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.
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.
