Using chemical genetics to investigate mechanisms of anesthetic action

描述(由申请人提供)： 自从一个多世纪前发现麻醉以来，这些药物的使用已经彻底改变了外科手术和围手术期护理领域。然而，尽管麻醉剂被广泛使用，人们对麻醉剂对中枢神经系统产生影响的机制知之甚少。例如，最近的工作已经证实，激酶参与了麻醉作用的许多方面，但由于缺乏明确的方法来研究激酶活性，这些途径的机制研究一直受到限制。这项建议描述了一种化学遗传学方法，用于在存在和不存在普通麻醉剂的情况下识别酪氨酸激酶c-Src的直接底物。C-Src在几个神经元信号通路中起着关键作用，包括那些被认为调节痛觉、记忆和神经元存活的通路。这种方法利用了一种不自然的ATP类似物(N6-苄基-ATP)，这种类似物不被野生型c-Src识别，但却是该酶突变形式c-Src-as1的特定底物。该模拟激酶将一种化学上独特的磷酸盐模拟物硫代磷酸转移到c-Src-as1的底物。这种独特的官能团可以被抗体修饰以识别，并将能够在模型神经元细胞系中纯化直接的c-Src-as1底物。我们将利用这种方法来研究c-Src-as1活性在几类关键麻醉剂存在或不存在的情况下是如何变化的。这里描述的工作将提供关于麻醉剂如何在神经元信号通路上发挥作用的有价值的信息。这项公共卫生研究的长期目标是利用这些信息来确定可能从特定麻醉剂中受益的群体以及麻醉并发症的高风险群体，例如手术中的意识。这项工作也可能为针对特定神经通路的新麻醉药的开发提供信息。

DESCRIPTION (provided by applicant): Since the discovery of anesthesia over a century ago, the use of these agents has revolutionized the fields of surgery and perioperative care. Despite their widespread use, however, relatively little is known about the mechanisms by which anesthetic agents exert their effects on the central nervous system. For example, recent work has established that kinases are involved in many aspects of anesthetic action, but mechanistic studies of these pathways have been limited by the lack of well defined methods for studying kinase activity. This proposal describes a chemical genetic approach for identifying direct substrates of the tyrosine kinase c-Src in the presence and absence of common anesthetic agents. C-Src is critical in several neuronal signalling pathways, including those believed to modulate pain perception, memory and neuron survival. This approach utilizes an unnatural ATP analog (N6-benzyl-ATP) that is not recognized by wild-type c-Src yet is a specific substrate of a mutant form of the enzyme, c-Src-as1. The analog kinase delivers a chemically unique phosphate mimic, phosphorothioate, to substrates of c-Src-as1. This unique functional group can be modified for recognition by an antibody and will enable purification of direct c-Src-as1 substrates in a model neuron cell line. We will utilize this methodology to examine how c-Src-as1 activity varies in the presence or absence of several key classes of anesthetic agents. The work described here will provide valuable information on how anesthetic agents exert their effects on neuronal signalling pathways. The long term goal of this research in public health is to utilize this information to identify groups that are likely to benefit from a particular anesthetic as well as groups that are at high risk for complications of anesthesia, such as awareness during surgery. This work may also provide information for the development of new anesthetics directed at a particular neural pathway.