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.

Transport and inhibition mechanisms of human VMAT2.

Vesicular monoamine transporter 2 (VMAT2) accumulates monoamines in presynaptic vesicles for storage and exocytotic release, and has a vital role in monoaminergic neurotransmission1-3. Dysfunction of monoaminergic systems causes many neurological and psychiatric disorders, including Parkinson's disease, hyperkinetic movement disorders and depression4-6. Suppressing VMAT2 with reserpine and tetrabenazine alleviates symptoms of hypertension and Huntington's disease7,8, respectively. Here we describe cryo-electron microscopy structures of human VMAT2 complexed with serotonin and three clinical drugs at 3.5-2.8 Å, demonstrating the structural basis for transport and inhibition. Reserpine and ketanserin occupy the substrate-binding pocket and lock VMAT2 in cytoplasm-facing and lumen-facing states, respectively, whereas tetrabenazine binds in a VMAT2-specific pocket and traps VMAT2 in an occluded state. The structures in three distinct states also reveal the structural basis of the VMAT2 transport cycle. Our study establishes a structural foundation for the mechanistic understanding of substrate recognition, transport, drug inhibition and pharmacology of VMAT2 while shedding light on the rational design of potential therapeutic agents.

Quantification of tramadol and serotonin by cobalt nickel tungstate in real biological samples to evaluate the effect of analgesic drugs on neurotransmitters.

In this work, CoNiWO4 nanocomposite was used as an electrochemical sensor for the simultaneous electrochemical detection of tramadol and serotonin. The nanocomposite was synthesized using a hydrothermal method and characterized via XRD, SEM, TGA, Zeta, UV, and FTIR. The sensor was developed by depositing CoNiWO4-NPs onto the glassy carbon electrode surface. Tramadol and serotonin were detected by employing cyclic voltammetry (CV), differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS), and chronoamperometry. Analytes were detected at different pH, concentrations, and scan rates. The prepared sensor showed a 0-60 µM linear range, with a LOD of 0.71 µM and 4.29 µM and LOQ of 14.3 µM and 2.3 µM for serotonin and tramadol, respectively. Finally, the modified electrode (CoNiWO4-GCE) was applied to determine tramadol and serotonin in biological samples.

Molecular Dynamics Refinement of Open State Serotonin 5-HT3A Receptor Structures.

Pentameric ligand-gated ion channels play an important role in mediating fast neurotransmissions. As a member of this receptor family, cation-selective 5-HT3 receptors are a clinical target for treating nausea and vomiting associated with chemotherapy and radiation therapy (Thompson and Lummis, 2006). Multiple cryo-electron microscopy (cryo-EM) structures of 5-HT3 receptors have been determined in distinct functional states (e.g., open, closed, etc.) (Basak et al., 2018; Basak et al., 2018; Polovinkin et al., 2018; Zhang et al., 2015). However, recent work has shown that the transmembrane pores of the open 5-HT3 receptor structures rapidly collapse and become artificially asymmetric in molecular dynamics (MD) simulations. To avoid this hydrophobic collapse, Dämgen and Biggin developed an equilibration protocol that led to a stable open state structure of the glycine receptor in MD simulations (Dämgen and Biggin, 2020). However, the protocol failed to yield open-like structures of the 5-HT3 receptor in our simulations. Here, we present a refined equilibration protocol that involves the rearrangement of the transmembrane helices to achieve stable open state structures of the 5-HT3 receptor that allow both water and ion permeation through the channel. Notably, channel gating is mediated through collective movement of the transmembrane helices, involving not only pore lining M2 helices but also their cross-talk with the adjacent M1 and M3 helices. Thus, the successful application of our refined equilibration protocol underscores the importance of the conformational coupling between the transmembrane helices in stabilizing open-like structures of the 5-HT3 receptor.

Evaluation of the Influence of Biological Factors during the Course of Treatment in Patients with Ovarian Cancer.

