The dopamine transporter (DAT) is linked to neuropsychiatric disorders including ADHD, Parkinson's disease, and substance use disorders. Accordingly, DAT is the target of illicit drugs and clinically important medicines. However, the number and function of ligand binding sites in DAT is enigmatic due to conflicting data from available structures and molecular pharmacology. Herein, we design force sensors with DAT ligands and measure their interaction forces with wild-type and mutated DATs, from which two distinct populations of unbinding strengths and off-rates are detected. The high-force population is reduced by V152I and S422A mutations, or by substituting Na+ with K+ or NMDG+. In contrast, several modifications including mutation G386H, acetylation of K92 and K384, mutation K92A, mutation K384A, or protonation of H477 decrease the low-force population. The present data delineate the threshold of binding strength, which may account for certain ligand binding sites to be imperceptible in crystal or cryo-EM structures. Furthermore, the force spectra provide the information on the position and kinetic rates of a herein detected ligand binding site in DAT.

Introduction Based on the teachings of Rudolf Steiner, anthroposophic medicine (AM) is practiced in more than 80 countries around the world today. AM blends physical, mental and spiritual dimensions of life into patient care, nevertheless regarding itself as highly scientific. As a comparable systematic review (SR) of AM conducted more than two decades ago is now outdated, this review aims to establish an updated summary of the existing clinical evidence on AM. Objectives The primary aim was to evaluate the efficacy of substance-based interventions in AM for patients with acute or chronic illnesses. Additionally, it assessed the safety and adverse drug reaction (ADR) profile of AM remedies. Methods We reviewed available evidence on substance-based AM, focusing on mortality, morbidity and safety. All randomized and non-randomized trials of AM-specific monotherapies were considered, excluding mistletoe due to existing recent high-quality reviews. A Cochrane-compliant search strategy was employed across Medline, EMBASE, Cochrane Central Register of Controlled Trials, CINAHL, PsycInfo, and Anthromedlit-Datenbank, along manual reference list searches including studies published until November 2024. Results Our searches yielded 360 hits of which 17 publications met inclusion criteria. Endpoints and diseases included allergic rhinitis, pain, stroke, and quality of life related outcomes. The only statistically significant result favoring AM was a post-hoc analysis of a neurasthenia trial-a vague diagnosis no longer included in the ICD-11. Another study assessing quality of life claimed clinically relevant improvements, but this was based on inappropriate statistical analyses. None of the other studies reported results unambiguously favoring AM. AM was well tolerated, with studies showing adverse drug reactions occurring in 0% to 3% of patients. Conclusion Twenty years after a similar systematic review, the evidence for AM treatments remains largely unchanged. We conclude that these therapies are not supported by sound evidence. Their use in routine care must therefore be questioned. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Protocols ### Funding Statement This study was supported by the Medical University of Vienna, Vienna, Austria. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This systematic review used only data of already published human trials from established data bases. (Medline, EMBASE, Cochrane Central Register of Controlled Trials, CINAHL, PsycInfo, and Anthromedlit-Datenbank) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Further data is available in the supplementary files. All data produced in the present study are available upon reasonable request to the authors.

The serotonin transporter (SERT) belongs to the family of neurotransmitter sodium symporters (NSS), together with other neurotransmitter transporters for norepinephrine, dopamine, glycine, and GABA. The main physiological role of SERT is the retrieval of previously released serotonin from the synaptic cleft. Thereby, SERT plays an important role in regulating the extracellular serotonin concentration and maintaining serotonergic neurotransmission. This process can be influenced by molecules acting as serotonin uptake inhibitors, like paroxetine. Here, we report the development of a novel photoswitchable paroxetine derivative and its pharmacological interaction profile with SERT as a tool compound for the light-induced control of SERT. Based on the azo-extension strategy, the photoswitchable moiety was formed at the former position of the fluoro substituent in paroxetine. The resulting azo-paroxetine (9) was easily and reversibly switched between active (Z) and inactive (E) configurations and remained stable between these configurations: serotonin uptake was inhibited more than 12 times more potently by the active (Z)-configuration having a sub μM IC50 value. This was supported by electrophysiological patch-clamp recordings in the whole-cell configuration and docking studies. No significant toxic impact of azo-paroxetine (9) and no off-target activity at the norepinephrine transporter (NET), human GABA transporter subtypes 1 and 3, and rat GAT1 were observed. Our results demonstrate that the activity of SERT can be reversibly manipulated by the optopharmacological agent azo-paroxetine (9). This compound can thus be applied as a tool for the selective manipulation of SERT in central or peripheral investigations, further benefiting from its low probability for compound-related off-target effects.

