Identification of the potassium-binding site in serotonin transporter.

Clearance of serotonin (5-hydroxytryptamine, 5-HT) from the synaptic cleft after neuronal signaling is mediated by serotonin transporter (SERT), which couples this process to the movement of a Na+ ion down its chemical gradient. After release of 5-HT and Na+ into the cytoplasm, the transporter faces a rate-limiting challenge of resetting its conformation to be primed again for 5-HT and Na+ binding. Early studies of vesicles containing native SERT revealed that K+ gradients can provide an additional driving force, via K+ antiport. Moreover, under appropriate conditions, a H+ ion can replace K+. Intracellular K+ accelerates the resetting step. Structural studies of SERT have identified two binding sites for Na+ ions, but the K+ site remains enigmatic. Here, we show that K+ antiport can drive substrate accumulation into vesicles containing SERT extracted from a heterologous expression system, allowing us to study the residues responsible for K+ binding. To identify candidate binding residues, we examine many cation binding configurations using molecular dynamics simulations, predicting that K+ binds to the so-called Na2 site. Site-directed mutagenesis of residues in this site can eliminate the ability of both K+ and H+ to drive 5-HT accumulation into vesicles and, in patch clamp recordings, prevent the acceleration of turnover rates and the formation of a channel-like state by K+ or H+. In conclusion, the Na2 site plays a pivotal role in orchestrating the sequential binding of Na+ and then K+ (or H+) ions to facilitate 5-HT uptake in SERT.

Carbazole derivatives as promising competitive and allosteric inhibitors of human serotonin transporter: computational pharmacology.

The human serotonin transporters (hSERTs) are neurotransmitter sodium symporters of the aminergic G protein-coupled receptors, regulating the synaptic serotonin and neuropharmacological processes related to neuropsychiatric disorders, notably, depression. Selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine and (S)-citalopram are competitive inhibitors of hSERTs and are commonly the first-line medications for major depressive disorder (MDD). However, treatment-resistance and unpleasant aftereffects constitute their clinical drawbacks. Interestingly, vilazodone emerged with polypharmacological (competitive and allosteric) inhibitions on hSERTs, amenable to improved efficacy. However, its application usually warrants adjuvant/combination therapy, another subject of critical adverse events. Thus, the discovery of alternatives with polypharmacological potentials (one-drug-multiple-target) and improved safety remains essential. In this study, carbazole analogues from chemical libraries were explored using docking and molecular dynamics (MD) simulation. Selectively, two IBScreen ligands, STOCK3S-30866 and STOCK1N-37454 predictively bound to the active pockets and expanded boundaries (extracellular vestibules) of the hSERTs more potently than vilazodone and (S)-citalopram. For instance, the two ligands showed docking scores of -9.52 and -9.59 kcal/mol and MM-GBSA scores of -92.96 and -65.66 kcal/mol respectively compared to vilazodone's respective scores of -7.828 and -59.27 against the central active site of the hSERT (PDB 7LWD). Similarly, the two ligands also docked to the allosteric pocket (PDB 5I73) with scores of -8.15 and -8.40 kcal/mol and MM-GBSA of -96.14 and -68.46 kcal/mol whereas (S)-citalopram has -6.90 and -69.39 kcal/mol respectively. The ligands also conferred conformational stability on the receptors during 100 ns MD simulations and displayed interesting ADMET profiles, representing promising hSERT modulators for MDD upon experimental validation.Communicated by Ramaswamy H. Sarma.

Molecular Photoswitches Regulating the Activity of the Human Serotonin Transporter.

Serotonin is an essential mediator regulating diverse neural processes, and its deregulation is related to the development of debilitating neurological diseases. In particular, the human serotonin transporter (hSERT) is fundamental in completing the synaptic neural cycle by allowing reuptake of serotonin. Its inhibition is particularly attractive, especially as a pharmacological target against depressive syndrome. Here, we analyze, by using long-range molecular dynamic simulations, the behavior of a molecular photoswitch whose cis- and trans-isomers inhibit the hSERT differently. In particular, we evidence the structural and molecular basis behind the higher inhibiting capacity of the cis-isomer, which blocks more efficiently the hSERT conformational cycle, leading to serotonin uptake.

