Structural basis for recognition of diverse antidepressants by the human serotonin transporter

Abstract

Selective serotonin reuptake inhibitors are clinically prescribed antidepressants that act by increasing the local concentration of neurotransmitter at synapses and in extracellular spaces via blockade of the serotonin transporter. Here we report x-ray structures of engineered thermostable variants of the human serotonin transporter bound to the antidepressants sertraline, fluvoxamine, and paroxetine. The drugs prevent serotonin binding by occupying the central substrate binding site and stabilizing the transporter in an outward-open conformation.These structures explain how residues within the central site orchestrate binding of chemically diverse inhibitors and mediate transporter-drug selectivity. MAIN TEXTIn the central nervous system the serotonin transporter (SERT) modulates serotoninergic signaling by carrying out the uptake of serotonin (5-HT) from the synaptic space into presynaptic . An extracellular vestibule in the outward-facing conformation 7,9,10 forms a secondary, allosteric site which, when occupied by ligands, can result . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/204859 doi: bioRxiv preprint first posted online Oct. 17, 2017; page 3 of 15 in modulation of transporter activity by altering the kinetics of ligand dissociation from the central site [11][12][13] . Addictive substances such as cocaine and amphetamine bind to monoamine transporters and can either inhibit NT transport or promote NT efflux, respectively 14,15 . Selective serotonin reuptake inhibitors (SSRIs) are a class of small molecules that are highly selective for SERT over DAT and NET and that inhibit 5-HT reuptake with nanomolar potency Fig. 1), and as a consequence these compounds bind with a range of affinities to SERT. Sertraline contains a tetrahydronaphthalene ring system linked to a secondary amine together with a meta and para substituted dichlorophenyl group. Fluvoxamine, by contrast, consists of a 2-aminoethyloxime moiety attached to a methyoxybutyl group, and a phenyl group containing a trifluoronated methyl at the para positon. Fluoxetine also contains a trifluoronated aromatic group but is instead coupled to a phenylpropylamine moiety. Paroxetine and citalopram also differ substantially in the structures of their aromatic, amine, and halogenated substitutents.Recently, we solved x-ray structures of a thermostabilized, transport-inactive construct of human SERT, deemed the ts3 construct, in complex with the SSRIs paroxetine and citalopram 8 .We employed the thermostabilized variant of SERT in order to facilitate purification and crystallization 19,20 . One of the thermostabilizing mutations, however, involves a residue (Thr439Ser) that is directly positioned within the central binding site, in close proximity to the bound SSRIs. Moreover, recent computational modeling of a wild-type SERT-paroxetine complex, in which the structure of the Drosophila DAT was used as a ...

Targeting Solute Carrier Transporters through Functional Mapping

“…Such mechanisms have been described for the elevator and gated-pore transport processes [14][15][16][17] and have been suggested for the rocker switch [18]. More specifically, some allosteric inhibitors have been identified for the glutamate transporter EAAT1 (SLC1A3) [14] and the SERT (SLC6A4) [15,17]. Interestingly, in both cases, the allosteric inhibitors bind at the interface between the scaffold and mobile domains, hence preventing the conformational change.…”

“…Of particular interest for medicinal chemistry are allosteric mechanisms, by which the binding of a molecule at a site distant from the ligand (orthosteric) binding site, modulates the function of the transporter. Such mechanisms have been described for the elevator and gated-pore transport processes [14][15][16][17] and have been suggested for the rocker switch [18]. More specifically, some allosteric inhibitors have been identified for the glutamate transporter EAAT1 (SLC1A3) [14] and the SERT (SLC6A4) [15,17].…”

The mechanism of a high-affinity allosteric inhibitor of the serotonin transporter

“…Based on computational modeling and experimental binding studies, we previously located the lowaffinity second binding site for S-CIT and clomipramine to the extracellular vestibule (EV), the entry pathway toward the S1 site 21 . Interestingly, in one of the recent hSERT crystal structures (PDB 5I73), two S-CIT molecules are bound to the protein: one in the S1 site (denoted as S1:S-CIT) and another bound to a binding site in the EV-the S2 site (denoted as S2:S-CIT)~13 Å above the S1 site 11,23 . This is consistent with previous findings that the EV of LeuT harbors a S2 site capable of binding ligands [24][25][26] .…”

Monoamine transporters: structure, intrinsic dynamics and allosteric regulation

“…MATs belong to the solute carrier 6 (SLC6) family of human transporters, which, in turn, is a subfamily of the broader neurotransmitter:sodium symporters (NSSs) that comprise transporters from prokaryotic to human. MATs comprise three main members -the dopamine (DA) transporter (DAT) 1-3 , serotonin transporter (SERT) [4][5][6] and norepinephrine transporter (NET) -which have critical roles in regulating neurotransmission at synapses; the SLC6 family also includes the glycine transporter, GABA transporter and neutral amino acid transporter subfamilies, in addition to the MAT subfamily. Dysregulation of MATs has been linked to depression, anxiety disorder, attention-deficit-hyperactivity disorder, obsessive-compulsive disorder, substance-use disorders 7 , epilepsy, Parkinson's disease and autism-spectrum disorder [8][9][10][11] ; thus, MATs serve as pharmacological targets for several neuropsychiatric and neurodegenerative disorders.…”