Fluorescent labeling techniques have enabled the visualization of various biomolecules, cellular structures, and their associated physiological processes. At the same time, there remains a demand for developing novel fluorescent compounds possessing unique chemical properties for biological imaging. A recently developed class of fluorophores, termed PyrAtes, displays optimal brightness and large Stokes shifts that are ideal for fluorescence microscopy. Herein, we report the development of PyrAte-based fluorescently labeled ligands that bind to the serotonin transporter (SERT), a membrane transport protein important for neurotransmitter homeostasis, which hitherto has not been visualized in its native environment using fluorescent small molecules. The design of a PyrAte fluorophore attached to (S)-citalopram, a selective serotonin reuptake inhibitor, resulted in the synthesis of two fluorescent drug conjugates varying in linker length: PYR-C6-CIT and PYR-C3-CIT. Docking and molecular dynamics experiments are performed to estimate their binding affinities to SERT. Our in vitro experiments confirm both compounds are effectively binding to SERT overexpressed in human embryonic kidney 293 cells, with the shorter conjugate displaying improved SERT affinity and membrane staining properties. Furthermore, ex vivo imaging of endogenous SERT was demonstrated in acute mouse brain slices using two-photon microscopy. The large Stokes shift of the PyrAte fluorophore enables simultaneous detection of its own fluorescence signal at 500 nm along with that of a yellow fluorescent protein-based serotonergic marker. Our findings provide novel tools for unprecedented SERT visualization and establish the utility of PyrAtes for the selective staining of membrane proteins in live cells and tissue.

The human γ-aminobutyric acid (GABA) transporter 1 (hGAT-1) plays a pivotal role in synaptic neurotransmission by facilitating the clearance of GABA from the synaptic cleft. Pathogenic mutations in the SLC6A1 gene encoding hGAT-1 have been implicated in a spectrum of neurodevelopmental disorders, including epilepsy, autism spectrum disorder, intellectual disability, and developmental delay. Here, we elucidate the molecular and functional consequences of disease-associated mutations affecting the highly conserved glycine residue at position 443 (G443) in hGAT-1. Through a combination of in vitro biochemical analyses, ion flux assays, and pharmacological profiling in HEK293 cells, alongside in vivo studies in Drosophila melanogaster, we demonstrate that substitutions of G443 to aspartate (G443D) or valine (G443V) result in complete abolishment of GABA transport. This severe impairment stems from distinct disruptions in protein folding and trafficking. In particular, G443V is fully retained in the endoplasmic reticulum (ER), as substantiated by de-glycosylation assays indicating exclusively core-glycosylated protein bands and confocal co-localization with the ER chaperone calnexin. The G443D variant, on the other hand, exhibits partial trafficking to the plasma membrane, confirmed by the presence of maturely glycosylated bands, albeit at significantly reduced expression levels relative to the wild type transporter. Treatment with glycerol and 4-phenylbutyrate (4-PBA) successfully restored both surface expression and GABA uptake activity of the G443 mutants. Our findings highlight the potential of small-molecule chaperones as interventions for ameliorating protein misfolding and functional deficits in hGAT-1-associated pathologies.