Structure-based discovery of conformationally selective inhibitors of the serotonin transporter.

The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has unusual anti-depressant and substance-withdrawal effects, and stabilizes the inward-open conformation. Unfortunately, ibogaine's promiscuity and cardiotoxicity limit the understanding of inward-open state ligands. We docked over 200 million small molecules against the inward-open state of the SERT. Thirty-six top-ranking compounds were synthesized, and thirteen inhibited; further structure-based optimization led to the selection of two potent (low nanomolar) inhibitors. These stabilized an outward-closed state of the SERT with little activity against common off-targets. A cryo-EM structure of one of these bound to the SERT confirmed the predicted geometry. In mouse behavioral assays, both compounds had anxiolytic- and anti-depressant-like activity, with potencies up to 200-fold better than fluoxetine (Prozac), and one substantially reversed morphine withdrawal effects.

Discovery and Development of Monoamine Transporter Ligands.

Monoamine transporters (MATs) are targets of a wide range of compounds that have been developed as therapeutic treatments for various neuropsychiatric and neurodegenerative disorders such as depression, ADHD, neuropathic pain, anxiety disorders, stimulant use disorders, epilepsy, and Parkinson's disease. The MAT family is comprised of three main members - the dopamine transporter (DAT), the norepinephrine transporter (NET), and the serotonin transporter (SERT). These transporters are through reuptake responsible for the clearance of their respective monoamine substrates from the extracellular space. The determination of X-ray crystal structures of MATs and their homologues bound with various substrates and ligands has resulted in a surge of structure-function-based studies of MATs to understand the molecular basis of transport function and the mechanism of various ligands that ultimately result in their behavioral effects. This review focusses on recent examples of ligand-based structure-activity relationship studies trying to overcome some of the challenges associated with previously developed MAT inhibitors. These studies have led to the discovery of unique and novel structurally diverse MAT ligands including allosteric modulators. These novel molecular scaffolds serve as leads for designing more effective therapeutic interventions by modulating the activities of MATs and ultimately their associated neurotransmission and behavioral effects.

Allosteric Binding of MDMA to the Human Serotonin Transporter (hSERT) via Ensemble Binding Space Analysis with ΔG Calculations, Induced Fit Docking and Monte Carlo Simulations.

Despite the recent promising results of MDMA (3,4-methylenedioxy-methamphetamine) as a psychotherapeutic agent and its history of misuse, little is known about its molecular mode of action. MDMA enhances monoaminergic neurotransmission in the brain and its valuable psychoactive effects are associated to a dual action on the 5-HT transporter (SERT). This drug inhibits the reuptake of 5-HT (serotonin) and reverses its flow, acting as a substrate for the SERT, which possesses a central binding site (S1) for antidepressants as well as an allosteric (S2) one. Previously, we characterized the spatial binding requirements for MDMA at S1. Here, we propose a structure-based mechanistic model of MDMA occupation and translocation across both binding sites, applying ensemble binding space analyses, electrostatic complementarity, and Monte Carlo energy perturbation theory. Computed results were correlated with experimental data (r = 0.93 and 0.86 for S1 and S2, respectively). Simulations on all hSERT available structures with Gibbs free energy estimations (ΔG) revealed a favourable and pervasive dual binding mode for MDMA at S2, i.e., adopting either a 5-HT or an escitalopram-like orientation. Intermediate ligand conformations were identified within the allosteric site and between the two sites, outlining an internalization pathway for MDMA. Among the strongest and more frequent interactions were salt bridges with Glu494 and Asp328, a H-bond with Thr497, a π-π with Phe556, and a cation-π with Arg104. Similitudes and differences with the allosteric binding of 5-HT and antidepressants suggest that MDMA may have a distinctive chemotype. Thus, our models may provide a framework for future virtual screening studies and pharmaceutical design and to develop hSERT allosteric compounds with a unique psychoactive MDMA-like profile.

Serotonin transporter: Recent progress of in silico ligand prediction methods and structural biology towards structure-guided in silico design of therapeutic agents.

