CANNABINOID RECEPTOR AS THERAPEUTIC TARGET

大麻素受体作为治疗靶点

• 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
• 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

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 该计划项目代表了一项全面的协作努力，最终的目标是开发新型的药物类似物，从而通过对大麻素受体作用来产生其治疗作用。 该程序的一个核心假设是，最近的这种受体的可用性为具有高度选择性的合理设计类似物提供了诱导大麻素的某些作用，包括镇痛，抑制呕吐和减少眼内压力和免疫调节而没有其不良的精神活性影响。 同样，将有机会开发新型配体，这些配体可以成功阻止大麻素的作用。 这样的过程将需要详细了解该受体的分子，生化和解剖特征及其与大麻素活性相关的亚型。 因此，可以将受体活动位点用作成功设计这些N OVEL类似物的模板。 要开发的配体组将涵盖与大麻活性相关的所有四类分子，包括经典大麻素（CCS），非经典大麻素（NCCS），氨基烷基糖（AAAIS）（AAIS）和Arachidonic Acid Acid Acid Aciids（AAAAS）。 （2）在病毒和细菌中，大麻素受体及其突变体的表达分离，纯化和重建。 （3）通过高亲和共价受体配体获得受体活性位点（S），并通过确定配体反应的氨基酸残基（使用Photaffication标记）。将使用高性能液相色谱和质谱法来实现受体（长度= 472个氨基酸）上特定位点（长度= 472个氨基酸）的测定。 使用了几种不同的质谱技术。在反应前后，受体蛋白水解消化的MALDI-TOF质谱图映射。 该测量将为结合位点提供特定蛋白水解肽内的定义。 II LC-ESI质谱受体的蛋白水解消化。 该测量将提供与上述I获得的类似信息。 但是，由于我们将分裂流量，因此将收集感兴趣的部分以进行进一步研究。 MALDI-ITMS和ESI-Triple四极杆串联肽的质谱法已被鉴定为通过感兴趣的配体（通过上面的I＆II中观察到的质量转移或放射性标记）进行了修饰。 在这种情况下，我们最近在同源重组过程中定义了大肠杆菌RECA的结合位点与单链DNA的结合位点。 在这种情况下，使用光叠链链接，并通过Edman测序独立确认由MALDI-ITMS/MS（在单个氨基酸水平上）确定的结合位点。 了解对受体的结构和功能的亲密知识将需要一种跨学科方法，这将通过几个实验室之间的一致合作努力来完成，并承诺大麻素研究和/或有效解决此问题所需的高度技术专长。 强烈的协作相互作用将以以下主要特定目的（1）开发受体的高亲和力配体的发展，该配体将用于在作用的大麻素位点获得分子信息。