Targeting of alpha7 nAChR for therapeutic effects

描述（由申请人提供）：a7烟碱乙酰胆碱受体正被大力追求作为多种疾病的药物靶点，从阿尔茨海默病到感染性休克。我们已经证明，至少有三种不同的结构基序可以用来修饰核心激动剂结构，如苦参碱或喹啉，以实现a7选择性。例如，选择性可以通过添加一个大的疏水侧基（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态阶段后产生稳定离子通道脱敏的能力。脱敏是由于与受体的长时间结合，而特定药物的脱敏特性可能会影响其对特定适应症的治疗效用。我们表明，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。我们将使用单独转染a7或与促炎细胞因子受体联合转染的哺乳动物细胞来验证诱导a7离子通道稳定脱敏的药物可能仍然有效地通过细胞内JAK/STAT途径介导离子通道独立的信号转导。相反，我们还将验证离子通道激活对于LTP（海马体中与记忆相关的过程）的增强至关重要的假设。我们已经建立了各种a7激动剂如何停靠在a7受体的配体结合区域的模型，并确定了氨基酸，我们假设这些氨基酸将与a7选择性激动剂疏水侧基上的取代基进行点对点相互作用。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。我们将用位点定向突变以及新的a7选择性配体来测试我们的假设，这些配体在形成特定点对点相互作用的能力上受到限制，例如，只能作为氢键供体或受体的药物。野生型和突变型受体将在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型正变构调制器PNU-120596来测量特定配体的脱敏特性，并克服a7受体固有的有限开放概率，使其单通道电流更易于研究。我们将使用一种新型的高选择性非竞争性拮抗剂tkP3BzPB来分离离子通道激活依赖和独立的信号转导形式，并进一步操纵离子通道打开概率。这些研究将为a7激动剂的设计提供重要的进展，这些激动剂具有针对特定适应症的优化的药理学特性。

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. PUBLIC HEALTH RELEVANCE: There are many types of nicotine receptors in the brain, and only some of them are related to why people become addicted to nicotine. One type of nicotine receptor that is not the cause of addiction is the alpha7-type receptor, and stimulation of this receptor combats conditions like schizophrenia, Alzheimer's disease, septic shock and other inflammatory diseases. We have identified drugs that will selectively stimulate alpha7 receptors in one of two different ways. One form of stimulation may help alleviate brain diseases; the other may help alleviate diseases like arthritis. We will use our new discoveries about how these drugs work to help make alpha7-stimulating drugs optimally designed to treat specific diseases.