Targeting alpha7 nAChR for Therapeutic Effects

描述（由申请人提供）：α 7神经元烟碱乙酰胆碱受体是重要的治疗靶点，与阿尔茨海默病、中风和精神分裂症相关。了解这种受体对天然和实验激动剂的反应对于a7的治疗靶向至关重要。我们已经表明，通过相对低的激动剂浓度刺激α 7受体可以支持激活和脱敏之间的平衡，从而产生我们假设为治疗效果基础的功能模式。在这一延续中，我们将研究α 7-选择性激动剂如何既激活这种受体，又在某些情况下通过次级抑制或脱敏作用产生功能下调。我们的实验利用结构上不同的α 7-选择性激动剂，包括大分子如苄叉假木贼碱（BA）和吲哚托烷（IT）以及用于受体选择性的较小探针如胆碱和托烷。我们的数据表明，BA化合物与残留的抑制作用与ACh结合位点的边缘上的疏水残基相互作用。我们将通过使用定点突变体和新的实验激动剂来验证这一假设。我们将开发新的化合物，包括额外的吲哚-托烷（IT）作为激动剂进行评估，并用作实验探针。IT化合物的原型是托烷司琼，一种5 HT 3受体拮抗剂和α 7选择性激动剂。我们的初步研究表明，虽然托烷司琼中的吲哚部分对5 HT 3受体具有活性，但托烷基团本身是α 7激动剂。吲哚结合似乎使托烷司琼成为一种比小激动剂托品酮更有效的α 7激动剂，尽管效果较差。我们假设，无论是基于分子内效应或特定的药物和氨基酸之间的相互作用或附近的激动剂结合位点，取代的吲哚将修改IT激动剂的性质的方式，将是可预测的基础上，我们以前的研究BA化合物和模型的受体结合位点。结构多样的BA和IT激动剂是比内源性激动剂ACh和胆碱大得多的分子，并且可能具有比ACh结合位点包括更多的受体接触点的结合位点。我们将利用选择性和非选择性激动剂以及竞争性拮抗剂进行扫描半胱氨酸可及性实验，以确定大分子和小分子探针的结合位点。我们将定义配体和蛋白质的基础，通过它某些药物实现对a7受体的选择性。总之，我们的研究将确定有效的方法来激活α 7受体与潜在的治疗药物，也将提供见解如何新的代理可能被设计成选择性地激活这种受体，由于受体脱敏或非选择性激活其他受体亚型的抑制性副作用最小化。

DESCRIPTION (provided by applicant): The a7 neuronal nicotinic acetylcholine receptor is an important therapeutic target, relevant to Alzheimer's Disease, stroke, and schizophrenia. Understanding how this receptor responds to natural and experimental agonists is crucial for the therapeutic targeting of a7. We have shown that stimulation of a7 receptors by relatively low agonist concentrations can support an equilibration between activation and desensitization, resulting in a functional modality that we hypothesize to be the basis for therapeutic effects. In this continuation we will study how a7-selective agonists both activate this receptor and, in some cases, produce down-regulation of function through secondary inhibitory or desensitizing effects. Our experiments utilize structurally diverse a7-selective agonists including large molecules such as benzylidene anabaseines (BA) and indole tropanes (IT) and smaller probes for receptor selectivity such as choline and tropane. Our data suggest that BA compounds with residual inhibitory effects interact with hydrophobic residues on the margins of the ACh binding site. We will test that hypothesis through the use of site-directed mutants and new experimental agonists. We will develop novel compounds including additional indole-tropanes (IT) to evaluate as agonists and use as experimental probes. The prototype for the IT compounds is tropisetron, a 5HT3 receptor antagonist and a7-selective agonist. Our preliminary studies show that while the indole moiety in tropisetron has activity with 5HT3 receptors, the tropane group is, on its own, an a7 agonist. Indole conjugation appears to make tropisetron a more potent, though less efficacious, agonist for a7 than the small agonist tropinone. We hypothesize that, based either on intramolecular effects or specific interactions between the drug and amino acids in or near the agonist binding site, substitutions of the indole will modify the properties of IT agonists in ways that will be predictable based on our previous studies of BA compounds and models of the receptor binding site. The structurally diverse BA and IT agonists are much larger molecules than the endogenous agonists, ACh and choline, and are likely to have binding sites that include more points of contact on the receptor than the binding site of ACh. We will conduct scanning cysteine accessibility experiments to identify the binding sites for large and small molecular probes, utilizing both selective and nonselective agonists as well as competitive antagonists. We will define both the ligand and protein basis through which certain drugs achieve selectivity for a7 receptors. Taken together, our studies will define effective ways to activate a7 receptors with potentially therapeutic agents and will also provide insights into how new agents may be designed to selectively activate this receptor with minimized inhibitory side-effects due to receptor desensitization or the non-selective activation of other receptor subtypes.