The aim of this study was to evaluate the influence of ß-endorphins and serotonin on the course of treatment, disease-free time, and overall survival of patients with ovarian cancer. This study may contribute to the identification of modifiable factors that may influence the treatment of ovarian cancer. The research was carried out in a group of 162 patients of which 139 respondents were included in the research; ovarian cancer was diagnosed in 78 of these patients. The study consisted of three stages. In the first stage of diagnostics, a survey among the patients was carried out. In the second stage-5 mL of blood was collected from each patient (n = 139) in the preoperative period to determine the concentration of ß-endorphin and serotonin. In the third stage-blood samples were collected from those patients who had completed chemotherapy treatment or had surgery. Concentrations of ß-endorphin and serotonin were measured by the Luminex method, using the commercial Luminex Human Discovery Assay kit. The average age of the patients was 62.99 years. The level of ß-endorphin significantly differs among patients diagnosed with ovarian cancer and among patients in the control group (202.86; SD-15.78 vs. 302.00; SD-24.49). A lower level of ß-endorphins was found in the patients with a recurrence of the neoplastic process compared to those without recurrence (178.84; SD-12.98 vs. 205.66; SD-13.37). On the other hand, the level of serotonin before chemotherapy was higher in the group of people with disease recurrence compared to those without recurrence (141.53; SD-15.33 vs. 134.99; SD-10.08). Statistically significantly positive correlations were found between the level of ß-endorphin and both disease-free time (ß-endorphin levels before chemotherapy: rho Spearman 0.379, p < 0.027; ß-endorphin levels after chemotherapy: rho Spearman 0.734 p < 0.001) and survival time (ß-endorphin levels before chemotherapy: rho Spearman 0.267, p < 0.018; ß-endorphin levels after chemotherapy: rho Spearman 0.654 p < 0.001). 1. The levels of serotonin and ß-endorphin levels are significantly related to ovarian cancer and change during treatment. 2. High mean preoperative concentrations of ß-endorphins were significantly related to overall survival and disease-free time.

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.

Structure-based discovery of nonhallucinogenic psychedelic analogs.

Drugs that target the human serotonin 2A receptor (5-HT2AR) are used to treat neuropsychiatric diseases; however, many have hallucinogenic effects, hampering their use. Here, we present structures of 5-HT2AR complexed with the psychedelic drugs psilocin (the active metabolite of psilocybin) and d-lysergic acid diethylamide (LSD), as well as the endogenous neurotransmitter serotonin and the nonhallucinogenic psychedelic analog lisuride. Serotonin and psilocin display a second binding mode in addition to the canonical mode, which enabled the design of the psychedelic IHCH-7113 (a substructure of antipsychotic lumateperone) and several 5-HT2AR ß-arrestin-biased agonists that displayed antidepressant-like activity in mice but without hallucinogenic effects. The 5-HT2AR complex structures presented herein and the resulting insights provide a solid foundation for the structure-based design of safe and effective nonhallucinogenic psychedelic analogs with therapeutic effects.

A Cornflower Extract Containing N-Feruloylserotonin Reduces Inflammation in Human Skin by Neutralizing CCL17 and CCL22 and Inhibiting COX-2 and 5-LOX.

Thymus and Activation-Regulated Chemokine (TARC/CCL17) and Macrophage-Derived Chemokine (MDC/CCL22) are two key chemokines exerting their biological effect via binding and activating a common receptor CCR4, expressed at the surface of type 2 helper T (Th2) cells. By recruiting Th2 cells in the dermis, CCL17 and CCL22 promote the development of inflammation in atopic skin. The aim of this research was to develop a plant extract whose biological properties, when applied topically, could be beneficial for people with atopic-prone skin. The strategy which was followed consisted in identifying ligands able to neutralize the biological activity of CCL17 and CCL22. Thus, an in silico molecular modeling and a generic screening assay were developed to screen natural molecules binding and blocking these two chemokines. N-Feruloylserotonin was identified as a neutraligand of CCL22 in these experiments. A cornflower extract containing N-feruloylserotonin was selected for further in vitro tests: the gene expression modulation of inflammation biomarkers induced by CCL17 or CCL22 in the presence or absence of this extract was assessed in the HaCaT keratinocyte cell line. Additionally, the same cornflower extract in another vehicle was evaluated in parallel with N-feruloylserotonin for cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymatic cellular inhibition. The cornflower extract was shown to neutralize the two chemokines in vitro, inhibited COX-2 and 5-LOX, and demonstrated anti-inflammatory activities due mainly to the presence of N-feruloylserotonin. Although these findings would need to be confirmed in an in vivo study, the in vitro studies lay the foundation to explain the benefits of the cornflower extract when applied topically to individuals with atopic-prone skin.

