Creatine transporter 1 (CT1, SLC6A8) regulates cellular energy levels. Mutations in CT1 cause severe neurological disorders, whereas CT1 overexpression has been associated with cancer progression. Rational development of CT1 inhibitors has been limited by the absence of structural data. Here, we constructed homology models of CT1 representing distinct transport states and integrated them into a structure-based virtual screening workflow. From this approach, 16 top-ranked compounds were tested in vitro. Among them, compound 11 showed inhibitory activity with an IC50 comparable to the reference ligand ompenaclid. We demonstrate that tiagabine (13) and its analogue 16 also inhibit CT1. Following the completion of this study, cryo-EM structures of CT1 were reported. Retrospective comparison confirmed good agreement between our models and the experimental structures. These findings provide a computational-experimental framework for CT1 inhibitor discovery and support future structure-based drug design.

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.

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.

In this chapter we will show how electrophysiological recordings were used to gain insights into the transport kinetics and pharmacology of monoamine transporters (MATs). We will discuss data obtained from whole cell patch clamp recordings that allow for real time monitoring of MAT function. A notable property of MATs is that they carry so-called uncoupled currents. We will begin this chapter by reviewing the experimental evidence that has led to the conclusion that the currents carried by MATs are largely uncoupled and, therefore, not directly related to substrate transport. We will discuss how this has made it difficult to understand the operation of MATs. We will also explain why the existence of these currents has led to the proposition that MATs do not operate by alternate access but rather by a single file diffusion mechanism. However, we will show that ultimately the uncoupled currents carried by MATs can be most parsimoniously explained within the framework of the alternate access mechanism. We will review the existing evidence that MATs, like most other transporters, undergo a cycle during which they visit outward and inward-facing conformations (i.e., the transport cycle). We will outline what we have learned about the transport cycle of MATs from electrophysiological recordings. Thereafter, we will describe how electrophysiological recordings can be utilized to understand how drugs that target MATs affect their operation. To this end, we will discuss the binding modes of three different MAT ligands: (i) amphetamines, (ii) ibogaine, and (iii) zinc.

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.

The creatine transporter-1 (CRT-1/SLC6A8) maintains the uphill transport of creatine into cells against a steep concentration gradient. Cellular creatine accumulation is required to support the ATP-buffering by phosphocreatine. More than 60 compounds have been explored in the past for their ability to inhibit cellular creatine uptake, but the number of active compounds is very limited. Here, we show that all currently known inhibitors are full alternative substrates. We analyzed their structure-activity relationship for inhibition of CRT-1 to guide a rational approach to the synthesis of novel creatine transporter ligands. Measurements of both inhibition of [3H]creatine uptake and transport associated currents allowed for differentiating between full and partial substrates and true inhibitors. This combined approach led to a refined understanding of the structural requirements for binding to CRT-1, which translated into the identification of three novel compounds- i.e., compound 1 (2-(N-benzylcarbamimidamido)acetic acid), MIPA572 (=carbamimidoylphenylalanine), and MIPA573 (=carbamimidoyltryptophane) that blocked CRT- 1 transport, albeit with low affinity. In addition, we found two new alternative full substrates, namely MIPA574 (carbamimidoylalanine) and GiDi1257 (1-carbamimidoylazetidine-3-carboxylic acid), which was superior in affinity to all known CTR-1 ligands, and one partial substrate, namely GiDi1254 (1-carbamimidoylpiperidine-4-carboxylic acid). SIGNIFICANCE STATEMENT The creatine transporter-1 (CRT-1) is required to maintain intracellular creatine levels. Inhibition of CRT-1 has been recently proposed as a therapeutic strategy for cancer, but pharmacological tools are scarce. In fact, all available inhibitors are alternative substrates. We tested existing and newly synthesized guanidinocarboxylic acids for CRT-1 inhibition and identified three blockers, one partial and two full substrates of CRT-1. Our results support a refined structural understanding of ligand binding to CRT-1 and provide a proof-of-principle for blockage of CRT-1.

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.

