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3,4-Methylenedioxymethamphetamine (MDMA, 'ecstasy') is re-emerging in clinical settings as a candidate for the treatment of specific neuropsychiatric 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 incubations, metabolic stability studies, 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, dopamine, and norepinephrine transporters (hSERT, hDAT, and hNET, respectively) but decreased agonist activity at 5-HT2A/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. In addition, TDMA showed an enhanced intrinsic clearance in comparison to its congeners. Additional screening for their interaction with human organic cation transporters (hOCTs) and plasma membrane monoamine transporter (hPMAT) revealed a weaker interaction of the MDMA analogs with hOCT1, hOCT2, and hPMAT. Our findings suggest that these new MDMA bioisosteres might constitute appealing therapeutic alternatives to MDMA, sparing the primary pharmacological activity at hSERT, hDAT, and hNET, but displaying a reduced activity at 5-HT2A/2B/2C receptors and alternative hepatic metabolism. Whether these MDMA bioisosteres may pose lower risk alternatives to the clinically re-emerging MDMA warrants further studies.
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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.
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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.
Assuntos
Simulação de Dinâmica Molecular , Potássio , Proteínas da Membrana Plasmática de Transporte de Serotonina , Sódio , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Potássio/metabolismo , Sítios de Ligação , Humanos , Sódio/metabolismo , Serotonina/metabolismo , Ligação Proteica , AnimaisRESUMO
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.
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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.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Serotonina , Serotonina , Humanos , Ligantes , Ansiedade , Transtornos de AnsiedadeRESUMO
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.
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Monoamine transporters are of great interest for their role in the physiological activity of the body and their link to mental and behavioural disorders. Currently, static well-plate assays or manual perfusion systems are used to characterize the interaction of psychostimulants, antidepressants and drugs of abuse with the transporters but still suffer from significant drawbacks caused by lack of automation, for example, low reproducibility, non-comparability of results. An automated microfluidic platform was developed to address the need for more standardized procedures for cell-based assays. An automated system was used to control and drive the simultaneous perfusion of 12 channels on a microfluidic chip, establishing a more standardized protocol to perform release assays to study monoamine transporter-mediated substrate efflux. D-Amphetamine, GBR12909 (norepinephrine transporter) and p-chloroamphetamine, paroxetine (serotonin transporter) were used as control compounds to validate the system. The platform was able to produce the expected releasing (D-Amphetamine, p-chloroamphetamine) or inhibiting (GBR12909, paroxetine) profiles for the two transporters. The reduction of manual operation and introduction of automated flow control enabled the implementation of stronger standardized protocols and the possibility of obtaining higher throughput by increasing parallelization.
Assuntos
Microfluídica , p-Cloroanfetamina , Paroxetina , Reprodutibilidade dos Testes , Proteínas de Membrana Transportadoras , Perfusão , DextroanfetaminaRESUMO
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., slow koff) of S-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.
Assuntos
Estimulantes do Sistema Nervoso Central , Cocaína , Metilfenidato , Camundongos , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Estimulantes do Sistema Nervoso Central/farmacologia , Cocaína/farmacologia , Cocaína/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Metilfenidato/farmacologiaRESUMO
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.
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Dopamina , Serotonina , Encéfalo , Proteínas de TransporteRESUMO
Epidemiological studies indicate a bidirectional association between metabolic disturbances, including obesity and related pathological states, and mood disorders, most prominently major depression. However, the biological mechanisms mediating the comorbid relationship between the deranged metabolic and mood states remain incompletely understood. Here, we tested the hypothesis that the enhanced activation of brown fat tissue (BAT), known to beneficially regulate obesity and accompanying dysfunctional metabolic states, is also paralleled by an alteration of affective behaviour. We used upstream stimulatory factor 1 (USF-1) knock-out (KO) mice as a genetic model of constitutively activated BAT and positive cardiometabolic traits and found a reduction of depression-like and anxiety-like behaviours associated with USF-1 deficiency. Surgical removal of interscapular BAT did not impact the behavioural phenotype of USF-1 KO mice. Further, the absence of USF-1 did not lead to alterations of adult hippocampal neural progenitor cell proliferation, differentiation, or survival. RNA-seq analysis characterised the molecular signature of USF-1 deficiency in the hippocampus and revealed a significant increase in the expression of several members of the X-linked lymphocyte-regulated (xlr) genes, including xlr3b and xlr4b. Xlr genes are the mouse orthologues of the human FAM9 gene family and are implicated in the regulation of dendritic branching, dendritic spine number and morphology. The transcriptional changes were associated with morphological alterations in hippocampal neurons, manifested in reduced dendritic length and complexity in USF-1 KO mice. Collectively these data suggest that the metabolic regulator USF-1 is involved in the control of affective behaviour in mice and that this modulation of mood states is unrelated to USF-1-dependent BAT activation, but reflected in structural changes in the brain.
