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

Abstract

J. Neurochem. (2010) 112, 1605–1618. 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 [3H] 2β‐carbomethoxy‐3β‐(4‐fluorophenyl)‐tropane (CFT) binding compared with DA itself; bivalent compounds with a 6‐carbon linker and heterologous combinations of DA‐, amphetamine‐ and β‐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.

Studies of the Biogenic Amine Transporters. 14. Identification of Low-Efficacy “Partial” Substrates for the Biogenic Amine Transporters

“…Releasers are BAT substrates, and the inward transport of a BAT substrate, together with Na ϩ and Cl Ϫ , leads to the countertransport of endogenous neurotransmitter out of the nerve terminal. Resolution of the structure of the bacterial LeuT Aa transporter (Yamashita et al, 2005;Shi et al, 2008) identified two substrate binding sites (S1 and S2), suggesting that the BATs might also possess two substrate binding sites, where the binding of a substrate to S2 triggers the release of substrate bound to S1 and Na ϩ into the intracellular compartment (Nyola et al, 2010;Schmitt et al, 2010). These findings emphasize the importance of conformational changes to transporter function (Ferrer and Javitch, 1998;Reith et al, 2001;Gether et al, 2006) Fig.…”

“…The increased concentration of internal cellular sodium at the transporter also facilitates reverse transport of dopamine (Goodwin et al, 2009;Pifl et al, 2009). Recent data suggest that the model developed on the basis of the LeuT Aa transporter may also apply to the biogenic amine transporters (Schmitt et al, 2010). In addition, other mechanisms, such as receptor phosphorylation and substrate-induced currents, can affect substrate-mediated neurotransmitter efflux (Sitte and Freissmuth, 2010).…”

Structure-Function Studies of the SLC17 Transporter Sialin Identify Crucial Residues and Substrate-induced Conformational Changes

“…The low affinity site could be a specific to a transition state, which would also explain the higher turnover rate for GlcUA transport. A two-binding site model with different binding affinities for different substrates is not unprecedented as it has been proposed for the bacterial LeuT transporter (45) and the human dopamine transporter (46). Interestingly, two substrate binding sites have been identified in VGLUT1 through docking studies and molecular dynamic stimulation (14).…”

Molecular determinants for selective recognition of antidepressants in the human serotonin and norepinephrine transporters

“…S3) (8, 10, 11). Several studies have shown that the equivalent site in monoamine transporters can accommodate inhibitors (17,18,25,26), and mutations of S2 residues in SERT and NET have been found to perturb inhibitor affinity (10,11). To explore whether talopram binds within the S2 site in NET, we introduced 32 single-point mutations into 16 different residues within or in close proximity of the S2 site (Fig.…”