Cannabinergic Ligands and Active Sites- Project 1

其他项目信息-摘要/摘要 该项目作为配体/药物开发的一个全面的合作努力，研究 CB 1和CB 2的结构和功能特性，这两种已知的大麻素受体。期间 在当前的资金资助期内，我们已经开发出高亲和力的共价和非共价CB 1/CB 2配体， 使我们能够探测结构上不同种类的配体相互作用和激活的方式， 使两种受体都失活。为了研究每个受体上单个配体的“足迹”，我们 开发了一种配体辅助蛋白质结构（LAPS）方法，该方法包括使用共价键， 能够不可逆地连接到受体活性位点的配体， 受体突变体和详细的LC/MS/MS方法。到目前为止，我们的研究结果提供了初步证据， 大麻能配体的种类通过不同的途径相互作用并激活/失活CB 1和CB 2受体 结合基序我们假设，我们已经确定的单个结合基序可能与 具有不同的可识别的信号传导途径，导致不同的配体依赖性药理学 特征（功能选择性/偏向激动作用）。该项目将为发展 新的功能选择性CB 1和CB 2配体，包括两种互补组分。这个项目 包括两个组成部分：1）第一个是配体开发方面，其提出设计和 CB 1和CB 2新型高亲和力光活化亲电共价配体的合成 其中反应性基团被策略性地引入到一个（单官能）或两个（双官能）位点中， 母体配体此外，该组件包括高亲和性可逆寡核苷酸的设计和合成。 功能上不同的配体和一个领先的优化方面的设计和合成的功能选择性 CB 2激动剂; 2）在第二组分中，策略性地设计CB 1的单位点和多位点突变体， 在HEK 293细胞中表达的CB 2（人和啮齿动物）将用于测试单个成功的配体， 使用cAMP测定作为初始筛选（在 核心2）。结合位点表征将基于a）受体的互补使用 突变体;和B）LC/MS/MS表征与配体结合的特定氨基酸残基 使用我们的LAPS方法。在该项目主持下产生的结果和配体将 作为使用具有改善的成瘾性和疼痛的新药物治疗的基础， 药理学特征。 本计画一将设计并合成新型配体，以探讨其与CB 1及CB 2的相互作用 大麻素受体研究结果可用于开发改善疼痛和成瘾的药物 紊乱

OTHER PROJECT INFORMATION - SUMMARY/ABSTRACT This project serves as the ligand/drug development component of a comprehensive collaborative effort to study the structural and functional properties of CB1 and CB2, the two known cannabinoid receptors. During the current funding period we have developed high-affinity covalent and non-covalent CB1/CB2 ligands that have allowed us to probe the manner in which structurally diverse classes of ligands interact and activate or deactivate each of the two receptors. To study the "foot-printing" of individual ligands on each receptor, we developed a Ligand Assisted Protein Structure (LAPS) approach which includes the use of covalent ligands capable of irreversibly attaching to the receptor active site(s) in conjunction with judiciously designed receptor mutants and detailed LC/MS/MS methods. To date, our results provide initial evidence that different classes of cannabinergic ligands interact and activate/deactivate the CB1 and CB2 receptors through distinct binding motifs. We have hypothesized that the individual binding motifs we have identified may be associated with distinct identifiable signaling pathway(s) leading to different ligand-dependent pharmacological profiles (functional selectivity/biased agonism). The project will provide the basis for the development of novel functionally selective CB1 and CB2 ligands and includes two complementary components. This project encompasses two components: 1) The first is a ligand development aspect which proposes the design and synthesis of novel high-affinity photoactivatable and electrophilic covalent ligands for CB1 and CB2 in which the reactive groups are strategically introduced in one (monofunctional) or two (bifunctional) sites within the parent ligand. Additionally, this component includes the design and synthesis of high-affinity reversible functionally distinct ligands and a lead optimization aspect for the design and synthesis of functionally selective CB2 agonists; 2) In the second component strategically designed single and multiple site mutants of CB1 and CB2 (human and rodent) expressed in HEK293 cells will be used to test individual successful ligands for covalent attachment and for their functional properties using the cAMP assay as an initial screen (under the auspices of Core 2). Binding site characterization will be based on the complementary use of a) receptor mutants; and b) LC/MS/MS characterization of the specific amino acid residue(s) to which the ligand is attached using our LAPS methodology. The results and ligands produced under the auspices of this project will serve as a basis for the treatment of addiction and pain using novel medications with improved pharmacological profiles. This Project 1 will design and synthesize novel ligands to probe their interactions with the CB1 and CB2 cannabinoid receptors. The results can be used to develop improved medications for pain and addictive disorders.