Assuntos
Encéfalo , Transtorno Depressivo Maior , Adulto , Humanos , Animais , Camundongos , Camundongos Knockout , Transtornos de Ansiedade , HipocampoRESUMO
Dopamine (DA), the most abundant human brain catecholaminergic neurotransmitter, modulates key behavioral and neurological processes in young and senescent brains, including motricity, sleep, attention, emotion, learning and memory, and social and reward-seeking behaviors. The DA transporter (DAT) regulates transsynaptic DA levels, influencing all these processes. Compounds targeting DAT (e.g., cocaine and amphetamines) were historically used to shape mood and cognition, but these substances typically lead to severe negative side effects (tolerance, abuse, addiction, and dependence). DA/DAT signaling dysfunctions are associated with neuropsychiatric and progressive brain disorders, including Parkinson's and Alzheimer diseases, drug addiction and dementia, resulting in devastating personal and familial concerns and high socioeconomic costs worldwide. The development of low-side-effect, new/selective medicaments with reduced abuse-liability and which ameliorate DA/DAT-related dysfunctions is therefore crucial in the fields of medicine and healthcare. Using the rat as experimental animal model, the present work describes the synthesis and pharmacological profile of (S)-MK-26, a new modafinil analogue with markedly improved potency and selectivity for DAT over parent drug. Ex vivo electrophysiology revealed significantly augmented hippocampal long-term synaptic potentiation upon acute, intraperitoneally delivered (S)-MK-26 treatment, whereas in vivo experiments in the hole-board test showed only lesser effects on reference memory performance in aged rats. However, in effort-related FR5/chow and PROG/chow feeding choice experiments, (S)-MK-26 treatment reversed the depression-like behavior induced by the dopamine-depleting drug tetrabenazine (TBZ) and increased the selection of high-effort alternatives. Moreover, in in vivo microdialysis experiments, (S)-MK-26 significantly increased extracellular DA levels in the prefrontal cortex and in nucleus accumbens core and shell. These studies highlight (S)-MK-26 as a potent enhancer of transsynaptic DA and promoter of synaptic plasticity, with predominant beneficial effects on effort-related behaviors, thus proposing therapeutic potentials for (S)-MK-26 in the treatment of low-effort exertion and motivational dysfunctions characteristic of depression and aging-related disorders.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina , Dopamina , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Motivação/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , RatosRESUMO
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.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Serotonina , Serotonina , Citalopram/química , Citalopram/farmacologia , Humanos , Simulação de Dinâmica Molecular , Domínios Proteicos , Estrutura Secundária de Proteína , Serotonina/química , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/química , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Relação Estrutura-AtividadeRESUMO
Many psychoactive compounds have been shown to primarily interact with high-affinity and low-capacity solute carrier 6 (SLC6) monoamine transporters for norepinephrine (NET; norepinephrine transporter), dopamine (DAT; dopamine transporter) and serotonin (SERT; serotonin transporter). Previous studies indicate an overlap between the inhibitory capacities of substances at SLC6 and SLC22 human organic cation transporters (SLC22A1-3; hOCT1-3) and the human plasma membrane monoamine transporter (SLC29A4; hPMAT), which can be classified as high-capacity, low-affinity monoamine transporters. However, interactions between central nervous system active substances, the OCTs, and the functionally-related PMAT have largely been understudied. Herein, we report data from 17 psychoactive substances interacting with the SLC6 monoamine transporters, concerning their potential to interact with the human OCT isoforms and hPMAT by utilizing radiotracer-based in vitro uptake inhibition assays at stably expressing human embryonic kidney 293 cells (HEK293) cells. Many compounds inhibit substrate uptake by hOCT1 and hOCT2 in the low micromolar range, whereas only a few substances interact with hOCT3 and hPMAT. Interestingly, methylphenidate and ketamine selectively interact with hOCT1 or hOCT2, respectively. Additionally, 3,4-methylenedioxymethamphetamine (MDMA) is a potent inhibitor of hOCT1 and 2 and hPMAT. Enantiospecific differences of R- and S-α-pyrrolidinovalerophenone (R- and S-α-PVP) and R- and S-citalopram and the effects of aromatic substituents are explored. Our results highlight the significance of investigating drug interactions with hOCTs and hPMAT, due to their role in regulating monoamine concentrations and xenobiotic clearance.
