Targeting of alpha7 nAChR for therapeutic effects

描述（由申请人提供）：α 7烟碱乙酰胆碱受体正被积极地用作从阿尔茨海默病到脓毒性休克的多种疾病的药物靶标。我们已经证明，至少有三种不同的结构基序可用于修饰核心激动剂结构，如阿那巴aseine或奎宁环，以实现α 7选择性。例如，选择性可以通过添加大的疏水侧基如苯环来修饰核心激动剂来实现。疏水侧基的精确化学结构决定了功效和效力，以及另一个关键特征，即在离子通道激活的瞬时阶段之后产生稳定离子通道脱敏的能力。脱敏是由于与受体的长期结合，并且特定药剂的脱敏性质可能影响其对特定适应症的治疗效用。我们发现，脱敏和不激活受体离子通道的药物仍然可以有效地治疗炎症性疾病。我们将使用单独用α 7转染或与促炎性细胞因子受体组合转染的哺乳动物细胞来测试诱导α 7离子通道稳定脱敏的药物可能仍然有效介导通过细胞内JAK/STAT途径的离子通道非依赖性信号转导的假设。我们还将测试的假设，离子通道激活，相反，是必不可少的增强LTP，在海马体中的记忆相关的过程。我们已经生成了各种α 7-激动剂如何对接在α 7受体的配体结合结构域中的模型，并且已经鉴定了我们假设将与α 7-选择性激动剂的疏水侧基上的取代基具有点对点相互作用的氨基酸。我们将研究氢键和疏水相互作用对特定受体/配体组合的结合，门控和脱敏特性的潜在重要性。我们将测试我们的假设与定点突变，以及与新的α 7-选择性配体，将限制在他们的能力，以形成特定的点对点的相互作用，例如，代理人只能是H-键供体或受体。野生型和突变型受体将在非洲爪蟾卵母细胞或转染的哺乳动物细胞中表达，我们将通过测量全细胞和单通道电流来研究离子通道特性。我们将使用2型正变构调节剂PNU-120596来测量特定配体的脱敏特性，并克服α 7受体固有的有限开放概率，使其单通道电流更适合研究。我们将使用一种新型的高选择性α 7非竞争性拮抗剂tkP 3BzPB来分离离子通道激活依赖性和非依赖性的信号转导形式，并进一步操纵离子通道的开放概率。这些研究一起将提供重要的进展，导致设计具有针对特定适应症的药理学性质的优化概况的α 7激动剂。

DESCRIPTION (provided by applicant): The a7 nicotinic acetylcholine receptor is being energetically pursued as a drug target for diverse disorders, from Alzheimer's disease to septic shock. We have demonstrated that there are at least three distinct structural motifs which can be used to modify a core agonist structure, such as anabaseine or quinuclidine, to achieve a7 selectivity. For example, selectivity can be achieved through modification of the core agonist with the addition of a large hydrophobic side group such as a benzene ring. The precise chemical structure of the hydrophobic side group determines efficacy and potency, as well as another key feature, the ability to produce stable ion channel desensitization following a transient phase of ion channel activation. The desensitization is due to prolonged binding to the receptor, and the desensitizing properties of specific agents are likely to impact their therapeutic utility for specific indications. We show that drugs which desensitize and do not activate the receptor ion channel can still be effective at treating inflammatory diseases. We will use mammalian cells transfected with a7 alone, or in combination with pro-inflammatory cytokine receptors to test the hypothesis that drugs which induce stable desensitization of the a7 ion channel may still be effective at mediating ion channel independent signal transduction through the intracellular JAK/STAT pathway. We will also test the hypothesis that ion channel activation, in contrast, is essential for the enhancement of LTP, a memory-related process in the hippocampus. We have generated models for how the various a7-agonists dock in the ligand- binding domain of the a7 receptor and have identified amino acids which we hypothesize will have point-to- point interactions with substituents on the hydrophobic side groups of the a7-selective agonists. We will investigate the potential importance of hydrogen bonding and hydrophobic interactions on the binding, gating, and desensitizing properties of the specific receptor/ligand combinations. We will test our hypotheses with site-directed mutations, as well as with novel a7-selective ligands that will be restricted in their ability to form specific point-to-point interactions, for example, agents which are only able to be H-bond donors or acceptors. Wild-type and mutant receptors will be expressed in either Xenopus oocytes or transfected mammalian cells, and we will study ion channel properties by measuring both whole-cell and single-channel currents. We will use the Type 2 positive allosteric modulator PNU-120596 to measure the desensitizing properties of specific ligands and to overcome the intrinsically limited open probability of a7 receptors, making their single-channel currents more amenable to study. We will use tkP3BzPB, a novel highly selective a7 noncompetitive antagonist, to separate ion channel activation dependent and independent forms of signal transduction, and to further manipulate ion channel open probability. Together these studies will provide important advancements leading to the design of a7 agonists with optimized profiles of pharmacological properties for specific indications.