Acylserotonins - a new class of plant lipids with antioxidant activity and potential pharmacological applications.

During analysis of components of baobab (Adansonia digitata) seed oil, several new fluorescent compounds were detected in HPLC chromatograms that were not found previously in any seed oils investigated so far. After preparative isolation of these compounds, structural analysis by NMR spectroscopy, UHPLC-HR-MS, GC-FID and spectroscopic methods were applied and allowed identification of these substances as series of N-acylserotonins containing saturated C22 to C26 fatty acids with minor contribution of C27 to C30 homologues. The main component was N-lignocerylserotonin and the content of odd carbon-atom-number fatty acids was unusually high among the homologues. The suggested structure of the investigated compounds was additionally confirmed by their chemical synthesis. Synthetic N-acylserotonins showed pronounced inhibition of membrane lipid peroxidation of liposomes prepared from chloroplast lipids, especially when the peroxidation was initiated by a water-soluble azo-initiator, AIPH. Comparative studies of the reaction rate constants of the N-acylserotonins and tocopherols with a stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) in solvents of different polarity revealed that N-acylserotonins showed similar activity to δ-tocopherol in this respect. The described compounds have been not reported before either in plants or in animals. This indicates that we have identified a new class of plant lipids with antioxidant properties that could have promising pharmacological activities.

Nano-analysis Reveals High Fraction of Serotonin Release during Exocytosis from a Gut Epithelium Model Cell.

Electrochemical methods were used to explore the exocytotic nature of serotonin (5-HT) release in human carcinoid BON cells, an in vitro human enterochromaffin cell model, to understand the mechanisms operating the release of gut-derived 5-HT in the intestinal mucosal epithelium. We show that the fractional vesicular 5-HT release in BON cells is 80 % compared to previous work in pancreatic beta cells (34 %). The fractional release increased from 80 % in control BON cells to 87 % with 5-HT preincubation and nearly 100 % with the combination of 5-HT and the 5-HT4 autoreceptor agonist, cisapride. Thus, partial release is the primary mechanism of exocytosis in BON cells, resulting in a variable amount of the vesicular content being released. Factors that control secretion of 5-HT from enterochromaffin cells or BON cells are important as partial release provides a mechanism for development of effective therapeutic strategies to treat gastrointestinal diseases.

Serotonin Promotes Serum Albumin Interaction with the Monomeric Amyloid ß Peptide.

Prevention of amyloid ß peptide (Aß) deposition via facilitation of Aß binding to its natural depot, human serum albumin (HSA), is a promising approach to preclude Alzheimer's disease (AD) onset and progression. Previously, we demonstrated the ability of natural HSA ligands, fatty acids, to improve the affinity of this protein to monomeric Aß by a factor of 3 (BBRC, 510(2), 248-253). Using plasmon resonance spectroscopy, we show here that another HSA ligand related to AD pathogenesis, serotonin (SRO), increases the affinity of the Aß monomer to HSA by a factor of 7/17 for Aß40/Aß42, respectively. Meanwhile, the structurally homologous SRO precursor, tryptophan (TRP), does not affect HSA's affinity to monomeric Aß, despite slowdown of the association and dissociation processes. Crosslinking with glutaraldehyde and dynamic light scattering experiments reveal that, compared with the TRP-induced effects, SRO binding causes more marked changes in the quaternary structure of HSA. Furthermore, molecular docking reveals distinct structural differences between SRO/TRP complexes with HSA. The disintegration of the serotonergic system during AD pathogenesis may contribute to Aß release from HSA in the central nervous system due to impairment of the SRO-mediated Aß trapping by HSA.

Extracellular loops of the serotonin transporter act as a selectivity filter for drug binding.