Assuntos
Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Transportador 1 de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico/metabolismo , Psicotrópicos/farmacologia , 3,4-Metilenodioxianfetamina/análogos & derivados , 3,4-Metilenodioxianfetamina/farmacologia , Linhagem Celular , Sistema Nervoso Central/efeitos dos fármacos , Citalopram/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Células HEK293 , Humanos , Pirrolidinas/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
The concentrative power of the transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT) is thought to be fueled by the transmembrane Na+ gradient, but it is conceivable that they can also tap other energy sources, for example, membrane voltage and/or the transmembrane K+ gradient. We have addressed this by recording uptake of endogenous substrates or the fluorescent substrate APP+(4-(4-dimethylamino)phenyl-1-methylpyridinium) under voltage control in cells expressing DAT, NET, or SERT. We have shown that DAT and NET differ from SERT in intracellular handling of K+. In DAT and NET, substrate uptake was voltage-dependent due to the transient nature of intracellular K+ binding, which precluded K+ antiport. SERT, however, antiports K+ and achieves voltage-independent transport. Thus, there is a trade-off between maintaining constant uptake and harvesting membrane potential for concentrative power, which we conclude to occur due to subtle differences in the kinetics of co-substrate ion binding in closely related transporters.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Potássio/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Compostos de Anilina/metabolismo , Transporte Biológico Ativo , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Células HEK293 , Humanos , Cinética , Potenciais da Membrana , Microscopia de Fluorescência , Microscopia de Vídeo , Modelos Biológicos , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/genética , Compostos de Piridínio/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/genéticaRESUMO
Organic cation transporters 1-3 (OCT1-3, SLC22A1-3) and the plasma membrane monoamine transporter (PMAT, SLC29A4) play a major role in maintaining monoaminergic equilibrium in the central nervous system. With many psychoactive substances interacting with OCT1-3 and PMAT, a growing literature focuses on characterizing their properties via in vitro and in vivo studies. In vitro studies mainly aim at characterizing compounds as inhibitors or substrates of murine, rat, and human isoforms. The preponderance of studies has put emphasis on phenylalkylamine derivatives, but ketamine and opioids have also been investigated. Studies employing in vivo (knockout) models mostly concentrate on the interaction of psychoactive substances and OCT3, with an emphasis on stress and addiction, pharmacokinetics, and sensitization to psychoactive drugs. The results highlight the importance of OCT3 in the mechanism of action of psychoactive compounds. Concerning in vivo studies, a veritable research gap concerning OCT1, 2, and PMAT exists. This review provides an overview and summary of research conducted in this field of research.