Serotonin transporter (SERT) is a membrane transporter which terminates neurotransmission of serotonin through its reuptake. This transporter as well as its substrate have long drawn attention as a key mediator and drug target in a variety of diseases including mental disorders. Accordingly, its structural basis has been studied by X-ray crystallography to gain insights into a design of ligand with high affinity and high specificity over closely related transporters. Recent progress in structural biology including single particle cryo-EM have made big strides also in determination of the structures of human SERT in complex with its ligands. Moreover, rapid progress in machine learning such as deep learning accelerates computer-assisted drug design. Here, we would like to summarize recent progresses in our understanding of SERT using these two rapidly growing technologies, limitations, and future perspectives.

The substrate import mechanism of the human serotonin transporter.

The serotonin transporter (SERT) initiates the reuptake of extracellular serotonin in the synapse to terminate neurotransmission. The cryogenic electron microscopy structures of SERT bound to ibogaine and the physiological substrate serotonin resolved in different states have provided a glimpse of the functional conformations at atomistic resolution. However, the conformational dynamics and structural transitions to intermediate states are not fully understood. Furthermore, the molecular basis of how serotonin is recognized and transported remains unclear. In this study, we performed unbiased microsecond-long simulations of the human SERT to investigate the structural dynamics to various intermediate states and elucidated the complete substrate import pathway. Using Markov state models, we characterized a sequential order of conformational-driven ion-coupled substrate binding and transport events and calculated the free energy barriers of conformation transitions associated with the import mechanism. We find that the transition from the occluded to inward-facing state is the rate-limiting step for substrate import and that the substrate decreases the free energy barriers to achieve the inward-facing state. Our study provides insights on the molecular basis of dynamics-driven ion-substrate recognition and transport of SERT that can serve as a model for other closely related neurotransmitter transporters.

Sodium Binding Stabilizes the Outward-Open State of SERT by Limiting Bundle Domain Motions.

The human serotonin transporter (hSERT) removes the neurotransmitter serotonin from the synaptic cleft by reuptake into the presynaptic nerve terminal. A number of neurologic diseases are associated with dysfunction of the hSERT, and several medications for their treatment are hSERT blockers, including citalopram, fluoxetine, and paroxetine. The substrate transport is energized by the high concentration of external NaCl. We showed through molecular dynamics simulations that the binding of NaCl stabilized the hSERT in the substrate-binding competent conformation, which was characterized by an open access path to the substrate-binding site through the outer vestibule. Importantly, the binding of NaCl reduced the dynamics of the hSERT by decreasing the internal fluctuations of the bundle domain as well as the movement of the bundle domain relative to the scaffold domain. In contrast, the presence of only the bound chloride ion did not reduce the high domain mobility of the apo state.

Occlusion of the human serotonin transporter is mediated by serotonin-induced conformational changes in the bundle domain.

The human serotonin transporter (hSERT) terminates neurotransmission by removing serotonin (5HT) from the synaptic cleft, an essential process for proper functioning of serotonergic neurons. Structures of the hSERT have revealed its molecular architecture in four conformations, including the outward-open and occluded states, and show the transporter's engagement with co-transported ions and the binding mode of inhibitors. In this study, we investigated the molecular mechanism by which the hSERT occludes and sequesters the substrate 5HT. This first step of substrate uptake into cells is a structural change consisting of the transition from the outward-open to the occluded state. Inhibitors such as the antidepressants citalopram, fluoxetine, and sertraline inhibit this step of the transport cycle. Using molecular dynamics simulations, we reached a fully occluded state, in which the transporter-bound 5HT becomes fully shielded from both sides of the membrane by two closed hydrophobic gates. Analysis of 5HT-triggered occlusion showed that bound 5HT serves as an essential trigger for transporter occlusion. Moreover, simulations revealed a complex sequence of steps and showed that movements of bundle domain helices are only partially correlated. 5HT-triggered occlusion is initially dominated by movements of transmembrane helix 1b, while in the final step, only transmembrane helix 6a moves and relaxes an intermediate change in its secondary structure.

The novel therapeutic strategy of vilazodone-donepezil chimeras as potent triple-target ligands for the potential treatment of Alzheimer's disease with comorbid depression.