Neurotransmitter transporters are essential regulators of synaptic neurotransmission, maintaining the delicate balance of neurotransmitters in the central and peripheral nervous systems. Dysfunction of these transporters is linked to a wide range of neurological and psychiatric disorders, including depression, Parkinson's disease, epilepsy, and substance use disorders. This special issue of The Journal of Neurochemistry brings together cutting-edge research that advances our understanding of the molecular mechanisms, structural dynamics, and physiological roles of neurotransmitter transporters. The contributions highlight key themes, including the regulation of transporter trafficking and surface expression through post-translational modifications, the structural dynamics underlying transporter function, and the role of glutamate transporters in excitotoxicity and stress resilience. Additionally, the issue explores pharmacological innovations, such as optopharmacological tools and bioisosteric analogs, that offer new avenues for therapeutic intervention. By integrating insights from structural biology, electrophysiology, genetic models, and computational approaches, this collection underscores the interdisciplinary nature of transporter research and its potential to inform the development of novel therapies for neurological and psychiatric conditions. The articles collectively emphasize the progress and challenges in the field while pointing toward future directions for translational research and precision medicine.

The human monoamine transporters (MATs) for serotonin (SERT), dopamine (DAT), and norepinephrine (NET) play a key role in neurotransmission by transporting neurotransmitters from the synaptic cleft back into the neuron. MATs are embedded in the cell membrane’s lipid bilayer, encompassing cholesterol, phospholipids, and sphingolipids as main components. Membrane cholesterol association has been shown for all MATs impacting transporter conformation, substrate affinity, transport velocity, and turnover rates. In the present study, we compared the regulatory impact of cholesterol on the uptake and efflux function, binding affinity, and transporter oligomerization across all three MATs. We observed that cholesterol depletion impairs transporter-mediated uptake in human transporter-transfected HEK293 cells and reduces the binding affinity of all MATs. Electrophysiological investigations in SERT-expressing cells revealed that cholesterol alterations affect the transition of the transporter from the outward to the inward-facing conformation in the presence of substrate. Upon cholesterol depletion, FRET imaging and single molecule microscopy studies indicated altered oligomerization behavior exclusively for SERT. Interestingly, reduction of membrane cholesterol selectively increased amphetamine-induced efflux via SERT, while efflux via DAT and NET was reduced. This effect was diminished in a mutant with reduced PIP2 binding capacity. Hence, the increased efflux at SERT due to cholesterol depletion appears to depend on the ability of PIP2 to bind to SERT. Thus, we hypothesize that the interaction profile between cholesterol and MATs may fine-tune the transporter functionality and influence MAT-dependent disorders.

As the first member of the solute carrier 6 (SLC6) protein family, the γ-aminobutyric acid (GABA) transporter 1 (GAT1, SLC6A1), plays a pivotal role in the uptake of GABA from the synaptic cleft into neurons and astrocytes. This process facilitates the subsequent storage of GABA in presynaptic vesicles. The human SLC6A1 gene is highly susceptible to missense mutations, leading to severe clinical outcomes, such as epilepsy, in the afflicted patients. The molecular mechanisms of SLC6A1-associated disorders are discerned to some degree; many SLC6A1 mutations are now known to impair protein folding, and consequently fail to reach the plasma membrane. Inherently, once inside the endoplasmic reticulum (ER), GAT1 abides by a complex cascade of events that enable efficient intracellular trafficking. This involves association with specialized molecular chaperones responsible for steering the protein folding process, oligomerization, sorting through the Golgi apparatus, and ultimately delivery to the cell surface. The entire process is subject to stringent quality control mechanisms at multiple checkpoints. While the majority of the existing loss-of-function SLC6A1 variants interfere with folding and membrane targeting, certain mutants retain abundant surface expression. In either scenario, suppressed GAT1 activity disrupts GABAergic neurotransmission, preceding the disease manifestation in individuals harboring these mutations. The nervous system is enthralling and calls for systematic, groundbreaking research efforts to dissect the precise molecular factors associated with the onset of complex neurological disorders, and uncover additional non-canonical therapeutic targets. Recent research has given hope for some of the misfolded SLC6A1 variants, which can be salvaged by small molecules, i.e., chemical and pharmacological chaperones, acting on multiple upstream targets in the secretory pathway. We here highlight the significance of pharmacochaperoning as a therapeutic strategy for the treatment of SLC6A1-related disorders.

