Department of Pharmacology, Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California-San Diego, La Jolla, California (G.A.C.-H., X.W., P.T.); Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida (C.S., R.L.P.); and Department of Pharmaceutical Sciences, School of Pharmacy, Bouvé College of Health Sciences, Northeastern University, Boston, Massachusetts (G.A.T.).
Department of Pharmacology, Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California-San Diego, La Jolla, California (G.A.C.-H., X.W., P.T.); Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida (C.S., R.L.P.); and Department of Pharmaceutical Sciences, School of Pharmacy, Bouvé College of Health Sciences, Northeastern University, Boston, Massachusetts (G.A.T.)
J Pharmacol Exp Ther. 2022 Nov;383(2):157-171. doi: 10.1124/jpet.122.001354. Epub 2022 Sep 2.
A series of dipicolyl amine pyrimidines (DPPs) were previously identified as potential 7 agonists by means of a calcium influx assay in the presence of the positive allosteric modulator (PAM) 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596). The compounds lack the quaternary or strongly basic nitrogens of typical nicotinic agonists. Although differing in structure from typical nicotinic agonists, based on crystallographic data with the acetylcholine binding protein, they appeared to engage the site shared by such typical orthosteric agonists. Using oocytes expressing human 7 receptors, we found that the DPPs were efficacious activators of the receptor, with currents showing rapid desensitization characteristic of 7 receptors. However, we note that the rate of recovery from this desensitization depends strongly on structural features within the DPP family. Although the activation of receptors by DPP was blocked by the competitive antagonist methyllycaconitine (MLA), MLA had no effect on the DPP-induced desensitization, suggesting multiple modes of DPP binding. As expected, the desensitized conformational states could be reactivated by PAMs. Mutants made insensitive to acetylcholine by the C190A mutation in the agonist binding site were weakly activated by DPPs. The observation that activation of C190A mutants by the DPP compounds was resistant to the allosteric antagonist (-)-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide supports the hypothesis that the activity of these noncanonical agonists in the orthosteric binding sites was not entirely dependent on the classic epitopes controlling activation by typical agonists and that perhaps they may access alternative modes for promoting the conformational changes associated with activation and desensitization. SIGNIFICANCE STATEMENT: This study reports a family of nicotinic acetylcholine receptor agonists that break the rules about what the structure of a nicotinic acetylcholine receptor agonist should be. It shows that the activity of these noncanonical agonists in the orthosteric binding sites is not dependent on the classical epitopes controlling activation by typical agonists and that through different binding poses, they promote unique conformational changes associated with receptor activation and desensitization.
先前，通过在正变构调节剂 (PAM) 1-(5-氯-2,4-二甲氧基-苯基)-3-(5-甲基异唑-3-基)-脲 (PNU-120596) 存在的情况下进行钙离子内流测定，鉴定出一系列二吡啶基胺嘧啶 (DPP) 为潜在的 7 型激动剂。这些化合物缺乏典型烟碱型乙酰胆碱受体激动剂的季铵或强碱性氮。尽管与典型烟碱型乙酰胆碱受体激动剂的结构不同，但基于与乙酰胆碱结合蛋白的晶体结构数据，它们似乎与典型的变构激动剂共享结合位点。使用表达人 7 型受体的卵母细胞，我们发现 DPP 是该受体的有效激活剂，电流表现出快速脱敏的特征，这是 7 型受体的特征。然而，我们注意到，从这种脱敏中恢复的速度强烈取决于 DPP 家族内的结构特征。尽管竞争性拮抗剂甲基藜芦碱 (MLA) 可阻断 DPP 对受体的激活，但 MLA 对 DPP 诱导的脱敏没有影响，这表明 DPP 具有多种结合方式。正如预期的那样，变构调节剂可以使失敏的构象状态重新激活。在激动剂结合位点用 C190A 突变使受体对乙酰胆碱不敏感的突变体被 DPP 弱激活。观察到 DPP 化合物对 C190A 突变体的激活对变构拮抗剂 (-)-4-(2,3,5,6-四甲基苯基)-3a,4,5,9b-四氢-3H-环戊[c]喹啉-8-磺酰胺具有抗性，支持了以下假设：这些非典型激动剂在变构结合位点的活性不完全依赖于控制典型激动剂激活的经典表位，并且它们可能通过替代模式促进与激活和脱敏相关的构象变化。意义陈述：本研究报告了一类烟碱型乙酰胆碱受体激动剂，它们打破了烟碱型乙酰胆碱受体激动剂结构应该是什么的规则。它表明，这些非典型激动剂在变构结合位点的活性不依赖于控制典型激动剂激活的经典表位，并且通过不同的结合构象，它们促进与受体激活和脱敏相关的独特构象变化。