The antidepressants trazodone and nefazodone were approved some 4 and 3 decades ago, respectively. Their action is thought to be mediated, at least in part, by inhibition of the serotonin transporter [SERT/solute carrier (SLC)-6A4]. Surprisingly, their mode of action on SERT has not been characterized. Here, we show that, similar to the chemically related drug vilazodone, trazodone and nefazodone are allosteric ligands: trazodone and nefazodone inhibit uptake by and transport-associated currents through SERT in a mixed-competitive and noncompetitive manner, respectively. Contrary to noribogaine and its congeners, all three compounds preferentially interact with the Na+-bound outward-facing state of SERT. Nevertheless, they act as pharmacochaperones and rescue the folding-deficient variant SERT-P601A/G602A. The vast majority of disease-associated point mutations of SLC6 family members impair folding of the encoded transporter proteins. Our findings indicate that their folding defect can be remedied by targeting allosteric sites on SLC6 transporters. SIGNIFICANCE STATEMENT: The serotonin transporter is a member of the solute carrier-6 family and is the target of numerous antidepressants. Trazodone and nefazodone have long been used as antidepressants. Here, this study shows that their inhibition of the serotonin transporter digressed from the competitive mode seen with other antidepressants. Trazodone and nefazodone rescued a folding-deficient variant of the serotonin transporter. This finding demonstrates that folding defects of mutated solute carrier-6 family members can also be corrected by allosteric ligands.

Orphan solute carrier (SLC) represents a group of membrane transporters whose exact functions and substrate specificities are not known. Elucidating the function and regulation of orphan SLC transporters is not only crucial for advancing our knowledge of cellular and molecular biology but can potentially lead to the development of new therapeutic strategies. Here, we provide evidence for the biological function of a ubiquitous orphan lysosomal SLC, the Major Facilitator Superfamily Domain-containing Protein 1 (MFSD1), which has remained phylogenetically unassigned. Targeted metabolomics revealed that dipeptides containing either lysine or arginine residues accumulate in lysosomes of cells lacking MFSD1. Whole-cell patch-clamp electrophysiological recordings of HEK293-cells expressing MFSD1 on the cell surface displayed transport affinities for positively charged dipeptides in the lower mM range, while dipeptides that carry a negative net charge were not transported. This was also true for single amino acids and tripeptides, which MFSD1 failed to transport. Our results identify MFSD1 as a highly selective lysosomal lysine/arginine/histidine-containing dipeptide exporter, which functions as a uniporter.

Python code for a global activation energy (Ea) fit to temperature dependent stopped-flow data (*.bka file): As shown in our manuscript, the accuracy of Ea estimation from stopped-flow data is greatly increased by a global fit to the data. Thereby, the ensemble of curves of a sample at different temperatures is fitted globally using the analytical solution (Eqn. 10), instead of evaluating permeabilities for different temperatures individually and independently. In our python code, osmotic water flux measurements of a sample (mixture of vesicles with and without water channels) recorded at different temperatures are simultaneously fitted using Eqn. 7, Eqn. 10, Eqn. 11 and Eqn. 14. Here, the only free fit parameter, which is evaluated for the different temperatures independently is coefficient b from Eqn. 7. The accuracy of the results increases with the span of the temperature values, number of performed measurements and a decreased empty vesicle fraction.

The human serotonin transporter hSERT, or SLC6A4, from the solute carrier 6 (SLC6) protein family, plays an important role in the central and peripheral nervous systems. Its major neurobiological task is the termination of serotonergic neurotransmission by reuptake of its endogenous substrate, serotonin, into presynaptic boutons after signaling. The transporter undergoes several conformational changes in each physiological substrate uptake cycle and interacts with protons, sodium, chloride, and potassium ions during these transitions. Over the years, a substantial body of knowledge about hSERT function and mechanism has been accumulated, significantly improving pharmacological treatment options especially for major depression by covering several chemical compound classes. Nevertheless, effective symptom amelioration, side effect profiles, and patient compliance are still far from ideal and require urgent action. For this reason, a fundamental approach is to tackle key unanswered questions regarding the transporter's function, including the mechanism of potassium and proton binding, whose roles are both interconnected and poorly understood. To this end, we have applied molecular simulation approaches at the coarse-grained and fully-atomistic levels, including free energy calculations. The simulations predict the locations of the potassium and proton binding sites and suggest the molecular origins of selectivity at each site. In addition to the gains in basic scientific understanding, the knowledge obtained paves the way for designing mechanism-based transporter ligands that are useful as highly specialized therapeutic agents.

