Targeting alpha7 nAChR for Therapeutic Effects

描述（由申请人提供）：a7神经元烟碱乙酰胆碱受体是一个重要的治疗靶点，与阿尔茨海默病、中风和精神分裂症有关。了解该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。我们已经证明，通过相对低浓度的激动剂刺激a7受体可以支持激活和脱敏之间的平衡，从而产生一种功能模式，我们假设这是治疗效果的基础。在这篇续文中，我们将研究a7选择性激动剂如何激活该受体，并在某些情况下通过继发性抑制或脱敏作用产生功能下调。我们的实验利用结构多样的a7选择性激动剂，包括大分子的如苄基苯基苯胺（BA）和吲哚托烷（IT），以及较小的受体选择性探针，如胆碱和托烷。我们的数据表明，具有残留抑制作用的BA化合物与ACh结合位点边缘的疏水残基相互作用。我们将通过使用位点导向突变体和新的实验性激动剂来检验这一假设。我们将开发新的化合物，包括额外的吲哚-托烷（IT），以评估作为激动剂和用作实验探针。IT化合物的原型是托司司琼，一种5HT3受体拮抗剂和a7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5HT3受体具有活性，但托烷基团本身是一种a7激动剂。吲哚偶联似乎使托咪司琼成为一种更有效的a7激动剂，尽管效果不如小激动剂托品酮。我们假设，基于分子内效应或药物与激动剂结合位点内或附近的氨基酸之间的特异性相互作用，吲哚的取代将以基于我们先前对BA化合物和受体结合位点模型的可预测的方式改变IT激动剂的性质。结构多样的BA和IT激动剂比内源性激动剂ACh和胆碱分子大得多，并且可能具有比ACh结合位点更多的受体接触点的结合位点。我们将进行扫描半胱氨酸接近性实验，以确定大分子和小分子探针的结合位点，利用选择性和非选择性激动剂以及竞争性拮抗剂。我们将定义配体和蛋白质基础，通过它们某些药物实现对a7受体的选择性。综上所述，我们的研究将确定使用潜在治疗药物激活a7受体的有效方法，并将为如何设计新药物以选择性地激活该受体提供见解，同时最小化由于受体脱敏或其他受体亚型的非选择性激活而产生的抑制副作用。

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.