CANNABINOID RECEPTOR AS THERAPEUTIC TARGET

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 该方案项目代表了一项全面的合作努力，其最终目标是开发通过作用于大麻素受体而产生治疗效果的新型药物类似物。该计划的一个中心假设是，这种受体(S)的最近上市提供了机会，合理地设计具有高度选择性的类似物，以诱导大麻类药物的某些作用，包括镇痛、抑制呕吐和降低眼压以及免疫调节，而不会产生不良的精神活性影响。同样，将有机会开发能够成功阻断大麻类药物作用的新型配体。这一过程需要详细了解这种受体及其亚型的分子、生化和解剖学特征，这些亚型与大麻素活性有关。因此，受体活性部位可以作为模板(S)，成功地设计出这些新的类似物。拟开发的配体将包括与大麻仿制活性有关的所有四类分子，包括经典大麻素(CCS)、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(S)及其突变体在病毒和细菌中的表达、分离、纯化和重组。(3)借助于高亲和力的共价受体配体并通过测定配体与之反应的氨基酸残基(光亲和标记法)获得受体活性部位(S)。光亲和标记的受体(长度=472个氨基酸)上的特定位置的确定将使用高效液相色谱和质谱仪来完成。几种不同的质谱学技术正在被使用。I.MALDI-TOF质谱图对反应前和反应后的受体的蛋白水解物进行绘制。这种测量将提供特定蛋白水解肽内结合部位的定义。II受体蛋白水解物的LC-ESI质谱分析。这一测量将提供与上文I中获得的信息类似的信息。然而，由于我们将分流，因此将收集感兴趣的部分以供进一步研究。MALDI-ITMS和ESI-三重四极串联质谱仪对已被感兴趣的配体(通过观察到的上述I和II的质量位移或通过放射性标记)修饰的多肽进行分析。在此背景下，我们最近确定了大肠杆菌RecA在同源重组过程中与单链DNA的结合位点。在这种情况下，使用了光交联，并通过Edman测序独立地确认了由MALDI-ITMS/MS确定的结合位置(在单一氨基酸水平上)。要深入了解受体的结构和功能，需要采取跨学科的方法，这将通过几个致力于大麻素研究和/或有效解决这一问题所需的高度技术专门知识的实验室之间的协调合作努力来实现。将强调强大的协作相互作用，主要具体目标如下：(1)开发受体(S)的高亲和力配体，用于获取大麻素作用部位(S)的分子信息。

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.