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 ef-fects, 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 sta-bilized an outward-closed state of the SERT with little activity against common off-targets. A cryo-EM struc-ture 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 fluox-etine (Prozac), and one substantially reversed morphine withdrawal effects.

Psychostimulants interacting with the dopamine transporter (DAT) can be used illicitly or for the treatment of specific neuropsychiatric disorders. However, they can also produce severe and persistent adverse events. Often, their pharmacological properties in vitro do not fully correlate to their pharmacological profile in vivo. Here, we investigated the pharmacological effects of enantiomers of pyrovalerone, α-pyrrolidinovalerophenone, and 3,4-methylenedioxypyrovalerone as compared to the traditional psychostimulants cocaine and methylphenidate, using a variety of in vitro, computational, and in vivo approaches. We found that in vitro drug-binding kinetics at DAT correlate with the time-course of in vivo psychostimulant action in mice. In particular, a slow dissociation (i.e., slowkoff) ofS-enantiomers of pyrovalerone analogs from DAT predicts their more persistent in vivo effects when compared to cocaine and methylphenidate. Overall, our findings highlight the critical importance of drug-binding kinetics at DAT for determining the in vivo profile of effects produced by psychostimulant drugs.

Large-scale drug screening is currently the basis for the identification of new chemical entities. This is a rather laborious approach, because a large number of compounds must be tested to cover the chemical space in an unbiased fashion. However, the structures of targetable proteins have become increasingly available. Thus, a new era has arguably been ushered in with the advent of methods, which allow for structure-based docking campaigns (i.e., virtual screens). Solute carriers (SLCs) are among the most promising drug targets. This claim is substantiated by the fact that a large fraction of the 400 solute carrier genes is associated with human diseases. The ability to dock large ligand libraries into selected structures of solute carriers has set the stage for rational drug design. In the present study, we show that these structure-based approaches can be refined by taking into account how solute carriers operate. We specifically address the feasibility of targeting solute carriers with allosteric modulators, because their actions differ fundamentally from those of ligands, which bind to the substrate binding site. For the pertinent analysis we used transition state theory in conjunction with the linear free energy relationship (LFER). These provide the theoretical framework to understand how allosteric modulators affect solute carrier function.

EDITORIAL article Front. Physiol., 08 November 2023Sec. Membrane Physiology and Membrane Biophysics Volume 14 - 2023 | https://doi.org/10.3389/fphys.2023.1320336

Typical and atypical dopamine uptake inhibitors (DUIs) prefer distinct conformations of the dopamine transporter (DAT) to form ligand-transporter complexes, resulting in markedly different effects on behavior, neurochemistry, and potential for addiction. Here we show that cocaine and cocaine-like typical psychostimulants elicit changes in DA dynamics distinct from those elicited by atypical DUIs, as measured via voltammetry procedures. While both classes of DUIs reduced DA clearance rate, an effect significantly related to their DAT affinity, only typical DUIs elicited a significant stimulation of evoked DA release, an effect unrelated to their DAT affinity, which suggests a mechanism of action other than or in addition to DAT blockade. When given in combination, typical DUIs enhance the stimulatory effects of cocaine on evoked DA release while atypical DUIs blunt them. Pretreatments with an inhibitor of CaMKIIα, a kinase that interacts with DAT and that regulates synapsin phosphorylation and mobilization of reserve pools of DA vesicles, blunted the effects of cocaine on evoked DA release. Our results suggest a role for CaMKIIα in modulating the effects of cocaine on evoked DA release without affecting cocaine inhibition of DA reuptake. This effect is related to a specific DAT conformation stabilized by cocaine. Moreover, atypical DUIs, which prefer a distinct DAT conformation, blunt cocaine’s neurochemical and behavioral effects, indicating a unique mechanism underlying their potential as medications for treating psychostimulant use disorder.