The presynaptic serotonin transporter (SERT) clears extracellular serotonin following vesicular release to ensure temporal and spatial regulation of serotonergic signalling and neurotransmitter homeostasis. Prescription drugs used to treat neurobehavioral disorders, including depression, anxiety, and obsessive-compulsive disorder, trap SERT by blocking the transport cycle. In contrast, illicit drugs of abuse like amphetamines reverse SERT directionality, causing serotonin efflux. Both processes result in increased extracellular serotonin levels. By combining molecular dynamics simulations with biochemical experiments and using a homologous series of serotonin analogues, we uncovered the coupling mechanism between the substrate and the transporter, which triggers the uptake of serotonin. Free energy analysis showed that only scaffold-bound substrates could initiate SERT occlusion through attractive long-range electrostatic interactions acting on the bundle domain. The associated spatial requirements define substrate and inhibitor properties, enabling additional possibilities for rational drug design approaches.

The human GABA transporter (GAT1) is a membrane transporter that mediates the reuptake of the neurotransmitter GABA from the synaptic cleft into neurons and glial cells. Dysregulation of the transport cycle has been associated with epilepsy and neuropsychiatric disorders, highlighting the crucial role of the transporter in maintaining homeostasis of brain GABA levels. GAT1 is a secondary active transporter that couples the movement of substrate to the simultaneous transport of sodium and chloride ions along their electrochemical gradients. Using MD simulations, we identified a novel sodium recruiting site at the entrance to the outer vestibule, which attracts positively charged ions and increases the local sodium concentration, thereby indirectly increasing sodium affinity. Mutations of negatively charged residues at the recruiting site slowed the binding kinetics, while experimental data revealed a change in sodium dependency of GABA uptake and a reduction of sodium affinity. Simulation showed that sodium displays a higher affinity for the sodium binding site NA2, which plays a role in stabilisation of the outward-open conformation. We directly show that the presence of a sodium ion bound to NA2 increases the stability of the closed inner gate and restrains motions of TM5. We find that sodium is only weakly bound to NA1 in the absence of GABA, while the presence of the substrate strengthens the interaction due to the completed ion coordinating shell, explaining cooperativity between GABA and sodium.

Recent studies have sparked renewed interest in the therapeutic potential of psychedelics for treating depression and other mental health conditions. Simultaneously, the novel psychoactive substances (NPS) phenomenon, with a huge number of NPS emerging constantly, has changed remarkably the illicit drug market, being their scientific evaluation an urgent need. Thus, this study aims to elucidate the impact of amino-terminal modifications to the 5-MeO-DMT molecule on its interactions with serotonin receptors and transporters, as well as its psychoactive and thermoregulatory properties. Our findings demonstrated, using radioligand binding methodologies, that all examined 5-MeO-tryptamines exhibited selectivity for 5-HT1AR over 5-HT2AR. In fact, computational docking analyses predicted a better interaction in the 5-HT1AR binding pocket compared to 5-HT2AR. Our investigation also proved the interaction of these compounds with SERT, revealing that the molecular size of the amino group significantly influenced their affinity. Subsequent experiments involving serotonin uptake, electrophysiology, and superfusion release assays confirmed 5-MeO-pyr-T as the most potent partial 5-HT releaser tested. All tested tryptamines elicited, to some degree, the head twitch response (HTR) in mice, indicative of a potential hallucinogenic effect and mainly mediated by 5-HT2AR activation. However, 5-HT1AR was also shown to be implicated in the hallucinogenic effect, and its activation attenuated the HTR. In fact, tryptamines that produced a higher hypothermic response, mediated by 5-HT1AR, tended to exhibit a lower hallucinogenic effect, highlighting the opposite role of both 5-HT receptors. Moreover, although some 5-MeO-tryptamines elicited very low HTR, they still act as potent 5-HT2AR agonists. In summary, this research offers a comprehensive understanding of the psychopharmacological profile of various amino-substituted 5-MeO-tryptamines, keeping structural aspects in focus and accumulating valuable data in the frame of NPS. Moreover, the unique characteristics of some 5-MeO-tryptamines render them intriguing molecules as mixed-action drugs and provide insight within the search of non-hallucinogenic but 5-HT2AR ligands as therapeutical agents.

