Cannabinergic Ligands and Active Sites- Project 1

其他项目信息--摘要/摘要 该项目是一个全面合作研究的配基/药物开发部分 已知的两种大麻素受体CB1和CB2的结构和功能特性。在.期间 在目前的资金期限内，我们开发了高亲和力的共价和非共价CB1/CB2配体 使我们能够探索结构上不同类别的配体相互作用和激活或 停用这两个感受器。为了研究每个受体上个别配体的“足迹”，我们 开发了一种配体辅助蛋白质结构(LAPS)方法，其中包括使用共价 能够与受体活性部位不可逆地结合的配体(S)与精心设计的 受体突变体和详细的LC/MS/MS方法。到目前为止，我们的结果提供了不同的初步证据 不同类别的大麻能配体通过不同的方式相互作用并激活/去激活CB1和CB2受体 具有约束力的主题。我们假设我们已经确定的单个结合基序可能与 不同的可识别信号通路(S)导致不同的配体依赖的药理作用 概况(功能选择性/偏向激动性)。该项目将为开发 新型功能选择性CB1和CB2配体，包括两个互补成分。这个项目 包括两个部分：1)第一个是配体开发方面，它提出了设计和 新型高亲和力CB1和CB2共价配体的合成 其中反应性基团被策略性地引入其中的一个(单官能团)或两个(双官能团)位 母体配体。此外，该成分还包括高亲和力可逆性的设计和合成 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 CB2激动剂；2)在第二组分中，策略性地设计了CB1和CB1的单点和多点突变 在HEK293细胞中表达的CB2(人和啮齿动物)将用于测试单个成功的配体 以cAMP分析为初始筛选的共价连接及其功能性质(见 核心2的赞助)。结合部位的描述将基于a)受体的互补使用 突变体；以及b)配体所与的特定氨基酸残基(S)的LC/MS/MS表征 使用我们的LAPS方法附加。在该项目的赞助下产生的成果和配体将 作为使用新型药物治疗成瘾和疼痛的基础 药理特征。 本项目1将设计和合成新的配体，以探索它们与CB1和CB2的相互作用 大麻素受体。这一结果可用于开发治疗疼痛和上瘾的改良药物 精神错乱。

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.