The serotonin transporter (SERT) shapes serotonergic neurotransmission by retrieving its eponymous substrate from the synaptic cleft. Ligands that discriminate between SERT and its close relative, the dopamine transporter DAT, differ in their association rate constant rather than their dissociation rate. The structural basis for this phenomenon is not known. Here we examined the hypothesis that the extracellular loops 2 (EL2) and 4 (EL4) limit access to the ligand-binding site of SERT. We employed an antibody directed against EL4 (residues 388-400) and the antibody fragments 8B6 scFv (directed against EL2 and EL4) and 15B8 Fab (directed against EL2) and analyzed their effects on the transport cycle of and inhibitor binding to SERT. Electrophysiological recordings showed that the EL4 antibody and 8B6 scFv impeded the initial substrate-induced transition from the outward to the inward-facing conformation but not the forward cycling mode of SERT. In contrast, binding of radiolabeled inhibitors to SERT was enhanced by either EL4- or EL2-directed antibodies. We confirmed this observation by determining the association and dissociation rate of the DAT-selective inhibitor methylphenidate via electrophysiological recordings; occupancy of EL2 with 15B8 Fab enhanced the affinity of SERT for methylphenidate by accelerating its binding. Based on these observations, we conclude that (i) EL4 undergoes a major movement during the transition from the outward to the inward-facing state, and (ii) EL2 and EL4 limit access of inhibitors to the binding of SERT, thus acting as a selectivity filter. This insight has repercussions for drug development.

Microscopic Interactions of Melatonin, Serotonin and Tryptophan with Zwitterionic Phospholipid Membranes.

The interactions at the atomic level between small molecules and the main components of cellular plasma membranes are crucial for elucidating the mechanisms allowing for the entrance of such small species inside the cell. We have performed molecular dynamics and metadynamics simulations of tryptophan, serotonin, and melatonin at the interface of zwitterionic phospholipid bilayers. In this work, we will review recent computer simulation developments and report microscopic properties, such as the area per lipid and thickness of the membranes, atomic radial distribution functions, angular orientations, and free energy landscapes of small molecule binding to the membrane. Cholesterol affects the behaviour of the small molecules, which are mainly buried in the interfacial regions. We have observed a competition between the binding of small molecules to phospholipids and cholesterol through lipidic hydrogen-bonds. Free energy barriers that are associated to translational and orientational changes of melatonin have been found to be between 10-20 kJ/mol for distances of 1 nm between melatonin and the center of the membrane. Corresponding barriers for tryptophan and serotonin that are obtained from reversible work methods are of the order of 10 kJ/mol and reveal strong hydrogen bonding between such species and specific phospholipid sites. The diffusion of tryptophan and melatonin is of the order of 10-7 cm2/s for the cholesterol-free and cholesterol-rich setups.

A serotonin voltammetric biosensor composed of carbon nanocomposites and DNA aptamer.

A sensitive and selective voltammetric biosensor composed of layer-by-layer (LbL) self-assembly of positively charged poly(diallyldimethylammonium)-wrapped oxidized single-walled carbon nanotubes (PDDA-oSWCNTs), negatively charged serotonin (5-hydroxytryptamine, 5-HT)-specific aptamer, and tyrosinase on Au nanoparticles deposited screen printed carbon electrode was developed for measurement of 5-HT. Surface characteristics of 5-HT biosensor were explored using scanning electron microscopy, X-ray photoelectron spectroscopy, and electrochemical impedance spectroscopy. The respective effects of 5-HT-specific aptamer and oSWCNTs on the detection of 5-HT were investigated by differential pulse voltammetry (DPV). The peak current at the potential of 0.29 V (vs. Ag/AgCl) increased with respect to 5-HT concentration resulting in two dynamic ranges from 0.05 to 0.5 and 1 to 20 µM with a limit of detection of 2 nM from the LbL biosensor in buffer solution, which were better than those without the LbL of aptamer and oSWCNTs. The developed biosensor was applied to the direct determination of 5-HT concentrations in undiluted healthy control and Internet gaming disorder serum samples. The results were verified by comparison with those from liquid chromatography-mass spectrometric analyses.

The Intestinal Fatty Acid-Enteroendocrine Interplay, Emerging Roles for Olfactory Signaling and Serotonin Conjugates.