Depression is one of the most frequent comorbid psychiatric symptoms of Alzheimer's disease (AD), and no efficacious drugs have been approved specifically for this purpose thus far. Herein, we proposed a novel therapeutic strategy that merged the key pharmacophores of the antidepressant vilazodone (5-HT1A receptor partial agonist and serotonin transporter inhibitor) and the anti-AD drug donepezil (acetylcholinesterase inhibitor) together to develop a series of multi-target-directed ligands for potential therapy of the comorbidity of AD and depression. Accordingly, 55 vilazodone-donepezil chimeric derivatives were designed and synthesized, and their triple-target activities against acetylcholinesterase, 5-HT1A receptor, and serotonin transporter were systematically evaluated. Among them, compound 5 displayed strong triple-target bioactivities in vitro, low hERG potassium channel inhibition and acceptable brain distribution. Importantly, oral intake of 5 mg/kg of the compound 5 dihydrochloride significantly alleviated the depressive symptoms and ameliorated cognitive dysfunction in mouse models. In brief, these results highlight vilazodone-donepezil chimeras as a prospective therapeutic approach for the treatment of the comorbidity of AD and depression.

Thermal Unfolding of the Human Serotonin Transporter: Differential Effect by Stabilizing and Destabilizing Mutations and Cholesterol on Thermodynamic and Kinetic Stability.

Folding-deficient mutants of solute carrier 6 (SLC6) family members have been linked to human diseases. The serotonin transporter [(SERT)/SLC6A4] is an important drug target in the treatment of depression, anxiety, and obsessive-compulsive disorders and-with structural information in several conformational states-one of the best understood transporters. Here, we surmised that thermal unfolding offered a glimpse on the folding energy landscape of SLC6 transporters. We carried out molecular dynamic (MD) simulations to understand the mechanistic basis for enhanced and reduced stability, respectively, of the thermostabilized variant SERT-Y110A/I291A/T439S, which had previously been used for crystallization of human SERT in the outward-facing state, and of the folding-deficient SERT-P601A/G602A. We also examined the hydrophobic mismatch caused by the absence of cholesterol to explore the contribution of cholesterol to protein stability. When compared with wild type SERT, the thermodynamic and kinetic stability of SERT-Y110A/I291A/T439S was enhanced. In the other instances, changes in these two components were not correlated: the mutations in SERT-P601A/G602A led to a drop in thermodynamic but an increase in kinetic stability. The divergence was even more pronounced after cholesterol depletion, which reduced thermodynamic stability but increased the kinetic stability of wild type SERT to a level comparable to that of SERT-Y110A/I291A/T439S. We conclude that the low cholesterol content of the endoplasmic reticulum facilitates progression of the folding trajectory by reducing the energy difference between folding intermediates and the native state. SIGNIFICANCE STATEMENT: Point mutations in solute carrier 6 (SLC6) family members cause folding diseases. The serotonin transporter [(SERT)/SLC6A4] is a target for antidepressants and the best understood SLC6. This study produced molecular dynamics simulations and examined thermal unfolding of wild type and mutant SERT variants to understand their folding energy landscape. In the folding-deficient SERT-P012A/G602A, changes in kinetic and thermodynamic stability were not correlated. Similarly, cholesterol depletion lowered thermodynamic but enhanced kinetic stability. These observations allow for rationalizing the action of pharmacochaperones.

Evaluation of i-Motif Formation in the Serotonin Transporter-Linked Polymorphic Region.

Neuropsychiatric disorders such as major depressive disorder (MDD) arise from a complex set of genetic and environmental factors. The serotonin transporter (SERT) is a key regulator of synaptic serotonin (5-HT), and its inhibition is an important pharmacological target for treating MDD. The SERT-linked polymorphic region (5-HTTLPR) contains two major variants (short and long) that have been implicated in modulating susceptibility to MDD by altering the level of expression of SERT. Both variants contain C-rich repeats that conform to consensus i-motif folding sequences. i-Motifs are quadruplex DNA structures that have been proposed to have a role in transcription regulation. With spectroscopic techniques, we demonstrate that both alleles are able to form i-motifs at acidic pH, and at neutral pH under conditions of molecular crowding. This highlights the potential for i-motif formation to contribute to transcriptional regulation of the serotonin transporter, with a potential role in the pathophysiology of neuropsychiatric disorders.

