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Binding site residues control inhibitor selectivity in the human norepinephrine transporter but not in the human dopamine transporter.

ABSTRACT: The transporters for norepinephrine and dopamine (NET and DAT, respectively) constitute the molecular targets for recreational drugs and therapeutics used in the treatment of psychiatric disorders. Despite a strikingly similar amino acid sequence and predicted topology between these transporters, some inhibitors display a high degree of selectivity between NET and DAT. Here, a systematic mutational analysis of non-conserved residues within the extracellular entry pathway and the high affinity binding site in NET and DAT was performed to examine their role for selective inhibitor recognition. Changing the six diverging residues in the central binding site of NET to the complementary residues in DAT transferred a DAT-like pharmacology to NET, showing that non-conserved binding site residues in NET are critical determinants for inhibitor selectivity. In contrast, changing the equivalent residues in the central site of DAT to the corresponding residues in NET had modest effects on the same inhibitors, suggesting that non-conserved binding site residues in DAT play a minor role for selective inhibitor recognition. Our data points towards distinct structural determinants governing inhibitor selectivity in NET and DAT, and provide important new insight into the molecular basis for NET/DAT selectivity of therapeutic and recreational drugs.

SUBMITTER: Andersen J

PROVIDER: S-EPMC4621520 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Binding site residues control inhibitor selectivity in the human norepinephrine transporter but not in the human dopamine transporter.

Andersen Jacob J Ringsted Kristoffer B KB Bang-Andersen Benny B Strømgaard Kristian K Kristensen Anders S AS Kristensen Anders S AS

Scientific reports 20151027

The transporters for norepinephrine and dopamine (NET and DAT, respectively) constitute the molecular targets for recreational drugs and therapeutics used in the treatment of psychiatric disorders. Despite a strikingly similar amino acid sequence and predicted topology between these transporters, some inhibitors display a high degree of selectivity between NET and DAT. Here, a systematic mutational analysis of non-conserved residues within the extracellular entry pathway and the high affinity bi ...[more]

PMID: 26503701

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Project description:Dopamine transporter (DAT) blockers like cocaine and many other abused and therapeutic drugs bind and stabilize an inactive form of the transporter inhibiting reuptake of extracellular dopamine (DA). The resulting increases in DA lead to the ability of these drugs to induce psychomotor alterations and addiction, but paradoxical findings in animal models indicate that not all DAT antagonists induce cocaine-like behavioral outcomes. How this occurs is not known, but one possibility is that uptake inhibitors may bind at multiple locations or in different poses to stabilize distinct conformational transporter states associated with differential neurochemical endpoints. Understanding the molecular mechanisms governing the pharmacological inhibition of DAT is therefore key for understanding the requisite interactions for behavioral modulation and addiction. Previously, we leveraged complementary computational docking, mutagenesis, peptide mapping, and substituted cysteine accessibility strategies to identify the specific adduction site and binding pose for the crosslinkable, photoactive cocaine analog, RTI 82, which contains a photoactive azide attached at the 2β position of the tropane pharmacophore. Here, we utilize similar methodology with a different cocaine analog N-[4-(4-azido-3-I-iodophenyl)-butyl]-2-carbomethoxy-3-(4-chlorophenyl)tropane, MFZ 2-24, where the photoactive azide is attached to the tropane nitrogen. In contrast to RTI 82, which crosslinked into residue Phe319 of transmembrane domain (TM) 6, our findings show that MFZ 2-24 adducts to Leu80 in TM1 with modeling and biochemical data indicating that MFZ 2-24, like RTI 82, occupies the central S1 binding pocket with the (+)-charged tropane ring nitrogen coordinating with the (-)-charged carboxyl side chain of Asp79. The superimposition of the tropane ring in the three-dimensional binding poses of these two distinct ligands provides strong experimental evidence for cocaine binding to DAT in the S1 site and the importance of the tropane moiety in competitive mechanisms of DA uptake inhibition. These findings set a structure-function baseline for comparison of typical and atypical DAT inhibitors and how their interactions with DAT could lead to the loss of cocaine-like behaviors.

Project description:PurposeA new PET radiotracer 18F-AF78 showing great potential for clinical application has been reported recently. It belongs to a new generation of phenethylguanidine-based norepinephrine transporter (NET)-targeting radiotracers. Although many efforts have been made to develop NET inhibitors as antidepressants, systemic investigations of the structure-activity relationships (SARs) of NET-targeting radiotracers have rarely been performed.MethodsWithout changing the phenethylguanidine pharmacophore and 3-fluoropropyl moiety that is crucial for easy labeling, six new analogs of 18F-AF78 with different meta-substituents on the benzene-ring were synthesized and evaluated in a competitive cellular uptake assay and in in vivo animal experiments in rats. Computational modeling of these tracers was established to quantitatively rationalize the interaction between the radiotracers and NET.ResultsUsing non-radiolabeled reference compounds, a competitive cellular uptake assay showed a decrease in NET-transporting affinity from meta-fluorine to iodine (0.42 and 6.51 µM, respectively), with meta-OH being the least active (22.67 µM). Furthermore, in vivo animal studies with radioisotopes showed that heart-to-blood ratios agreed with the cellular experiments, with AF78(F) exhibiting the highest cardiac uptake. This result correlates positively with the electronegativity rather than the atomic radius of the meta-substituent. Computational modeling studies revealed a crucial influence of halogen substituents on the radiotracer-NET interaction, whereby a T-shaped π-π stacking interaction between the benzene-ring of the tracer and the amino acid residues surrounding the NET binding site made major contributions to the different affinities, in accordance with the pharmacological data.ConclusionThe SARs were characterized by in vitro and in vivo evaluation, and computational modeling quantitatively rationalized the interaction between radiotracers and the NET binding site. These findings pave the way for further evaluation in different species and underline the potential of AF78(F) for clinical application, e.g., cardiac innervation imaging or molecular imaging of neuroendocrine tumors.

Dataset Information

Binding site residues control inhibitor selectivity in the human norepinephrine transporter but not in the human dopamine transporter.

Publications

Binding site residues control inhibitor selectivity in the human norepinephrine transporter but not in the human dopamine transporter.

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