Intestinal enteroendocrine cells (EECs) respond to fatty acids from dietary and microbial origin by releasing neurotransmitters and hormones with various paracrine and endocrine functions. Much has become known about the underlying signaling mechanisms, including the involvement of G-protein coupled receptors (GPCRs), like free fatty acids receptors (FFARs). This review focusses on two more recently emerging research lines: the roles of odorant receptors (ORs), and those of fatty acid conjugates in gut. Odorant receptors belong to a large family of GPCRs with functional roles that only lately have shown to reach beyond the nasal-oral cavity. In the intestinal tract, ORs are expressed on serotonin (5-HT) and glucagon-like-peptide-1 (GLP-1) producing enterochromaffin and enteroendocrine L cells, respectively. There, they appear to function as chemosensors of microbiologically produced short-, and branched-chain fatty acids. Another mechanism of fatty acid signaling in the intestine occurs via their conjugates. Among them, conjugates of unsaturated long chain fatty acids and acetate with 5-HT, N-acyl serotonins have recently emerged as mediators with immune-modulatory effects. In this review, novel findings in mechanisms and molecular players involved in intestinal fatty acid biology are highlighted and their potential relevance for EEC-mediated signaling to the pancreas, immune system, and brain is discussed.

Structural and Molecular Dynamics Analysis of Plant Serotonin N-Acetyltransferase Reveal an Acid/Base-Assisted Catalysis in Melatonin Biosynthesis.

Serotonin N-acetyltransferase (SNAT) is the key rate-limiting enzyme in melatonin biosynthesis. It mediates melatonin biosynthesis in plants by using serotonin and 5-methoxytryptamine (5-MT), but little is known of its underlying mechanisms. Herein, we present a detailed reaction mechanism of a SNAT from Oryza sativa through combined structural and molecular dynamics (MD) analysis. We report the crystal structures of plant SNAT in the apo and binary/ternary complex forms with acetyl-CoA (AcCoA), serotonin, and 5-MT. OsSNAT exhibits a unique enzymatically active dimeric fold not found in the known structures of arylalkylamine N-acetyltransferase (AANAT) family. The key residues W188, D189, D226, N220, and Y233 located around the active pocket are important in catalysis, confirmed by site-directed mutagenesis. Combined with MD simulations, we hypothesize a novel plausible catalytic mechanism in which D226 and Y233 function as catalytic base and acid during the acetyl-transfer reaction.

Restructuring of Membrane Water and Phospholipids in Direct Interaction of Neurotransmitters with Model Membranes Associated with Synaptic Signaling: Interface-Selective Vibrational Sum Frequency Generation Study.

Comprehensive molecular-level understanding of the role of interfacial water and phospholipids associated with synaptic membranes during their direct interaction with neurotransmitters is essential because of their involvement in synaptic signaling. Herein, the interfacial regions of the synaptic membranes mimicking anionic and zwitterionic phospholipids are probed in the presence of dopamine and serotonin neurotransmitters using surface-specific vibrational sum frequency generation technique. Neurotransmitters intrude into the headgroup region of both zwitterionic and anionic lipids by restructuring the interfacial water associated with the phospholipids, although the restructuring mechanism is different for both lipids. Neurotransmitters also decrease the overall ordering of both the phospholipids probably by creating gauche defects. Neurotransmitters restructure the surface water, conformation, and the ordering of the hydrocarbon chains of the zwitterionic and anionic phospholipids associated with synaptic membranes, which could be potentially an important step for synaptic signaling.

Discovery of a new class of multi-target heterocycle piperidine derivatives as potential antipsychotics with pro-cognitive effect.

A series of benzoisoxazoleylpiperidine derivatives were synthesized by using the multi-target strategies and their potent affinities for dopamine (DA), serotonin (5-HT) and human histamine H3 receptors have been evaluated. Of these compounds, the promising candidate 4w displayed high affinities for D2, D3, 5-HT1A, 5-HT2A and H3, a moderate affinity for 5-HT6, negligible effects on the human ether-a-go-go-related gene (hERG) channel, low affinities for off-target receptors (5-HT2C, adrenergic α1 and H1). In addition, the animal behavioral study revealed that, compared to risperidone, compound 4w significantly inhibited apomorphine-induced climbing and MK-801-induced movement behaviors with a high threshold for catalepsy and low liabilities for weight gain and hyperprolactinemia. Results from the conditioned avoidance response test and novel object recognition task demonstrated that 4w had pro-cognitive effects. Thus, the antipsychotic drug-like activities of 4w indicate that it may be a potential polypharmacological antipsychotic candidate drug.