Assuntos
Proteínas de Transporte de Cátions Orgânicos , Transportador 1 de Cátions Orgânicos , Animais , Transporte Biológico , Cátions , Humanos , Camundongos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , RatosRESUMO
While classical cathinones, such as methcathinone, have been shown to be monoamine releasing agents at human monoamine transporters, the subgroup of α-pyrrolidinophenones has thus far solely been characterized as monoamine transporter reuptake inhibitors. Herein, we report data from previously undescribed α-pyrrolidinopropiophenone (α-PPP) derivatives and compare them with the pharmacologically well-researched α-PVP (α-pyrrolidinovalerophenone). Radiotracer-based in vitro uptake inhibition assays in HEK293 cells show that the investigated α-PPP derivatives inhibit the human high-affinity transporters of dopamine (hDAT) and norepinephrine (hNET) in the low micromolar range, with α-PVP being ten times more potent. Similar to α-PVP, no relevant pharmacological activity was found at the human serotonin transporter (hSERT). Unexpectedly, radiotracer-based in vitro release assays reveal α-PPP, MDPPP and 3Br-PPP, but not α-PVP, to be partial releasing agents at hNET (EC50 values in the low micromolar range). Furthermore, uptake inhibition assays at low-affinity monoamine transporters, i.e., the human organic cation transporters (hOCT) 1-3 and human plasma membrane monoamine transporter (hPMAT), bring to light that all compounds inhibit hOCT1 and 2 (IC50 values in the low micromolar range) while less potently interacting with hPMAT and hOCT3. In conclusion, this study describes (i) three new hybrid compounds that efficaciously block hDAT while being partial releasers at hNET, and (ii) highlights the interactions of α-PPP-derivatives with low-affinity monoamine transporters, giving impetus to further studies investigating the interaction of drugs of abuse with OCT1-3 and PMAT.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Norepinefrina/efeitos dos fármacos , Norepinefrina/metabolismo , Propiofenonas/farmacologia , Pirróis/farmacologia , Pirrolidinas/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Proteínas de Transporte de Nucleosídeo Equilibrativas/efeitos dos fármacos , Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Proteínas de Transporte de Cátions Orgânicos/efeitos dos fármacos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 1 de Cátions Orgânicos/efeitos dos fármacos , Transportador 1 de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico/efeitos dos fármacos , Transportador 2 de Cátion Orgânico/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismoRESUMO
Mephedrone is a largely abused psychostimulant. It elicits the release of monoamines via the high affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). Stereoselective metabolic reactions are involved in the inactivation and the elimination of its chemical structure. However, during these processes, several structures are generated and some of them have been reported to be still pharmacologically active. In this study 1) we have newly synthetized several putative mephedrone metabolites, 2) compared their activity at monoamine transporters, 3) generated quantitative structure activity relationships, and 4) exploited the chemical structure of the putative metabolites to screen a urine sample from a drug user and dissect mephedrone metabolism. We have found that most of the tested metabolites are weak inhibitors of monoamine transporters and that all of them are more potent at DAT and NET in comparison to SERT. The only exception is represented by the COOH-metabolite which shows no pharmacological activity at all three monoamine transporters. The enantioselectivity of mephedrone and its metabolites is present mainly at SERT, with only minor effects at DAT and NET being introduced when the ß-keto group is reduced to an OH-group. Importantly, while at DAT the putative metabolites did not show changes in inhibitory potencies, but rather changes in their substrate/blocker profile, at SERT they showed mainly changes in inhibitory potencies. Molecular modeling suggests that the hydrophobic nature of a specific SERT subpocket may be involved in such loss of affinity. Finally, the assessment of the putative metabolites in one urine sample of mephedrone user displayed two previously uncharacterized metabolites, 4-COOH-nor-mephedrone (4-COOH-MC) and dihydro-4- nor-mephedrone (dihydro-4-MC). These results confirm and expand previous studies highlighting the importance of the stereochemistry in the pharmacodynamics of phase-1 metabolites of mephedrone, established their structure-activity relationships at DAT, NET and SERT and pave the way for a systematic dissection of mephedrone metabolic routes. Given the number of structures found having residual and modified pharmacological profiles, these findings may help in understanding the complex subjective effects of administered mephedrone. Moreover, the dissection of mephedrone metabolic routes may help in developing new therapies for treating psychostimulants acute intoxications.