Role of the E3 ubiquitin ligase HRD1 in the regulation of serotonin transporter function.

To elucidate the regulation of serotonin transporter (SERT) function via its membrane trafficking, we investigated the involvement of the ubiquitin E3 ligase HRD1 (HMG-CoA reductase degradation protein), which participates in endoplasmic reticulum (ER)-associated degradation (ERAD), in the functional regulation of SERT. Cells transiently expressing wild-type SERT or a SERT C-terminal deletion mutant (SERTΔCT), a SERT protein predicted to be misfolded, were used for experiments. Studies using HRD1-overexpressing or HRD1-knockdown cells demonstrated that HRD1 is involved in SERT proteolysis. Overexpression of HRD1 promoted SERT ubiquitination, the effect of which was augmented by treatment with the proteasome inhibitor MG132. Immunoprecipitation studies revealed that HRD1 interacts with SERT in the presence of MG132. In addition, HRD1 was intracellularly colocalized with SERT, especially with aggregates of SERTΔCT in the ER. HRD1 also affected SERT uptake activity in accordance with the expression levels of the SERT protein. These results suggest that HRD1 contributes to the membrane trafficking and functional regulation of SERT through its involvement in ERAD-mediated SERT degradation.

Pharmacological Characterization of 4-Methylthioamphetamine Derivatives.

Amphetamine derivatives have been used in a wide variety of pathologies because of their pharmacological properties as psychostimulants, entactogens, anorectics, and antidepressants. However, adverse cardiovascular effects (sympathomimetics) and substance abuse problems (psychotropic and hallucinogenic effects) have limited their use. 4-Methylthioamphetamine (MTA) is an amphetamine derivative that has shown to inhibit monoamine uptake and monoamine oxidase. However, the pharmacological characterization (neurochemical, behavioral, and safety) of its derivatives 4-ethylthioamphetamine (ETA) and 4-methylthio-phenil-2-butanamine (MT-But) have not been studied. In the current experiments, we show that ETA and MT-But do not increase locomotor activity and conditioned place preference with respect to MTA. At the neurochemical level, ETA and MT-But do not increase in vivo DA release in striatum, but ETA and MT-But affect the nucleus accumbens bioaccumulation of DA and DOPAC. Regarding cardiovascular effects, the administration of MTA and ETA increased the mean arterial pressure and only ETA significantly increases the heart rate. Our results show that the pharmacological and safety profiles of MTA are modulated by changing the methyl-thio group or the methyl group of the aminoethyl chain.

Chemical and structural investigation of the paroxetine-human serotonin transporter complex.

Antidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand binding of small-molecules to conformationally dynamic transporters like SERT often require thermostabilizing mutations and antibodies to stabilize a specific conformation, leading to questions about relationships of these structures to the bonafide conformation and inhibitor binding poses of wild-type transporter. To address these concerns, we determined the structures of ∆N72/∆C13 and ts2-inactive SERT bound to paroxetine analogues using single-particle cryo-EM and x-ray crystallography, respectively. We synthesized enantiopure analogues of paroxetine containing either bromine or iodine instead of fluorine. We exploited the anomalous scattering of bromine and iodine to define the pose of these inhibitors and investigated inhibitor binding to Asn177 mutants of ts2-active SERT. These studies provide mutually consistent insights into how paroxetine and its analogues bind to the central substrate-binding site of SERT, stabilize the outward-open conformation, and inhibit serotonin transport.

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

The serotonin transporter (SERT) terminates serotonin signaling by rapid presynaptic reuptake. SERT activity is modulated by antidepressants, e.g., S-citalopram and imipramine, to alleviate symptoms of depression and anxiety. SERT crystal structures reveal two S-citalopram binding pockets in the central binding (S1) site and the extracellular vestibule (S2 site). In this study, our combined in vitro and in silico analysis indicates that the bound S-citalopram or imipramine in S1 is allosterically coupled to the ligand binding to S2 through altering protein conformations. Remarkably, SERT inhibitor Lu AF60097, the first high-affinity S2-ligand reported and characterized here, allosterically couples the ligand binding to S1 through a similar mechanism. The SERT inhibition by Lu AF60097 is demonstrated by the potentiated imipramine binding and increased hippocampal serotonin level in rats. Together, we reveal a S1-S2 coupling mechanism that will facilitate rational design of high-affinity SERT allosteric inhibitors.

