Synthesis of specific bivalent probes that functionally interact with 5-HT4 receptor dimers

G-protein-coupled receptor dimerization directs the design of new drugs that specifically bind to receptor dimers. Here, we generated a targeted series of homobivalent ligands for serotonin 5-HT4 receptor (5-HT4R) dimers composed of two 5-HT4R-specific ML10302 units linked by a spacer. The design of spacers was assisted by molecular modeling using our previously described 5-HT4R dimer model. Their syntheses were based on Sonogashira-Linstrumelle coupling methods. All compounds retained high-affinity binding to 5-HT4R but lost the agonistic character of the monomeric ML10302 compound. Direct evidence for the functional interaction of both pharmacophores of bivalent ligands with the 5-HT4R was obtained using a bioluminescence resonance energy transfer (BRET) based assay that monitors conformational changes within 5-HT4R dimers. Whereas the monovalent ML10302 was inactive in this assay, several bivalent derivatives dose-dependently increased the BRET signal, indicating that both pharmacophores functionally interact with the 5-HT4R dimer. These bivalent ligands may serve as a new basis for the synthesis of potential drugs for 5-HT4R-associated disorders.