Bivalent phenethylamines as novel dopamine transporter inhibitors: evidence for multiple substrate-binding sites in a single transporter.
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.
Schmitt
Kyle C
KC
Department of Pharmacology, New York University School of Medicine, New York City, New York 10016, USA.
Mamidyala
Sreeman
S
Biswas
Swati
S
Dutta
Aloke K
AK
Reith
Maarten E A
ME
D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D002461:Cell Line, Transformed; D003198:Computer Simulation; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020128:Inhibitory Concentration 50; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010627:Phenethylamines / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D011869:Radioligand Assay; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000378:metabolism
D050483:Dopamine Plasma Membrane Transport Proteins; D008024:Ligands; D010627:Phenethylamines; D014316:Tritium; D004298:Dopamine