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Neurotransmitter transporters (NTTs) are involved in the fine-tuning of brain neurotransmitter homeostasis. As such, they are implicated in a plethora of complex behaviors, including reward, movement, and cognition. During recent decades, compounds that modulate NTT functions have been developed. Some of them are in clinical use for the management of different neuropsychiatric conditions. The majority of these compounds have been found to selectively interact with the orthosteric site of NTTs. Recently, diverse allosteric sites have been described in a number of NTTs, modulating their function. A more complex NTT pharmacology may be useful in the development of novel therapeutics. Here, we summarize current knowledge on such modulatory allosteric sites, with specific focus on their pharmacological and therapeutic potential.
Assuntos
Regulação Alostérica , Neurotransmissores , Proteínas de Transporte de Neurotransmissores , Sítio Alostérico , Homeostase , Humanos , Proteínas de Transporte de Neurotransmissores/efeitos dos fármacosRESUMO
The transporters for dopamine (DAT) and serotonin (SERT) are important targets in the treatment of psychiatric disorders including major depression, anxiety and attention-deficit hyperactivity disorder. Drugs acting at these transporters can act as inhibitors or as releasers. In addition, it has been recently appreciated that some compounds are less efficacious releasers than amphetamine. Thus, they are classified as partial releasers. Compounds can act on both SERT and DAT or display exquisite selectivity for either SERT or DAT, but the structural basis for selectivity is poorly understood. The trifluoromethyl-substitution of methcathinone in the para-position has been shown to dramatically shift the selectivity of methcathinone (MCAT) towards SERT. Here, we examined MCAT, para-trifluoromethyl-methcathinone (pCF3MCAT) and other analogues to understand (i) the determinants of selectivity and (ii) the effects of the para-CF3-substitution of MCAT on the transport cycle. We systematically tested different para-substituted MCATs by biochemical, computational and electrophysiological approaches: addition of the pCF3group, but not of other substituents with larger van der Waal's volume, lipophilicity or polarity, converted the DAT-selective MCAT into a SERT-selective partial releaser. Electrophysiological and superfusion experiments, together with kinetic modelling, showed that pCF3MCAT, but not MCAT, trapped a fraction of SERTs in an inactive state by occupying the S2-site. These findings define a new mechanism of action for partial releasers, which is distinct from the other two known binding modes underlying partial release. Our observations highlight the fact that the substrate permeation pathway of monoamine transporters supports multiple binding modes, which can be exploited for drug design. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
Assuntos
Propiofenonas/farmacologia , Serotoninérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Sítios de Ligação , Simulação por Computador , Proteínas da Membrana Plasmática de Transporte de Dopamina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Simulação de Acoplamento Molecular , Relação Estrutura-AtividadeRESUMO
Synthetic cathinones are derivatives of the naturally occurring compound cathinone, the main psychoactive ingredient in the khat plant Catha edulis. Cathinone is the ß-keto analog of amphetamine, and all synthetic cathinones display a ß-keto moiety in their structure. Several synthetic cathinones are widely prescribed medications (e.g., bupropion, Wellbutrin®), while others are problematic drugs of abuse (e.g., 4-methylmethcathinone, mephedrone). Similar to amphetamines, synthetic cathinones are psychomotor stimulants that exert their effects by impairing the normal function of plasma membrane transporters for dopamine (DAT), norepinephrine (NET), and 5-HT (SERT). Ring-substituted cathinones like mephedrone are transporter substrates that evoke neurotransmitter release by reversing the normal direction of transporter flux (i.e., releasers), whereas pyrrolidine-containing cathinones like 3,4-methylenedioxypyrovalerone (MDPV) are potent transporter inhibitors that block neurotransmitter uptake (i.e., blockers). Regardless of molecular mechanism, all synthetic cathinones increase extracellular monoamine concentrations in the brain, thereby enhancing cell-to-cell monoamine signaling. Here, we briefly review the mechanisms of action, structure-activity relationships, and in vivo pharmacology of synthetic cathinones. Overall, the findings show that certain synthetic cathinones are powerful drugs of abuse that could pose significant risk to users.