Bivalent phenethylamines as novel dopamine transporter inhibitors: evidence for multiple substrate-binding sites in a single transporter

Abstract

Bivalent ligands--compounds incorporating two receptor-interacting moieties linked by a flexible chain--often exhibit profoundly enhanced binding affinity compared with their monovalent components, implying concurrent binding to multiple sites on the target protein. It is generally assumed that neurotransmitter sodium symporter (NSS) proteins, such as the dopamine transporter (DAT), contain a single domain responsible for recognition of substrate molecules. In this report, we show that molecules possessing two substrate-like phenylalkylamine moieties linked by a progressively longer aliphatic spacer act as progressively more potent DAT inhibitors (rather than substrates). One compound bearing two dopamine (DA)-like pharmacophoric 'heads' separated by an 8-carbon linker achieved an 82-fold gain in inhibition of [(3)H] 2beta-carbomethoxy-3beta-(4-fluorophenyl)-tropane (CFT) binding compared with DA itself; bivalent compounds with a 6-carbon linker and heterologous combinations of DA-, amphetamine- and beta-phenethylamine-like heads all resulted in considerable and comparable gains in DAT affinity. A series of short-chain bivalent-like compounds with a single N-linkage was also identified, the most potent of which displayed a 74-fold gain in binding affinity. Computational modelling of the DAT protein and docking of the two most potent bivalent (-like) ligands suggested simultaneous occupancy of two discrete substrate-binding domains. Assays with the DAT mutants W84L and D313N--previously employed by our laboratory to probe conformation-specific binding of different structural classes of DAT inhibitors--indicated a bias of the bivalent ligands for inward-facing transporters. Our results strongly indicate the existence of multiple DAT substrate-interaction sites, implying that it is possible to design novel types of DAT inhibitors based upon the 'multivalent ligand' strategy.

Figure 1

Chemical structures of the substrate-like DAT ligands investigated in the present study. (A) Classical monovalent phenethylamine substrates and monovalent analogues bearing an N-alkyl substituent. (B) Bivalent spacer-linked diphenethylamines, bivalent ligands possessing two independent substrate moieties tethered by a variable length N,N’-polymethylene chain. (C) Bivalent-like monoamines, short-chain bivalent compounds bearing two aromatic substrate-like “heads” linked at a shared nitrogen atom.

Figure 2

Representative energy-minimized poses of model DAT/ligand complex following docking of dopamine (A, B) and selected bivalent (-like) ligands (C, D). Selected DAT residues from respective ligand-binding pockets are labeled and rendered as sticks (transmembrane helices lacking interacting residues are hidden for visual clarity). The ligand molecules are highlighted with yellow-colored carbon atoms. For each pose, the inset panel depicts all residues located within 4.5 Å of the ligand; the strongest DAT/ligand interactions are indicated with dotted lines and a symbol depicting the chemistry of the interaction. (A, B) DA docked at both the S1 and S2 sites. At each site, the protonated amine of DA is anchored by a combination of hydrogen bonding and cation-π interactions and the ring hydroxyl moieties participate in hydrogen bonding. (C) Docking configuration of the most potent spacer-linked diphenethylamine ligand, D-362. In this representative low-energy pose, the catecholamine pharmacophores are oriented such that they interact with the transporter at both the S1 and S2 sites simultaneously. A number of major molecular interactions are similar to those found for DA (Asp79, Tyr156 and Ser149 at the S1 site and Lys92 and Asp313 at the S2 site). Cooperative binding to these two sites may underlie the dramatically higher binding affinity (82-fold) of D-362 compared to monovalent DA. (D) Docking configuration of D-278 (hydroxydobutamine), the most potent of the bivalent-like monoamine ligand series.

Figure 2

Representative energy-minimized poses of model DAT/ligand complex following docking of dopamine (A, B) and selected bivalent (-like) ligands (C, D). Selected DAT residues from respective ligand-binding pockets are labeled and rendered as sticks (transmembrane helices lacking interacting residues are hidden for visual clarity). The ligand molecules are highlighted with yellow-colored carbon atoms. For each pose, the inset panel depicts all residues located within 4.5 Å of the ligand; the strongest DAT/ligand interactions are indicated with dotted lines and a symbol depicting the chemistry of the interaction. (A, B) DA docked at both the S1 and S2 sites. At each site, the protonated amine of DA is anchored by a combination of hydrogen bonding and cation-π interactions and the ring hydroxyl moieties participate in hydrogen bonding. (C) Docking configuration of the most potent spacer-linked diphenethylamine ligand, D-362. In this representative low-energy pose, the catecholamine pharmacophores are oriented such that they interact with the transporter at both the S1 and S2 sites simultaneously. A number of major molecular interactions are similar to those found for DA (Asp79, Tyr156 and Ser149 at the S1 site and Lys92 and Asp313 at the S2 site). Cooperative binding to these two sites may underlie the dramatically higher binding affinity (82-fold) of D-362 compared to monovalent DA. (D) Docking configuration of D-278 (hydroxydobutamine), the most potent of the bivalent-like monoamine ligand series.