Mephedrone (4-methylmethcathinone) is a cathinone derivative that is recreationally consumed for its energizing and empathogenic effects. The stimulating properties are believed to arise from the ability of mephedrone to interact with the high-affinity transporters for dopamine (DA) (DAT) and norepinephrine (NET), whereas the entactogenic effect presumably relies on its activity at the serotonin (5-HT) transporter (SERT). Early studies found that mephedrone acts as a releaser at NET, DAT and SERT, and thus promotes efflux of the respective monoamines. Evidence linked drug-induced reverse transport of 5-HT via SERT to prosocial effects, whereas activity at DAT is strongly correlated with abuse liability. Consequently, we sought to evaluate the pharmacology of mephedrone at human (h) DAT and SERT, heterologously expressed in human embryonic kidney 293 cells, in further detail. In line with previous studies, we report that mephedrone evokes carrier-mediated release via hDAT and hSERT. We found this effect to be sensitive to the protein kinase C inhibitor GF109203X. Electrophysiological recordings revealed that mephedrone is actively transported by hDAT and hSERT. However, mephedrone acts as a full substrate of hSERT but as a partial substrate of hDAT. Furthermore, when compared to fully efficacious releasing agents at hDAT and hSERT (i.e. S(+)-amphetamine and para-chloroamphetamine, respectively) mephedrone displays greater efficacy as a releaser at hSERT than at hDAT. In summary, this study provides additional insights into the molecular mechanism of action of mephedrone at hDAT and hSERT.

The serotonin transporter (SERT/SLC6A4) is arguably the most extensively studied solute carrier (SLC). During its eponymous action - that is, the retrieval of serotonin from the extracellular space - SERT undergoes a conformational cycle. Typical inhibitors (antidepressant drugs and cocaine), partial and full substrates (amphetamines and their derivatives), and atypical inhibitors (ibogaine analogues) bind preferentially to different states in this cycle. This results in competitive or non-competitive transport inhibition. Here, we explored the action of N-formyl-1,3-bis (3,4-methylenedioxyphenyl)-prop-2-yl-amine (ECSI#6) on SERT: inhibition of serotonin uptake by ECSI#6 was enhanced with increasing serotonin concentration. Conversely, the KM for serotonin was lowered by augmenting ECSI#6. ECSI#6 bound with low affinity to the outward-facing state of SERT but with increased affinity to a potassium-bound state. Electrophysiological recordings showed that ECSI#6 preferentially interacted with the inward-facing state. Kinetic modeling recapitulated the experimental data and verified that uncompetitive inhibition arose from preferential binding of ECSI#6 to the K+-bound, inward-facing conformation of SERT. This binding mode predicted a pharmacochaperoning action of ECSI#6, which was confirmed by examining its effect on the folding-deficient mutant SERT-PG601,602AA: preincubation of HEK293 cells with ECSI#6 restored export of SERT-PG601,602AA from the endoplasmic reticulum and substrate transport. Similarly, in transgenic flies, the administration of ECSI#6 promoted the delivery of SERT-PG601,602AA to the presynaptic specialization of serotonergic neurons. To the best of our knowledge, ECSI#6 is the first example of an uncompetitive SLC inhibitor. Pharmacochaperones endowed with the binding mode of ECSI#6 are attractive, because they can rescue misfolded transporters at concentrations, which cause modest transport inhibition.

Increasing extracellular levels of serotonin (5-HT) in the brain ameliorates symptoms of depression and anxiety-related disorders, e.g., social phobias and post-traumatic stress disorder. Recent evidence from preclinical and clinical studies established the therapeutic potential of drugs inducing the release of 5-HT via the 5-HT-transporter. Nevertheless, current 5-HT releasing compounds under clinical investigation carry the risk for abuse and deleterious side effects. Here, we demonstrate that S-enantiomers of certain ring-substituted cathinones show preference for the release of 5-HT ex vivo and in vivo, and exert 5-HT-associated effects in preclinical behavioral models. Importantly, the lead cathinone compounds (1) do not induce substantial dopamine release and (2) display reduced off-target activity at vesicular monoamine transporters and 5-HT2B-receptors, indicative of low abuse-liability and low potential for adverse events. Taken together, our findings identify these agents as lead compounds that may prove useful for the treatment of disorders where elevation of 5-HT has proven beneficial.