Three studies of the human dopamine transporter reveal how it binds to molecules such as dopamine and cocaine. Dopamine imbalances underlie some brain conditions and these data will aid targeted drug design. Insights into how molecules bind to the human dopamine transporter.

The deployment of small-molecule fluorescent agents plays an ever-growing role in medicine and drug development. Herein, we complement the portfolio of powerful fluorophores, reporting the serendipitous discovery and development of a novel class with an imidazo[1,2-a]pyridinium triflate core, which we term PyrAtes. These fluorophores are synthesized in a single step from readily available materials (>60 examples) and display Stokes shifts as large as 240 nm, while also reaching NIR-I emissions at λmax as long as 720 nm. Computational studies allow the development of a platform for the prediction of λmax and λEm. Furthermore, we demonstrate the compatibility of these novel fluorophores with live cell imaging in HEK293 cells, suggesting PyrAtes as potent intracellular markers.

3,4-Methylenedioxymethamphetamine (MDMA, ' ecstasy' ) is re-emerging in clinical settings as a candidate for the treatment of specific psychiatric disorders (e.g. post-traumatic stress disorder) in combination with psychotherapy. MDMA is a psychoactive drug, typically regarded as an empathogen or entactogen, which leads to transporter-mediated monoamine release. Despite its therapeutic potential, MDMA can induce dose-, individual-, and context-dependent untoward effects outside safe settings. In this study, we investigated whether three new methylenedioxy bioisosteres of MDMA improve its off-target profile. In vitro methods included radiotracer assays, transporter electrophysiology, bioluminescence resonance energy transfer and fluorescence-based assays, pooled human liver microsome/S9 fraction incubation with isozyme mapping, and liquid chromatography coupled to high-resolution mass spectrometry. In silico methods included molecular docking. Compared with MDMA, all three MDMA bioisosteres (ODMA, TDMA, and SeDMA) showed similar pharmacological activity at human serotonin and dopamine transporters (hSERT and hDAT, respectively) but decreased activity at 5-HT 2A/2B/2C receptors. Regarding their hepatic metabolism, they differed from MDMA, with N -demethylation being the only metabolic route shared, and without forming phase II metabolites. Additional screening for their interaction with human organic cation transporters (hOCTs) and plasma membrane transporter (hPMAT) revealed a weaker interaction of the MDMA analogs with hOCT1, hOCT2, and hPMAT. Our findings suggest that these new MDMA analogs might constitute appealing therapeutic alternatives to MDMA, sparing the primary pharmacological activity at hSERT and hDAT, but displaying a reduced activity at 5-HT 2A/2B/2C receptors and reduced hepatic metabolism. Whether these MDMA bioisosteres may pose lower risk alternatives to the clinically re-emerging MDMA warrants further studies.

AbstractDer Einsatz kleiner fluoreszierender Moleküle spielt in der Medizin und Arzneimittelentwicklung eine immer größere Rolle. Mit diesem Beitrag ergänzen wir das Portfolio leistungsstarker Fluorophore durch die zufällige Entdeckung und Entwicklung einer neuen Klasse mit einem Imidazo[1,2‐a]pyridiniumtriflat‐Kern, die wir „PyrAt‐Verbindungen“ nennen. Diese Fluorophore werden in einem einzigen Schritt aus leicht verfügbaren Materialien synthetisiert (>60 Beispiele) und zeigen Stokes‐Verschiebungen bis zu 240 nm, während sie gleichzeitig NIR−I‐Emissionen bei λmax bis zu 720 nm erreichen. Computergestützte Studien ermöglichen die Entwicklung einer Plattform für die Vorhersage von λmax und λEm. Darüber hinaus demonstrieren wir die Kompatibilität dieser neuartigen Fluorophore mit der Lebendzellbildgebung in HEK293‐Zellen, was darauf hindeutet, dass sie potente intrazelluläre Marker sind.