Photolabeling of alcohol binding sites L1:

描述（由申请方提供）：我们的总体假设是，乙醇诱导的细胞粘附抑制（发育中神经系统损伤的一个可能原因）是由L1细胞粘附分子上的乙醇结合位点介导的。不同的醇显示出显著的结构特异性，乙醇抑制细胞粘附（激动剂作用）。其它醇（例如，1-辛醇）非竞争性拮抗乙醇对L1介导的细胞粘附和对小鼠全胚胎培养物发育的影响。我们假设这些位点位于L1，因为L1突变儿童的大脑与胎儿酒精谱系障碍（FASD）儿童的大脑相似。该提议的新颖方面是使用最近开发的光亲和醇类似物3-azibutanol（激动剂）和3-azioctanol（拮抗剂）分别光标记激动剂和拮抗剂结合位点。特定目的1通过用3-azibutanol光标记L1粘附分子上的醇激动剂位点来检验这一假设，3-azibutanol对细胞粘附的作用类似于乙醇。将用[3 H]3-azibutanol光标记的纯化L1消化，并通过HPLC分离光掺入醇的片段。光掺入的化学计量将通过质谱法测量。此外，靶片段的消化将产生适于通过质谱法测序的片段。为了评估药理学相关性，将测定每个光掺入位点的表观解离常数和药理学，并与从平行细胞粘附实验中获得的表观解离常数和药理学进行比较。具体目标2类似地测试假设，即使用光亲和标记3-azioctanol在L1上存在单独的醇拮抗剂位点，其在微摩尔浓度下抑制乙醇诱导的细胞粘附抑制。我们期望在LI上每个具有良好特征的位点的结合口袋中定义至少一个氨基酸。激动剂和拮抗剂位点的鉴定将分别有助于理解FASD的分子基础，并加速阻断乙醇对发育中的神经系统的毒性作用的药物的开发。

DESCRIPTION (provided by applicant): Our overall hypothesis is that ethanol-induced inhibition of cell adhesion, one possible cause of damage in the developing neural system, is mediated by alcohol binding sites on the L1 cell adhesion molecule. Different alcohols show remarkable structural specificity for alcohol inhibition of cell adhesion (agonist action). Other alcohols (e.g., 1-octanol) noncompetitively antagonize the effects of ethanol on Ll-mediated cell adhesion and on the development of mouse whole embryo cultures. We hypothesize that these sites are on L1 because the brains of children with L1 mutations resemble those of children with fetal alcohol spectrum disorder (FASD). The novel aspect of this proposal is the use of recently developed photoaffinity alcohol analogs, 3-azibutanol (an agonist) and 3-azioctanol (an antagonist) to photolabel the agonist and antagonist binding sites respectively. Specific Aim 1 tests the hypothesis that there are alcohol agonist sites on the L1 adhesion molecule by photolabeling them with 3-azibutanol, which acts similarly to ethanol on cell adhesion. Purified L1 photolabeled with [3H]3-azibutanol will be digested and fragments photoincorporating alcohol separated by HPLC. The stoichiometry of photoincorporation will be measured by mass spectrometry. Further, digestion of target fragments will produce fragments suitable for sequencing by mass spectrometry. To assess pharmacological relevance, the apparent dissociation constant and pharmacology of each photoincorporation site will be determined and compared to those obtained from parallel cell adhesion experiments. Specific Aim 2 similarly tests the hypothesis that there are separate alcohol antagonist sites on L1 using the photoaffinity label, 3-azioctanol, which inhibits ethanol-induced inhibition of cell adhesion at micromolar concentrations. We expect to define at least one amino acid in the binding pocket of each pharmacologically well-characterized site on LI. Identification of the agonist and of the antagonist site will, respectively, aid in understanding the molecular basis for FASD and accelerate the development of drugs that block the toxic effects of ethanol on the developing nervous system.