些挑战，本研究开发并描述了一种使用固定化受体作为色谱固定相快速发现M3R配体的方法。我们在M3R的c端融合了卤代烷烃脱卤酶（Halo）作为标签。通过halo标签与连接物之间的特异性共价反应，将融合M3R固定在6-氯己酸活化的氨基微球上。采用扫描电镜、x射线光电子能谱对固定化的M3R进行了全面表征，并对三种特定配体与受体的结合进行了研究。通过筛选止食口服液中的生物活性化合物，用区隔洗脱法评估筛选的化合物与受体的相互作用，以及评估目标化合物的体内活性，来验证复合基质中固定化M3R的可行性。结果表明，固定化的M3R具有高特异性、良好的稳定性和从复杂基质中分离M3R配体的能力。这使我们能够确定柚皮苷、橙皮苷、甘草素、桔梗苷D和甘草酸作为M3R的潜在配体。5种化合物与M3R的缔合常数分别为4.44 × 10.4、1.11 × 10.4、7.20 × 10.4、4.15 × 10.4和3.36 × 10.4 M-1。五种化合物的协同应用显示出与原配方相当的祛痰活性。我们认为，目前的方法有可能为发现受体配体提供一种高效的策略。(c) 2021 Elsevier B.V.版权所有上标或下标可用
The pursuit of new ligands binding to muscarinic-3 acetylcholine receptor (M3R) is viewed as challeng-ing due to the lack of screening methods with high efficiency. To address such challenges, this work developed and characterized an approach to the rapid discovery of M3R ligands using the immobilized receptor as the chromatographic stationary phase. We fused haloalkane dehalogenase (Halo) as a tag at the C-terminus of M3R. The fusion M3R was immobilized on 6-chlorocaproic acid-activated ammino-microspheres by the specific covalent reaction between the Halo-tag and the linker. Comprehensive char-acterizations of the immobilized M3R were performed by scanning electron microscope, X-ray photo-electron spectroscopy, and the investigation on the binding of three specific ligands to the receptor. The feasibility of the immobilized M3R in complex matrices was tested by screening the bioactive compounds in Zhisou oral liquid, assessing the interaction between the screened compounds and the receptor using zonal elution, and evaluating the in vivo activity of the targeted compounds. The results evidenced that the immobilized M3R has high specificity, good stability, and the capacity to separate M3R ligands from complex matrices. These allowed us to identify naringin, hesperidin, liquiritigenin, platycodin D, and gly-cyrrhizic acid as the potential ligands of M3R. The association constants of the five compounds to M3R were 4.44 x 10 4 , 1.11 x 10 4 , 7.20 x 10 4 , 4.15 x 10 4 , and 3.36 x 10 4 M-1 . The synergistic application of the five compounds exhibited an equivalent expectorant activity to the original formula. We reasoned that the current method is possible to provide a highly efficient strategy for the discovery of receptor ligands. (c) 2021 Elsevier B.V. All rights reserved.Superscript/Subscript Available