Nicotinic Receptor Template-guided Drug Design

描述(申请人提供)：这项拟议的研究源于夏普莱斯和泰勒实验室的合作，利用新颖的“点击化学”，利用乙酰胆碱结合蛋白作为反应容器，开发烟碱型乙酰胆碱受体的选择性配体。前体被固定在受体上的两个位置上，并含有延伸的叠氮和乙炔部分，它们通过环加成反应形成三唑。前体配体与蛋白质有适度的亲和力，在溶液中反应极慢。在蛋白质表面，反应物基团的结合和蛋白质的两亲性环境迫使蛋白质发生快速反应，形成高亲和力的复合体。合成采用组合反应物，形成单一的或主要的产物，用Dios质谱仪进行表征。有趣的是，这种“冷冻”过程捕捉到了意想不到的构象中的复合体，使得高亲和力的区域异构体从低丰度构象形成。最初，来自Lymneea和Aplysia的乙酰胆碱结合蛋白被用作模板来形成选择性烟碱剂。通过突变，模板被修改为包括A7受体的结合决定因素，然后是在大脑中发现的异源受体亚型。由于这实际上是一种“定格”反应，结合模板的构象柔性将在单独的研究中通过荧光各向异性的衰减来分析。络合物还为X射线结晶学研究提供了可能性。部分激动剂和拮抗剂将从组合文库中产生，这些文库以尼古丁、表巴替丁或其他杂环或双环为活性中心和外围锚定位点。将检查环加成产物对各自结合模板的亲和力和特异性。它们的选择性随后将在由转基因受体亚单位组合形成的尼古丁受体上进行检测。调查的最后阶段将包括在啮齿动物身上进行研究，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.