Nicotinic Receptor Template-guided Drug Design

描述（由申请人提供）：所提出的研究是由Sharpless & Taylor实验室的合作发展而来的，该合作采用新的“点击化学”，以乙酰胆碱结合蛋白作为反应容器来开发烟碱乙酰胆碱受体的选择性配体。前体锚定在受体上的两个位点，并含有延长的叠氮化物和乙炔部分，其通过环加成反应形成三唑。前体配体对蛋白质具有适度的亲和力，并且在溶液中反应极其缓慢。在蛋白质表面上，反应物基团的并置和蛋白质的两亲性环境迫使快速反应，导致形成高亲和力复合物。合成采用组合反应物，并且形成通过DIOS质谱表征的单一或主要产物。有趣的是，这种“冷冻框”程序捕获了处于意外构象的复合物，使得高亲和力区域异构体从低丰度构象形成。最初乙酰胆碱结合蛋白从拟南芥属和Aesthesia作为模板，以形成选择性的烟碱剂。通过诱变，模板被修饰以掺入α 7受体的结合决定簇，然后掺入在脑中发现的异源受体亚型。由于这是一个有效的“冻结框架”反应，结合模板的构象灵活性将在单独的研究中通过荧光各向异性的衰减进行分析。配合物还提供了X射线晶体学研究的潜力。部分激动剂和拮抗剂将从使用尼古丁、地棘蛙素或其他杂环或双环作为活性中心和外周位点锚定基因座的组合文库产生。将检查环加成产物对相应结合模板的亲和力和特异性。然后将在由转染的受体亚基组合形成的烟碱受体上检查它们的选择性。研究的最后阶段将包括在啮齿动物中进行的研究，以确定疗效和毒性。

DESCRIPTION (provided by applicant): The proposed study has grown from a collaboration of the Sharpless & Taylor laboratories to employ novel "click chemistry" to develop selective ligands for nicotinic acetylcholine receptor using the acetylcholine binding protein as the reaction vessel. The precursors are anchored to two sites on the receptor and contain extended azide and acetylene moieties that react by cycloaddition to form a triazole. The precursor ligands have modest affinity for the protein and react extremely slowly in solution. On the protein surface, the apposition of the reactant groups and the amphiphilic environment of the protein forces a rapid reaction leading to formation of high affinity complex. Synthesis employs combinatorial reactants, and a single or predominant product is formed that is characterized by DIOS mass spectrometry. Interestingly, this "freezeframe" procedure catches the complex in unanticipated conformations such that the high affinity regioisomer forms from a low abundance conformation. Initially acetylcholine binding proteins from Lymnaea and Aplysia are employed as templates to form selective nicotinic agents. By mutagenesis the template is modified to incorporate binding determinants of the a7 receptor and then the heterologous receptor subtypes found in the brain. Since this is effectively a "freeze-frame" reaction, conformational flexibility of the binding templates will be analyzed by decay of fluorescence anisotropy in separate studies. Complexes also offer the potential for X-ray crystallographic studies. Partal agonists and antagonists will be generated from combinatorial libraries using nicotine, epibatidine or other heterocycles or bicyclic rings as the active center and peripheral site anchoring loci. Cycloaddition products will be examined for affinity and specificity for the respective binding templates. Their selectivities will then be examined on nicotinic receptors formed from transfected receptor subunit combinations. The final stages of investigation will include studies in rodents to determine efficacy and toxicity.