CANNABINOID RECEPTOR AS THERAPEUTIC TARGET

大麻素受体作为治疗靶点

基本信息

批准号：
7722182
负责人：
Alexandros Makriyannis
金额：
$ 0.11万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-03-01 至 2009-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7722182
关键词：
Absence of pain sensation Active Sites Affinity Amides Amino Acids Analgesics Arachidonic Acids Bacteria Binding Sites Biochemical Cannabinoids Class Computer Retrieval of Information on Scientific Projects Database Development Escherichia coli Funding Goals Grant Institution Knowledge Label Laboratories Length Ligands Maps Measurement Molecular Peptides Performance Pharmaceutical Preparations Photoaffinity Labels Physiologic Intraocular Pressure Process Radioactive Reaction Research Research Personnel Resources Single-Stranded DNA Site Source Spectrometry, Mass, Electrospray Ionization Technical Expertise Techniques Therapeutic Effect United States National Institutes of Health Virus Vomiting analog cannabinoid receptor design homologous recombination interdisciplinary approach interest liquid chromatography mass spectrometry mutant novel programs receptor receptor structure function reconstitution tandem mass spectrometry therapeutic target

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The program project represents a comprehensive collaborative effort the ultimate goal of which is to develop novel drug analogs which produce their therapeutic effects by acting on the cannabinoid receptors. A central hypothesis of this program is that the recent availability of such receptor(s) offers the opportunity to rationally design analogs with a high degree of selectivity for inducing certain actions of cannabinoids including analgesia, inhibition of vomiting and reduction of intraocular pressure and immuno-modulation without their undesirable psychoactive effects. Similarly, there will be an opportunity for developing novel ligands which can successfully block the actions of cannabinoids. Such a process will require detailed knowledge of the molecular, biochemical and anatomical features of this receptor and its subtypes which are associated with cannabinoid activity. The receptor active sites could thus be used as template(s) for the successful design of these n ovel analogs. The group of ligands to be developed will encompass all four classes of molecules which are associated with cannabimimetic activity including classical cannabinoids (CCs), non-classical cannabinoids (NCCs), aminoalkylindoles (AAIs) and arachidonic acid amides (AAAs). (2) The expression isolation, purification and reconstitution of the cannabinoid receptor(s) and its mutants in viruses and bacteria. (3) Obtaining receptor active sites(s) with the help of high affinity covalent receptor ligands and by determining the amino acid residues with which the ligands reacted (using photoaffinity labeling). Determination of the specific sites on the receptor (length = 472 amino acids) that are photoaffinity labeled will be accomplished using high performance liquid chromatography and mass spectrometry. Several different mass spectrometric techniques are being used I. MALDI-TOF mass spectrometric mapping of proteolytic digests of the receptor prior to and after reaction. This measurement will provide definition of the binding site to within a particular proteolytic peptide. II LC-ESI mass spectrometry of proteolytic digests of the receptor. This measurement will provide similar information to that obtained in I above. However, since we will split the flow, fractions of interest will be collected for further study. MALDI-ITMS and ESI-triple quadrupole tandem mass spectrometry of peptides that have been identified to be modified by the ligands of interest (either through observed mass shifts in I & II above or by radioactive labeling). In this context, we have recently defined the binding site of E. coli RecA to single stranded DNA during the process of homologous recombination. In this case, photocrosslinking was used and the binding site determined by MALDI-ITMS/MS (at the single amino acid level) was independently confirmed by Edman sequencing. Obtaining intimate knowledge of the receptors' structure and function will require an interdisciplinary approach which will be accomplished through concerted collaborative efforts between several laboratories with a commitment for cannabinoid research and/or the high degree of technical expertise required for an effective approach to this problem. Strong collaborative interactions will be emphasized with the following major specific aims (1) the development of high affinity ligands for the receptor(s) which will be used for obtaining molecular information on the cannabinoid site(s) of action.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目及研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是对于中心来说，它不一定是研究者的机构。该计划项目代表了一项全面的合作努力，其最终目标是开发新型药物类似物，通过作用于大麻素受体来产生治疗效果。该计划的一个中心假设是，最近此类受体的可用性提供了合理设计具有高度选择性的类似物的机会，用于诱导大麻素的某些作用，包括镇痛、抑制呕吐和降低眼内压和免疫-调制而不会产生不良的精神作用。同样，将有机会开发能够成功阻断大麻素作用的新型配体。这样的过程将需要详细了解该受体及其与大麻素活性相关的亚型的分子、生化和解剖学特征。因此，受体活性位点可以用作成功设计这些新型类似物的模板。待开发的配体组将涵盖与大麻模拟活性相关的所有四类分子，包括经典大麻素（CC）、非经典大麻素（NCC）、氨基烷基吲哚（AAI）和花生四烯酸酰胺（AAA）。 (2) 大麻素受体及其突变体在病毒和细菌中的表达分离、纯化和重建。 (3) 借助高亲和力共价受体配体并通过确定与配体反应的氨基酸残基（使用光亲和标记）来获得受体活性位点。将使用高效液相色谱法和质谱法来确定受体上光亲和标记的特定位点（长度= 472 个氨基酸）。正在使用几种不同的质谱技术 I.反应之前和之后受体的蛋白水解消化物的 MALDI-TOF 质谱图谱。该测量将提供特定蛋白水解肽内结合位点的定义。 II 受体蛋白水解消化物的 LC-ESI 质谱分析。该测量将提供与上面 I 中获得的信息类似的信息。然而，由于我们将分割流量，因此将收集感兴趣的部分以供进一步研究。对已确定被感兴趣的配体修饰的肽进行 MALDI-ITMS 和 ESI-三重四极杆串联质谱分析（通过观察到上述 I 和 II 中的质量变化或通过放射性标记）。在此背景下，我们最近定义了同源重组过程中大肠杆菌 RecA 与单链 DNA 的结合位点。在这种情况下，使用了光交联，并通过 Edman 测序独立确认了 MALDI-ITMS/MS 确定的结合位点（在单个氨基酸水平）。获得对受体结构和功能的深入了解需要采用跨学科方法，这将通过几个实验室之间的协调一致努力来完成，这些实验室致力于大麻素研究和/或有效解决该问题所需的高度技术专业知识。将强调强有力的协作相互作用，并实现以下主要具体目标（1）开发受体的高亲和力配体，该配体将用于获得大麻素作用位点的分子信息。