Nawaratne Vindhya, Leach Katie, Suratman Nur, Loiacono Richard E, Felder Christian C, Armbruster Blaine N, Roth Bryan L, Sexton Patrick M, Christopoulos Arthur
Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences, Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia.
Mol Pharmacol. 2008 Oct;74(4):1119-31. doi: 10.1124/mol.108.049353. Epub 2008 Jul 15.
The M4 muscarinic acetylcholine (ACh) receptor (mAChR) is a potential therapeutic target but characterized by a lack of subtype-selective ligands. We recently generated "designer receptors exclusively activated by a designer drug" (DREADDs), which contained mutations of two conserved orthosteric-site residues (Y113C/A203G in the M4 mAChR) that caused a loss of ACh activity but a gain in responsiveness to clozapine-N-oxide (CNO). The current study characterized the interactions of the wild type and the M4 DREADD with a range of agonists, antagonists, and the recently discovered M4 mAChR allosteric potentiator, 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298). LY2033298 displayed positive binding cooperativity with ACh, neutral cooperativity with the antagonist, [3H]quinuclidinyl benzilate, and agonism for activation of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 at the wild-type M4 mAChR. LY2033298's cooperativity with clozapine or CNO was weakly positive with respect to binding but profoundly negative with respect to LY2033298 signaling. Although the DREADD mutations increased the binding and function of clozapine-like compounds, all other agonists lost the ability to activate the mutant; for the orthosteric agonists ACh and pilocarpine, this was due partly to a reduced affinity, whereas the affinity of LY2033298 or the atypical agonist 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride was unaltered. The interaction between LY2033298 and clozapine-like compounds reverted to neutral cooperativity on the DREADD, whereas LY2033298 caused a striking functional rescue of ACh potency and efficacy at the DREADD. These results provide conclusive evidence for the retention of a functional allosteric site on the M4 DREADD and highlight a role for residues Tyr113 and Ala203 in the transmission of cooperativity.
M4毒蕈碱型乙酰胆碱（ACh）受体（mAChR）是一个潜在的治疗靶点，但缺乏亚型选择性配体。我们最近构建了“仅由设计药物激活的设计受体”（DREADD），其包含两个保守的正构位点残基（M4 mAChR中的Y113C/A203G）的突变，这导致ACh活性丧失，但对氯氮平氮氧化物（CNO）的反应性增加。本研究表征了野生型和M4 DREADD与一系列激动剂、拮抗剂以及最近发现的M4 mAChR变构增强剂3-氨基-5-氯-6-甲氧基-4-甲基-噻吩并[2,3-b]吡啶-2-羧酸环丙基酰胺（LY2033298）之间的相互作用。LY2033298与ACh表现出正结合协同性，与拮抗剂[3H]喹核醇基苯甲酸酯表现出中性协同性，并且在野生型M4 mAChR上对磷酸化细胞外信号调节激酶（ERK）1/2的激活具有激动作用。LY2033298与氯氮平或CNO的协同性在结合方面呈弱阳性，但在LY2033298信号传导方面呈显著阴性。尽管DREADD突变增加了氯氮平样化合物的结合和功能，但所有其他激动剂均失去了激活突变体的能力；对于正构激动剂ACh和毛果芸香碱，这部分是由于亲和力降低，而LY2033298或非典型激动剂4-I-[3-氯苯基]氨基甲酰氧基）-2-丁炔基三甲基氯化铵的亲和力未改变。LY2033298与氯氮平样化合物之间的相互作用在DREADD上恢复为中性协同性，而LY2033298在DREADD上对ACh的效力和效能产生了显著的功能挽救。这些结果为M4 DREADD上功能性变构位点的保留提供了确凿证据，并突出了酪氨酸113和丙氨酸203残基在协同性传递中的作用。