The binding mode of vilazodone in the human serotonin transporter elucidated by ligand docking and molecular dynamics simulations.

Vilazodone is a novel antidepressant used for the treatment of major depressive disorder (MDD) with a primary action mechanism of inhibiting the human serotonin reuptake transporter (hSERT) and acting as a 5-HT1A receptor partial agonist. The interaction between vilazodone and the 5-HT1A receptor has been reported, however, the binding mode of vilazodone in the hSERT remains elusive. In the current study, to elucidate the molecular mechanism of vilazodone binding in the hSERT, the drug and its five analogs were docked into the hSERT crystal structure as initial conformations and were sampled by 400 ns molecular dynamics (MD) simulations. Through the analysis of the profiles of protein-ligand binding free energies, interaction fingerprints, and conformational rearrangements, the binding mode of vilazodone in the hSERT was revealed. As a result, unlike the classical antidepressants located in the S1 site of the hSERT, vilazodone adopted a linear pose in the binding pocket. Its arylpiperazine fragment occupies the central site (S1) and interacts with Y95, D98, I172, Y176, F335, F341, S438, and T439, while the indole fragment extends to the allosteric site (S2) via interacting with the ionic switch (R104/E403) between the two sites. The new insights obtained are not only helpful in understanding the binding mode of vilazodone in the hSERT, but also provide valuable guidance to the discovery of novel antidepressant drugs.

Evaluation of anti-depressant effects of phthalazinone-based triple-acting small molecules against 5-HT2A, 5-HT2C, and the serotonin transporter.

Development of highly effective, safe, and fast-acting anti-depressants is urgently required for the treatment of major depressive disorder. It has been suggested that targeting 5-HT2A and 5-HT2C in addition to inhibition of serotonin reuptake may be beneficial in generating anti-depressant agents with better pharmacology and less adverse effects. We have developed phthalazinone-based compounds that potently bind to 5-HT2A, 5-HT2C, and the serotonin transporter. The representative compounds 11j and 11l displayed strong binding affinities against these targets, and showed favorable toxicity profiles as determined by hERG binding and CYP inhibition assays. Furthermore, these compounds presented promising anti-depressant effects comparable to fluoxetine and also synergistic effects with fluoxetine in forced swimming test, which implicates these compounds can be developed to help the treatment of major depressive disorder.

Synthesis of Novel Nicotinic Ligands with Multimodal Action: Targeting Acetylcholine α4ß2, Dopamine and Serotonin Transporters.

Nicotinic acetylcholine receptors (nAChRs), serotonin transporters (SERT) and dopamine transporters (DAT) represent targets for the development of novel nicotinic derivatives acting as multiligands associated with different health conditions, such as depressive, anxiety and addiction disorders. In the present work, a series of functionalized esters structurally related to acetylcholine and nicotine were synthesized and pharmacologically assayed with respect to these targets. The synthesized compounds were studied in radioligand binding assays at α4ß2 nAChR, h-SERT and h-DAT. SERT experiments showed not radioligand [3H]-paroxetine displacement, but rather an increase in the radioligand binding percentage at the central binding site was observed. Compound 20 showed Ki values of 1.008 ± 0.230 µM for h-DAT and 0.031 ± 0.006 µM for α4ß2 nAChR, and [3H]-paroxetine binding of 191.50% in h-SERT displacement studies, being the only compound displaying triple affinity. Compound 21 displayed Ki values of 0.113 ± 0.037 µM for α4ß2 nAChR and 0.075 ± 0.009 µM for h-DAT acting as a dual ligand. Molecular docking studies on homology models of α4ß2 nAChR, h-DAT and h-SERT suggested potential interactions among the compounds and agonist binding site at the α4/ß2 subunit interfaces of α4ß2 nAChR, central binding site of h-DAT and allosteric modulator effect in h-SERT.