Bivalent ligands as molecular probes for CB1/OX1 receptor heterodimers

描述（由申请人提供）：人们普遍认为，许多G蛋白偶联受体，包括大麻素和食欲素受体，形成二聚体或寡聚体，这对其表达和活性至关重要。此外，受体异源二聚体/寡聚体显示出与单个单体不同的药理学和信号传导特性，这提供了可以调节受体功能的另一种机制，从而为寻找新的药物靶点开辟了一个全新的领域。为了充分利用异二聚体的独特药理学，必须首先实现对体内药理学的更基本的理解。有趣的是，GPCR异聚体在体内的重要性仍有待开发和认识，这主要是由于缺乏选择性的药理学工具和免疫学试剂。如果二价配体具有合适的单体受体亲和力和功能，则预期它们以大大增强的亲和力选择性地结合异源二聚体和寡聚体上的配体识别位点，这是由于在结合第一药效团之后第二药效团的容纳体积小并且形成在药理学上更稳定的复合物。进一步理解异源二聚体或寡聚体的一种方法是通过开发优先与CB 1/OX 1异源二聚体相互作用的小分子。虽然二价配体的一些物理性质，如高分子量，是值得关注的，二价配体已经被用作异源二聚体功能在体内的分子探针。特别地，使用u-阿片样物质（MOP）激动剂/d-阿片样物质（DOP）拮抗剂二价配体的工作显示在全身施用后是有效的镇痛剂，但不产生传统单价阿片样物质镇痛剂所见的耐受性或依赖性。这为我们所提出的方法的实用性提供了一个很好的例子。认识到受体异源二聚体/寡聚体与其单个单体相比显示出不同的药理学和信号传导特性，支持CB 1/OX 1异源二聚体作为治疗干预的潜在靶点的重要性。该R21建议概述了通过开发二价配体作为体内CB 1/OX 1受体异二聚体的探针来更好地理解受体异二聚化所需的初始步骤。特别是，我们建议合成和评估一系列的二价配体靶向大麻素/食欲素异源二聚体。亲和力和效力的优化将通过改变连接药效团的间隔区的长度来实现。所有化合物将在竞争性结合试验中使用表达CB 1或OX 1受体的单转染细胞系进行检测。然后，在双方面，即表达CB 1和OX 1受体的细胞中筛选具有合理亲和力的化合物。将使用GTP-γ- [35 S]试验评估高亲和力配体的有效性。最后，将在大鼠脑膜制备物中证实具有最高亲和力的化合物的亲和力和功效。公共卫生相关性：G蛋白偶联受体可以形成异源二聚体/寡聚体，并显示出与单个单体不同的药理学和信号传导特性，这提供了另一种可以调节受体功能的机制。我们的努力在开发的二价配体作为大麻素/食欲素异源二聚体的分子探针，将进一步我们的受体异源二聚化的理解，并可能最终导致新的异源二聚体为基础的药物治疗。

DESCRIPTION (provided by applicant): It is widely accepted that many G-protein coupled receptors, including the cannabinoid and orexin receptors, form dimers or oligomers, which are crucial for their expression and activity. Moreover, receptor hetero-dimer/oligomers display distinct pharmacological and signaling properties than the individual monomers, which present another mechanism that could modulate receptor function and thus opens a complete new field to search for novel drug targets. In order to take full advantage of the unique pharmacology of heterodimers, a more basic understanding of the in vivo pharmacology must first be achieved. Interestingly, the importance of GPCR heteromers in vivo remains to be exploited and appreciated, largely due to a lack of selective pharmacological tools and immunological reagents. Bivalent ligands, provided they have suitable monomeric receptor affinities and function, are expected to selectively bind with greatly enhanced affinity to ligand recognition sites on heterodimers and oligomers, due to the small containment volume for the second pharmacophore after the binding of the first one and the formation of thermodynamically more stable complex. One means of furthering our understanding of heterodimer or oligomers is through the development of small molecules which preferentially interact with CB1/OX1 heterodimers. Although some physical properties of bivalent ligands, such as high molecular weight, are of concern, bivalent ligands have already been useful as molecular probes of heterodimer function in vivo. In particular, the work with u-opioid (MOP) agonist/d-opioid (DOP) antagonist bivalent ligands were shown to be potent analgesics after systemic administration, but did not produce the tolerance or dependence seen with traditional monovalent opioid analgesics. This provides an excellent example of the utility of our proposed approach. A recognition that receptor hetero-dimer/oligomers display distinct pharmacological and signaling properties when compared to their individual monomers supports the significance of CB1/OX1 heterodimers as potential targets for therapeutic intervention. This R21 proposal outlines the initial steps necessary to better understand receptor heterodimerization by developing bivalent ligands as probes for CB1/OX1 receptor heterodimers in vivo. In particular, we propose to synthesize and evaluation a series of bivalent ligands targeting cannabinoid/orexin heterodimers. Optimization of affinity and potency will be accomplished by varying the length of the spacer that links the pharmacophores. All the compounds will be tested in competitive binding assays using single transfected cell lines, expressing either CB1 or OX1 receptors. Compounds with reasonably affinity will then be screened in dual transfects, that is, cells expressing both CB1 and OX1 receptors. Efficacy of high affinity ligands will be assessed using GTP-y- [35S] assays. Finally proof of affinity and efficacy with the compounds of highest affinity will be confirmed in rat brain membrane preparations. PUBLIC HEALTH RELEVANCE: G protein-coupled receptors can form hetero-dimer/oligomers and display distinct pharmacological and signaling properties than the individual monomers, which present another mechanism that could modulate receptor function. Our efforts in development of bivalent ligands as molecular probes for cannabinoid/orexin heterodimers will further our understanding of receptor heterodimerization and may ultimately lead to novel heterodimer based pharmacotherapies.