Interrogating the Molecular Basis for Substrate Recognition in Serotonin and Dopamine Transporters with High-Affinity Substrate-Based Bivalent Ligands

The transporters for the neurotransmitters serotonin and dopamine (SERT and DAT, respectively) are targets for drugs used in the treatment of mental disorders and widely used drugs of abuse. Studies of prokaryotic homologues have advanced our structural understanding of SERT and DAT, but it still remains enigmatic whether the human transporters contain one or two high-affinity substrate binding sites. We have designed and employed 24 bivalent ligands possessing a highly systematic combination of substrate moieties (serotonin and/or dopamine) and aliphatic or poly(ethylene glycol) spacers to reveal insight into substrate recognition in SERT and DAT. An optimized bivalent ligand comprising two serotonin moieties binds SERT with 3,800-fold increased affinity compared to that of serotonin, suggesting that the human transporters have two distinct substrate binding sites. We show that the bivalent ligands are inhibitors of SERT and an experimentally validated docking model suggests that the bivalent compounds b...