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Structural basis for action by diverse antidepressants on biogenic amine transporters.

ABSTRACT: The biogenic amine transporters (BATs) regulate endogenous neurotransmitter concentrations and are targets for a broad range of therapeutic agents including selective serotonin reuptake inhibitors (SSRIs), serotonin-noradrenaline reuptake inhibitors (SNRIs) and tricyclic antidepressants (TCAs). Because eukaryotic BATs are recalcitrant to crystallographic analysis, our understanding of the mechanism of these inhibitors and antidepressants is limited. LeuT is a bacterial homologue of BATs and has proven to be a valuable paradigm for understanding relationships between their structure and function. However, because only approximately 25% of the amino acid sequence of LeuT is in common with that of BATs, and as LeuT is a promiscuous amino acid transporter, it does not recapitulate the pharmacological properties of BATs. Indeed, SSRIs and TCAs bind in the extracellular vestibule of LeuT and act as non-competitive inhibitors of transport. By contrast, multiple studies demonstrate that both TCAs and SSRIs are competitive inhibitors for eukaryotic BATs and bind to the primary binding pocket. Here we engineered LeuT to harbour human BAT-like pharmacology by mutating key residues around the primary binding pocket. The final LeuBAT mutant binds the SSRI sertraline with a binding constant of 18?nM and displays high-affinity binding to a range of SSRIs, SNRIs and a TCA. We determined 12 crystal structures of LeuBAT in complex with four classes of antidepressants. The chemically diverse inhibitors have a remarkably similar mode of binding in which they straddle transmembrane helix (TM) 3, wedge between TM3/TM8 and TM1/TM6, and lock the transporter in a sodium- and chloride-bound outward-facing open conformation. Together, these studies define common and simple principles for the action of SSRIs, SNRIs and TCAs on BATs.

SUBMITTER: Wang H

PROVIDER: S-EPMC3904662 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Structural basis for action by diverse antidepressants on biogenic amine transporters.

Wang Hui H Goehring April A Wang Kevin H KH Penmatsa Aravind A Ressler Ryan R Gouaux Eric E

Nature 20131013 7474

The biogenic amine transporters (BATs) regulate endogenous neurotransmitter concentrations and are targets for a broad range of therapeutic agents including selective serotonin reuptake inhibitors (SSRIs), serotonin-noradrenaline reuptake inhibitors (SNRIs) and tricyclic antidepressants (TCAs). Because eukaryotic BATs are recalcitrant to crystallographic analysis, our understanding of the mechanism of these inhibitors and antidepressants is limited. LeuT is a bacterial homologue of BATs and has ...[more]

PMID: 24121440

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Project description:RationaleNew psychoactive substances (NPSs), including substituted cathinones and other stimulants, are synthesized, sold on the Internet, and ingested without knowledge of their pharmacological activity and/or toxicity. In vitro pharmacology plays a role in therapeutic drug development, drug-protein in silico interaction modeling, and drug scheduling.ObjectivesThe goal of this research was to determine mechanisms of action that may indicate NPS abuse liability.MethodsAffinities to displace the radioligand [125I]RTI-55 and potencies to inhibit [3H]neurotransmitter uptake for 22 cathinones, 6 benzofurans and another stimulant were characterized using human embryonic kidney cells stably expressing recombinant human transporters for dopamine, norepinephrine, or serotonin (hDAT, hNET, or hSERT, respectively). Selected compounds were tested for potencies and efficacies at inducing [3H]neurotransmitter release via the transporters. Computational modeling was conducted to explain plausible molecular interactions established by NPS and transporters.ResultsMost α-pyrrolidinophenones had high hDAT potencies and selectivities in uptake assays, with hDAT/hSERT uptake selectivity ratios of 83-360. Other substituted cathinones varied in their potencies and selectivities, with N-ethyl-hexedrone and N-ethyl-pentylone having highest hDAT potencies and N-propyl-pentedrone having highest hDAT selectivity. 4-Cl-ethcathinone and 3,4-methylenedioxy-N-propylcathinone had higher hSERT selectivity. Benzofurans generally had low hDAT selectivity, especially 1-(2,3-dihydrobenzofuran-5-yl)-N-methylpropan-2-amine, with 25-fold higher hSERT potency. Consistent with this selectivity, the benzofurans were releasers at hSERT. Modeling indicated key amino acids in the transporters' binding pockets that influence drug affinities.ConclusionsThe α-pyrrolidinophenones, with high hDAT selectivity, have high abuse potential. Lower hDAT selectivity among benzofurans suggests similarity to methylenedioxymethamphetamine, entactogens with lower stimulant activity.

Project description:Novel allosteric modulators of the dopamine transporter (DAT) have been identified. We have shown previously that SRI-9804 [N-(diphenylmethyl)-2-phenyl-4-quinazolinamine], SRI-20040 [N-(2,2-diphenylethyl)-2-phenyl-4-quinazolinamine], and SRI-20041 [N-(3,3-diphenylpropyl)-2-phenyl-4-quinazolinamine] partially inhibit [(125)I]RTI-55 ([(125)I]3β-(4'-iodophenyl)tropan-2β-carboxylic acid methyl ester) binding and [(3)H]dopamine ([(3)H]DA) uptake, slow the dissociation rate of [(125)I]RTI-55 from the DAT, and allosterically modulate d-amphetamine-induced, DAT-mediated DA release. We synthesized and evaluated the activity of >500 analogs of these ligands and report here on 36 selected compounds. Using synaptosomes prepared from rat caudate, we conducted [(3)H]DA uptake inhibition assays, DAT binding assays with [(3)H]WIN35428 ([(3)H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane), and DAT-mediated release assays with either [(3)H]MPP(+) ([(3)H]1-methyl-4-phenylpyridinium) or [(3)H]DA. We observed three groups of [(3)H]DA uptake inhibitors: 1) full-efficacy agents with a one-site fit, 2) full-efficacy agents with a two-site fit, and 3) partial-efficacy agents with a one-site fit-the focus of further studies. These agents partially inhibited DA, serotonin, and norepinephrine uptake, yet were much less potent at inhibiting [(3)H]WIN35428 binding to the DAT. For example, SRI-29574 [N-(2,2-diphenylethyl)-2-(imidazo[1,2-a]pyridin-6-yl)quinazolin-4-amine] partially inhibited DAT uptake, with an IC50 = 2.3 ± 0.4 nM, without affecting binding to the DAT. These agents did not alter DAT-mediated release of [(3)H]MPP(+) in the absence or presence of 100 nM d-amphetamine. SRI-29574 had no significant effect on the d-amphetamine EC50 or Emax value for DAT-mediated release of [(3)H]MPP(+). These studies demonstrate the existence of potent DAT ligands that partially block [(3)H]DA uptake, without affecting DAT binding or d-amphetamine-induced [(3)H]MPP(+) release. These compounds may prove to be useful probes of biogenic amine transporter function as well as novel therapeutics.

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Structural basis for action by diverse antidepressants on biogenic amine transporters.

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Structural basis for action by diverse antidepressants on biogenic amine transporters.

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