Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia
Mol Pharmacol. 2018 Nov;94(5):1298-1309. doi: 10.1124/mol.118.112490. Epub 2018 Sep 13.
Muscarinic acetylcholine receptors (mAChRs) are exemplar models for understanding G protein-coupled receptor (GPCR) allostery, possessing a "common" allosteric site in an extracellular vestibule (ECV) for synthetic modulators including gallamine, strychnine, and brucine. In addition, there is intriguing evidence of endogenous peptides/proteins that may target this region at the M mAChR. A common feature of synthetic and endogenous M mAChR negative allosteric modulators (NAMs) is their cationic nature. Using a structure-based approach, we previously designed a mutant M mAChR (N410K+T423K) to specifically abrogate binding of ECV cationic modulators (Dror et al., 2013). Herein, we used this "allosteric site-impaired" receptor to investigate allosteric interactions of synthetic modulators as well as basic peptides (poly-l-arginine, endogenously produced protamine, and major basic protein). Using [H]-methylscopolamine equilibrium and kinetic binding and functional assays of guanosine 5'--[-thio]triphosphate [S] binding and extracellular signal-regulated kinases 1 and 2 phosphorylation, we found modest effects of the mutations on potencies of orthosteric antagonists and an increase in the affinity of the cognate agonist, acetylcholine, likely reflecting the effect of the mutations on the access/egress of these ligands into the orthosteric pocket. More importantly, we noted a significant abrogation in affinity for all synthetic or peptidic modulators at the mutant mAChR, validating their allosteric nature. Collectively, these findings provide evidence for a hitherto-unappreciated role of endogenous cationic peptides interacting allosterically at the M mAChR and identify the allosteric site-impaired GPCR as a tool for validating NAM activity as well as a potential candidate for future chemogenetic strategies to understand the physiology of endogenous allosteric substances.
毒蕈碱型乙酰胆碱受体 (mAChR) 是理解 G 蛋白偶联受体 (GPCR) 变构作用的典型模型，在细胞外前庭 (ECV) 中具有一个“共同”的变构位点，可用于合成调节剂，包括箭毒碱、士的宁和马钱子碱。此外，还有令人着迷的证据表明内源性肽/蛋白可能靶向 M mAChR 的该区域。合成和内源性 M mAChR 负变构调节剂 (NAM) 的一个共同特征是它们的阳离子性质。我们之前使用基于结构的方法设计了一种突变 M mAChR（N410K+T423K），专门用于阻断 ECV 阳离子调节剂的结合（Dror 等人，2013 年）。在此，我们使用这种“变构位点受损”的受体来研究合成调节剂以及碱性肽（多聚精氨酸、内源性产生的鱼精蛋白和主要碱性蛋白）的变构相互作用。使用[H]-甲基东莨菪碱平衡和动力学结合以及鸟苷 5'--[硫]三磷酸 [S] 结合和细胞外信号调节激酶 1 和 2 磷酸化的功能测定，我们发现突变对正位拮抗剂的效力有适度影响，并且配体进入正位口袋的能力增加，这可能反映了突变对这些配体的进入/退出的影响。更重要的是，我们注意到突变型 mAChR 对所有合成或肽类调节剂的亲和力显著降低，验证了它们的变构性质。总的来说，这些发现为内源性阳离子肽通过变构相互作用与 M mAChR 相互作用提供了证据，并确定了变构位点受损的 GPCR 是验证 NAM 活性的工具，以及未来理解内源性变构物质生理学的化学遗传策略的潜在候选者。
