Tricyclic antidepressants exert their pharmacological effect -inhibiting the reuptake of serotonin, norepinephrine and dopamine -by directly blocking neurotransmitter transporters (SERT, NET and DAT, respectively) in the presynaptic membrane. The drug-binding site and the mechanism of this inhibition are poorly understood. We determined the crystal structure at 2.9 Å of the bacterial leucine transporter (LeuT), a homolog of SERT, NET and DAT, in complex with leucine and the antidepressant desipramine. Desipramine binds at the inner end of the extracellular cavity of the transporter and is held in place by a hairpin loop and by a salt bridge. This binding site is separated from the leucine-binding site by the extracellular gate of the transporter. By directly locking the gate, desipramine prevents conformational changes and blocks substrate transport. Mutagenesis experiments on human SERT and DAT indicate that both the desipramine-binding site and its inhibition mechanism are probably conserved in the human neurotransmitter transporters. Na + /Cl − -dependent neurotransmitter transporters for serotonin (SERT), norepinephrine (NET) and dopamine (DAT) in the presynaptic plasma membrane terminate neuronal signal transmission in the central nervous system through a reuptake mechanism (1-6). These systems have been shown to modulate mood, emotion, sleep and appetite (7). Depression, arguably the most prevalent psychiatric disorder, is directly associated with perturbation of serotonergic neurotransmission (8, 9), and drugs blocking serotonin reuptake have been used successfully for its treatment. One class of these drugs, tricyclic antidepressants (TCAs) such as desipramine and imipramine, binds to serotonin and norepinephrine transporters with affinities of nanomolar to tens of nanomolar concentrations and blocks transport activity (10). The response rate of patients to TCAs is typically 60-70% (11). More recently, highly selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine (Prozac) have also been developed and are increasingly prescribed to treat depression (12). The molecular The human SERT, DAT and NET proteins all belong to a family of transporters for amino acids and their derivatives, the Neurotransmitter:Sodium Symporter (NSS) family (2)(3)(4)(5)14). Whilst the dopamine transporters from human, bovine or rat are inhibited by TCAs at a K i of micromolar concentrations, the DAT proteins from C. elegans (15) and D. melanogaster (16) are inhibited by TCAs at a K i of nanomolar and sub-micromolar concentrations, respectively (17). As bacterial NSS proteins share up to 30 % sequence identity with human SERT and NET as well as worm and fly DATs, we hypothesized that bacterial NSS proteins also possess high binding affinity to TCAs and could provide opportunities for studying proteindrug interactions. We therefore chose a bacterial NSS protein, the leucine transporter (LeuT) from Aquifex aeolicus, to study the molecular mechanism of neurotransmitter transporter binding to TCAs (18). LeuT shares 2...

Sertraline and fluoxetine are selective serotonin reuptake inhibitors (SSRIs) widely-prescribed to treat depression. They exert their effects by inhibiting the presynaptic plasma membrane serotonin transporter (SERT). All SSRIs possess at specific positions halogen atoms, which are key determinants for the drugs’ specificity for SERT. For the SERT protein, however, the structural basis of its specificity for SSRIs is poorly understood. Here we report the crystal structures of LeuT, a bacterial SERT homolog, in complex with sertraline, R-fluoxetine or S-fluoxetine. The SSRI halogens all bind to exactly the same pocket within LeuT. Mutation at this halogen-binding pocket (HBP) in SERT dramatically reduces the transporter's affinity for SSRIs but not for tricyclic antidepressants. Conversely, when the only non-conserved HBP residue in both norepinephrine and dopamine transporters is mutated into that found in SERT, their affinities for all the three SSRIs increase uniformly. Thus, the specificity of SERT for SSRIs is dependent largely on interaction of the drug halogens with the protein's halogen-binding pocket.

RNA helicase DDX21 plays vital roles in ribosomal RNA biogenesis, transcription, and the regulation of host innate immunity during virus infection. How DDX21 recognizes and unwinds RNA and how DDX21 interacts with virus remain poorly understood. Here, crystal structures of human DDX21 determined in three distinct states are reported, including the apo‐state, the AMPPNP plus single‐stranded RNA (ssRNA) bound pre‐hydrolysis state, and the ADP‐bound post‐hydrolysis state, revealing an open to closed conformational change upon RNA binding and unwinding. The core of the RNA unwinding machinery of DDX21 includes one wedge helix, one sensor motif V and the DEVD box, which links the binding pockets of ATP and ssRNA. The mutant D339H/E340G dramatically increases RNA binding activity. Moreover, Hill coefficient analysis reveals that DDX21 unwinds double‐stranded RNA (dsRNA) in a cooperative manner. Besides, the nonstructural (NS1) protein of influenza A inhibits the ATPase and unwinding activity of DDX21 via small RNAs, which cooperatively assemble with DDX21 and NS1. The structures illustrate the dynamic process of ATP hydrolysis and RNA unwinding for RNA helicases, and the RNA modulated interaction between NS1 and DDX21 generates a fresh perspective toward the virus–host interface. It would benefit in developing therapeutics to combat the influenza virus infection.

Inhibition of human serotonin transporter (hSERT) has been reported to be a potent strategy for the treatment for depression. To discover novel selective serotonin reuptake inhibitors (SSRIs), a structure-based pharmacophore model (SBPM) was developed using the docked conformations of six highly active SSRIs. The best SBPM, consisting of four chemical features: two ring aromatics (RAs), one hydrophobic (HY), and one positive ionizable (PI), was further validated using Gunner-Henry (GH) scoring and receiver operating characteristic (ROC) curve methods. This well-validated SBPM was then used as a 3D-query in virtual screening to identify potential hits from National Cancer Institute (NCI) database. These hits were subsequently filtered by absorption, distribution, metabolism, excretion, and toxicity (ADMET) prediction and molecular docking, and their binding stabilities were validated by 20-ns MD simulations. Finally, only two compounds (NSC175176 and NSC705841) were identified as potential leads, which exhibited higher binding affinities in comparison with the paroxetine. Our results also suggest that cation-π interaction plays a crucial role in stabilizing the hSERT-inhibitor complex. To our knowledge, the present work is the first structure-based virtual screening study for new SSRI discovery, which should be a useful guide for the rapid identification of novel therapeutic agents from chemical database.

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