Characterization of the molecular biological effects of M1 allosteric modulators

M1 变构调节剂分子生物学效应的表征

• 批准号: 8267047
• 负责人: Meredith Noetzel
• 金额: $ 5.39万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8267047
• 关键词: Acetylcholine; Address; Adverse effects; Agonist; Allosteric Site; Alzheimer' s Disease; Binding; Binding Sites; Biological; Carboxylic Acids; Cells; Chemicals; Chimera organism; Cholinergic Agonists; Clinical Trials; Cognition; Coupling; Data; Development; Disease; Disease Progression; Dose-Limiting; Drug Design; Electrophysiology (science); G-Protein-Coupled Receptors; Imidazole; Lead; Ligands; Location; Medial; Membrane; Molecular; Molecular Conformation; Muscarinic Acetylcholine Receptor; Muscarinic M1 Receptor; Mutagenesis; Neurobehavioral Manifestations; Neurodegenerative Disorders; Patients; Pharmaceutical Preparations; Physiological; Play; Prefrontal Cortex; Property; Quinolones; Receptor Activation; Receptor Signaling; Regulation; Research; Role; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Specificity; System; Testing; Therapeutic; Therapeutic Effect; Therapeutic Intervention; basal forebrain; cholinergic; cognitive function; hippocampal pyramidal neuron; human CHRM1 protein; insight; mouse model; novel; novel strategies; patch clamp; receptor; receptor coupling; receptor expression; response; slow potential; success; tissue slice preparation; tool; transmission process

Agents that enhance cholinergic transmission or activate muscarinic acetylcholine receptors have been developed to ameliorate the loss of cognitive function in patients with Alzheimer's disease (AD) and have been suggested to have modest success in slowing disease progression. The development of selective orthosteric agonists for the M1 receptor has been relatively unsuccessful due to the high conservation of the orthosteric binding site across all five muscarinic receptor subtypes (M1-M5). We have discovered several different classes of allosteric modulators of G protein-coupled receptors: allosteric agonists and positive allosteric modulators (PAMs). Our preliminary data suggest that targeting allosteric sites on the receptor is a viable approach for developing highly selective activators of the M1 muscarinic acetylcholine receptor, since these allosteric sites are thought to be less conserved between receptor subtypes. Furthermore, evidence suggests that there are multiple allosteric binding sites on the M1 receptors. However, the locations of these secondary binding sites and the residues that are critical for binding have not been fully characterized. We propose to determine and characterize the location of the allosteric binding sites for BQCA (PAM), and TBPB and VU0357017 (allosteric agonists) using a combination of chimeric receptors and site-directed mutagenesis. We will then determine if these compounds display agonist-directed signaling, a phenomenon in which there is selective activation of particular signaling cascades, downstream of the M1 muscarinic acetylcholine receptor in cortical tissue slice preparations. Furthermore, we will determine the ability of these compounds to alter the electrophysiological properties of pyramidal neurons in the medial prefrontal cortex using whole-cell patch clamp recordings. In addition, we will utilize a mouse model of AD to try to understand how muscarinic receptor activation might be altered in pathological states. These studies will provide greater insight into the mechanism of action of allosteric ligands at the M1 receptor and potentially the possibility of selectively targeting activation of particular signaling cascades that are beneficial for disease treatment, while avoiding the activation of other cascades that may result in dose-limiting or adverse side effects.

已经开发了增强胆碱能传播或激活毒蕈碱乙酰胆碱受体的药物，以改善阿尔茨海默氏病（AD）患者认知功能的丧失，并被认为在减慢疾病进展方面取得了适度的成功。由于在所有五个毒蕈碱受体亚型（M1-M5）中，正构型结合位点的高度保守性高（M1-M5），因此，M1受体的选择性直角激动剂的发展相对不成功。我们发现了G蛋白偶联受体的几个不同类别的变构调节剂：变构激动剂和阳性变构调节剂（PAM）。我们的初步数据表明，靶向受体上的变构位点是一种可行的方法，用于开发M1毒蕈碱乙酰胆碱受体的高度选择性激活剂，因为这些变构位点在受体亚型之间被认为较少保守。此外，有证据表明，M1受体上有多个变构结合位点。但是，这些次要结合位点的位置和对结合至关重要的残基尚未完全表征。我们建议使用嵌合受体和定向诱变的组合来确定和表征BQCA（PAM）以及TBPB和VU0357017（变构激动剂）的变构结合位点的位置。然后，我们将确定这些化合物是否显示了以激动剂为导向的信号传导，这种现象是在皮质组织切片制剂中选择性激活特定信号级联反应的现象。此外，我们将确定这些化合物使用全细胞贴剂记录的内侧前额叶皮层中锥体神经元的电生理特性的能力。此外，我们将利用AD的小鼠模型来尝试了解病理状态中如何改变毒蕈碱受体激活。这些研究将提供更深入地了解变构配体在M1受体上的作用机理，并有可能选择靶向特定信号级联对疾病治疗的特定信号级联的可能性，同时避免激活其他可能导致剂量限制或不良副作用的级联反应。