Lanzafame Alfred A, Sexton Patrick M, Christopoulos Arthur
Drug Discovery Biology Laboratory, Department of Pharmacology, Building 13E, Monash University, Clayton 3800, Victoria, Australia.
Mol Pharmacol. 2006 Aug;70(2):736-46. doi: 10.1124/mol.106.024711. Epub 2006 May 18.
This study investigated the reciprocal cross-interactions between two distinct allosteric sites on the M(4) muscarinic acetylcholine receptor (mAChR) in the absence or presence of different orthosteric ligands. Initial studies revealed that two novel benzimidazole allosteric modulators, 17-beta-hydroxy-17-alpha-ethy nyl-delta(4)-androstano[3,2-b]pyrimido[1,2-a]benzimidazole (WIN 62,577) and 17-beta-hydroxy-17-alpha-ethynyl-5-alpha-androstano[3,2-b]pyrimido[1,2-a]benzimidazole (WIN 51,708), exhibited different degrees of positive, negative, or close-to-neutral cooperativity with the orthosteric site on M(1) or M(4) mAChRs, depending on the chemical nature of the orthosteric radioligand that was used [[(3)H]N-methylscopolamine ([(3)H]NMS) versus [(3)H]quinuclidinylbenzilate ([(3)H]QNB)]. The largest window for observing an effect (negative cooperativity) was noted for the combination of WIN 62,577 and [(3)H]QNB at the M(4) mAChR. Experiments involving the combination of these two ligands with unlabeled agonists [acetylcholine, 4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium (McN-A-343), or xanomeline] revealed low degrees of negative cooperativity between WIN 62,577 and each agonist, whereas stronger negative cooperativity was observed against atropine. It is interesting that when these experiments were repeated using the prototypical modulators heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl)-ammonium bromide (C(7)/3-phth), alcuronium, or brucine (which act at a separate allosteric site), WIN 62,577 exhibited negative cooperativity with each modulator when the orthosteric site was unoccupied, but this switched to neutral cooperativity when the receptor was occupied by [(3)H]QNB. Dissociation kinetic experiments using [(3)H]NMS and combination of C(7)/3-phth with WIN 62,577 also provided evidence for neutral cooperativity between the two allosteric sites when the orthosteric site is occupied. Together, these results provide insight into the nature of the interaction between two distinct allosteric sites on the M(4) mAChR and how this interaction is perturbed upon occupancy of the orthosteric site.
本研究调查了在不存在或存在不同正构配体的情况下，M(4)毒蕈碱型乙酰胆碱受体（mAChR）上两个不同变构位点之间的相互交叉作用。初步研究表明，两种新型苯并咪唑变构调节剂，17-β-羟基-17-α-乙炔基-δ(4)-雄甾烷[3,2-b]嘧啶并[1,2-a]苯并咪唑（WIN 62,577）和17-β-羟基-17-α-乙炔基-5-α-雄甾烷[3,2-b]嘧啶并[1,2-a]苯并咪唑（WIN 51,708），与M(1)或M(4) mAChR上的正构位点表现出不同程度的正协同、负协同或接近中性的协同作用，这取决于所使用的正构放射性配体的化学性质[[(3)H]N-甲基东莨菪碱([(3)H]NMS)与[(3)H]喹核醇基苯甲酸酯([(3)H]QNB)]。在M(4) mAChR上，WIN 62,577与[(3)H]QNB组合时观察到的最大效应窗口（负协同作用）。涉及这两种配体与未标记激动剂[乙酰胆碱、4-(间氯苯基氨甲酰氧基)-2-丁炔基三甲基铵（McN-A-343）或西尼莫林]组合的实验表明，WIN 62,577与每种激动剂之间的负协同作用程度较低，而对阿托品则观察到更强的负协同作用。有趣的是，当使用原型调节剂庚烷-1,7-双（二甲基-3'-邻苯二甲酰亚胺丙基）溴化铵（C(7)/3-邻苯二甲酰亚胺）、阿库氯铵或马钱子碱（作用于另一个变构位点）重复这些实验时，当正构位点未被占据时，WIN 62,577与每种调节剂表现出负协同作用，但当受体被[(3)H]QNB占据时，这种协同作用转变为中性协同作用。使用[(3)H]NMS以及C(7)/3-邻苯二甲酰亚胺与WIN 62,577组合的解离动力学实验也提供了证据，表明当正构位点被占据时，两个变构位点之间存在中性协同作用。这些结果共同深入了解了M(4) mAChR上两个不同变构位点之间相互作用的性质，以及这种相互作用在正构位点被占据时是如何受到干扰的。
