Bivalent ligands as molecular probes for CB1/OX1 receptor heterodimers

描述(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，这对它们的表达和活性至关重要。此外，受体异二聚体/低聚物具有不同于单一单体的药理和信号特性，这为调节受体功能提供了另一种机制，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独特的药理作用，首先必须对体内药理有更基本的了解。有趣的是，GPCR异构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试剂。二价配体，只要它们具有合适的单体受体亲和力和功能，由于第一个药效团结合后对第二个药效团的包裹体积较小，并形成热力学上更稳定的络合物，因此有望选择性地结合到杂二聚体和低聚物上的配体识别位点，亲和力大大增强。通过开发优先与CB1/OX1杂二聚体相互作用的小分子来加深我们对杂二聚体或低聚物的理解。虽然二价配体的一些物理性质，如高分子质量，引起了人们的关注，但在体内，二价配体已经被用作杂二聚体功能的分子探针。特别是，使用u-阿片(MOP)激动剂/d-阿片(DOP)拮抗剂的二价配体在全身给药后被证明是有效的镇痛剂，但不会产生传统单价阿片类止痛药的耐受性或依赖性。这为我们提议的方法的实用性提供了一个很好的例子。认识到受体异二聚体/低聚物与其各自的单体相比具有不同的药理和信号特性，这支持了CB1/OX1异二聚体作为潜在治疗干预靶点的重要性。这份R21提案概述了通过开发双价配体作为体内CB1/OX1受体异二聚体的探针来更好地了解受体异二聚所需的初始步骤。具体地说，我们建议合成和评价一系列针对大麻素/食欲素异二聚体的二价配体。亲和力和效价的优化将通过改变连接药效团的间隔物的长度来实现。所有化合物都将在竞争结合分析中使用单个表达CB1或OX1受体的转基因细胞株进行测试。具有合理亲和力的化合物将在双转染中筛选，即同时表达CB1和OX1受体的细胞。高亲和力配体的有效性将使用GTP-y-[35S]分析进行评估。最后，与亲和力最高的化合物的亲和力和有效性的证据将在大鼠脑膜制剂中得到证实。与公共健康相关：G蛋白偶联受体可以形成异二聚体/低聚物，并显示出与单独单体不同的药理和信号特性，这提供了另一种可能调节受体功能的机制。我们努力开发二价配体作为大麻素/食欲素异二聚体的分子探针，这将加深我们对受体异二聚化的理解，并最终可能导致基于异二聚体的新型药物治疗。

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.