Institute of Biomedicine, School of Life Science and Technology, Jinan University, Guangzhou, China.
National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou, China.
Acta Biochim Biophys Sin (Shanghai). 2019 Jun 20;51(6):627-637. doi: 10.1093/abbs/gmz045.
Class B G-protein coupled receptors (GPCR) PAC1-R is a neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP)-preferring receptor that mediates the effective neuroprotective activity. Based on our previous data showing that doxycycline and minocycline work as the positive allosteric modulator (PAM) of PAC1-R, we used computer molecular docking and isothermal titration calorimetry assay to further determine the bindings of doxycycline/minocycline's derivatives including tetracycline/tigecycline with the N-terminal extracellular domain of PAC1-R (PAC1-EC1). Then the cAMP assay combined with the PAC1-R natural agonist PACAP27 was used to confirm the possible PAM roles of the small-molecule antibiotics. The results showed that tetracycline/tigecycline had significant lower affinity to PAC1-EC1 than doxycycline/minocycline, which was consistent with their non-positive allosteric modulation activity on PAC1-R. Furthermore, by comparing the key residues contributing to the PAM binding with the predicted allosteric site in PAC1-EC1, we characterized four motifs contributing to PAM binding in PAC1-EC1. The site-directed mutation results showed that ASN60 played the most important role in the PAM binding of the small-molecule antibiotics, while ASP116 played a sensitive marginal role in the PAM binding. These results not only help to explain the clinical and experimental neuroprotective effects of doxycycline/minocycline, but also help to characterize the PAM binding site in PAC1-EC1, which will promote the screening and characterization of novel small-molecule PAMs targeting PAC1-EC1 with drug development potency in nerve system disease.
B 类 G 蛋白偶联受体 (GPCR) PAC1-R 是一种神经肽垂体腺苷酸环化酶激活多肽 (PACAP) 优先受体，介导有效的神经保护活性。基于我们之前的数据表明强力霉素和米诺环素作为 PAC1-R 的正变构调节剂 (PAM)，我们使用计算机分子对接和等温滴定量热法测定进一步确定强力霉素/米诺环素的衍生物，包括四环素/替加环素与 PAC1-R 的 N 端细胞外结构域 (PAC1-EC1) 的结合。然后，使用 cAMP 测定法结合 PAC1-R 天然激动剂 PACAP27 来确认小分子抗生素的可能 PAM 作用。结果表明，四环素/替加环素与 PAC1-EC1 的亲和力明显低于强力霉素/米诺环素，这与它们对 PAC1-R 的非正变构调节活性一致。此外，通过比较对 PAM 结合有贡献的关键残基与 PAC1-EC1 中预测的变构结合位点，我们确定了 PAC1-EC1 中对 PAM 结合有贡献的四个基序。定点突变结果表明，ASN60 在小分子抗生素的 PAM 结合中起最重要作用，而 ASP116 在 PAM 结合中起敏感的边缘作用。这些结果不仅有助于解释强力霉素/米诺环素的临床和实验神经保护作用，还有助于表征 PAC1-EC1 中的 PAM 结合位点，这将促进针对 PAC1-EC1 的新型小分子 PAM 的筛选和表征，从而为神经系统疾病的药物开发提供潜力。
