目的:基于亲和力的选择性毒蕈碱乙酰胆碱受体(mAChR)正构配体由于五种受体亚型的高度序列同源性而难以开发。选择性也可以通过特定亚型或信号传导途径的选择性激活来实现。有希望的,先前的研究将化合物6A和7A鉴定为在M2mAChR处的功能选择性和Gi偏向的化合物。这里,我们研究了M2mAChR上单个G蛋白亚家族的激活以及6A和7A的下游信号通路。
方法:用TRUPATH测定法在M2mAChRFlpInCHO细胞中测量G蛋白活化。使用cAMPCAMYELBRET传感器和细胞外信号调节激酶1/2磷酸化测定来测定下游信号传导途径中的活性。
结果:M2mAChR与G44%i1耦合,G◎oA和G◎s,但不是G187q,响应规范的正构激动剂。化合物6A和7A不引起任何G蛋白活化,cAMP抑制或刺激,或ERK1/2磷酸化。相反,Schild分析表明竞争激烈,化合物6A和7A与ACh的拮抗相互作用在Gαi1激活测定中。M2mAChR的过表达可能提示6A和7A的表达依赖性活化谱。
结论:这些数据验证了M2mAChR优先与G‰i/o偶联,并在较小程度上与G‰偶联,以响应规范的正构配体。然而,本研究未能检测到化合物6A和7A的Gαi偏倚,这突出了分类新配体时细胞背景的重要性.
OBJECTIVE: Affinity-based, selective orthosteric ligands for the muscarinic acetylcholine receptors (mAChRs) are difficult to develop due to high sequence homology across the five subtypes. Selectivity can also be achieved via the selective activation of a particular subtype or signalling pathway. Promisingly, a prior study identified compounds 6A and 7A as functionally selective and Gi biased compounds at the M2 mAChR. Here, we have investigated the activation of individual G protein subfamilies and the downstream signalling profiles of 6A and 7A at the M2 mAChR.
METHODS: G protein activation was measured with the TRUPATH assay in M2 mAChR FlpIn CHO cells. Activity in downstream signalling pathways was determined using the cAMP CAMYEL BRET sensor and assay of ERK 1/2 phosphorylation.
RESULTS: M2 mAChRs coupled to Gɑi1, GɑoA and Gɑs, but not Gɑq, in response to canonical orthosteric agonists. Compounds 6A and 7A did not elicit any G protein activation, cAMP inhibition or stimulation, or ERK 1/2 phosphorylation. Instead, a Schild analysis indicates a competitive, antagonistic interaction of compounds 6A and 7A with ACh in the Gɑi1 activation assay. Overexpression of the M2 mAChR may suggest an expression-dependent activation profile of compounds 6A and 7A.
CONCLUSIONS: These data confirm that the M2 mAChR preferentially couples to Gɑi/o and to a lesser extent to Gɑs in response to canonical orthosteric ligands. However, this study was not able to detect Gɑi bias of compounds 6A and 7A, highlighting the importance of cellular background when classifying new ligands.
BACKGROUND: This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc.