Bioisosteric analogs of MDMA: Improving the pharmacological profile?

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.

Unveiling the crucial role of betaine: modulation of GABA homeostasis via SLC6A1 transporter (GAT1).

Betaine is an endogenous osmolyte that exhibits therapeutic potential by mitigating various neurological disorders. However, the underlying cellular and molecular mechanisms responsible for its neuroprotective effects remain puzzling.In this study, we describe a possible mechanism behind the positive impact of betaine in preserving neurons from excitotoxicity. Here we demonstrate that betaine at low concentration modulates the GABA uptake by GAT1 (slc6a1), the predominant GABA transporter in the central nervous system. This modulation occurs through the temporal inhibition of the transporter, wherein prolonged occupancy by betaine impedes the swift transition of the transporter to the inward conformation. Importantly, the modulatory effect of betaine on GAT1 is reversible, as the blocking of GAT1 disappears with increased extracellular GABA. Using electrophysiology, mass spectroscopy, radiolabelled cellular assay, and molecular dynamics simulation we demonstrate that betaine has a dual role in GAT1: at mM concentration acts as a slow substrate, and at µM as a temporal blocker of GABA, when it is below its K0.5. Given this unique modulatory characteristic and lack of any harmful side effects, betaine emerges as a promising neuromodulator of the inhibitory pathways improving GABA homeostasis via GAT1, thereby conferring neuroprotection against excitotoxicity.

Bioisosteric analogs of MDMA with improved pharmacological profile.

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.

A comparative review on the well-studied GAT1 and the understudied BGT-1 in the brain.

γ-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system (CNS). Its homeostasis is maintained by neuronal and glial GABA transporters (GATs). The four GATs identified in humans are GAT1 (SLC6A1), GAT2 (SLC6A13), GAT3 (SLC6A11), and betaine/GABA transporter-1 BGT-1 (SLC6A12) which are all members of the solute carrier 6 (SLC6) family of sodium-dependent transporters. While GAT1 has been investigated extensively, the other GABA transporters are less studied and their role in CNS is not clearly defined. Altered GABAergic neurotransmission is involved in different diseases, but the importance of the different transporters remained understudied and limits drug targeting. In this review, the well-studied GABA transporter GAT1 is compared with the less-studied BGT-1 with the aim to leverage the knowledge on GAT1 to shed new light on the open questions concerning BGT-1. The most recent knowledge on transporter structure, functions, expression, and localization is discussed along with their specific role as drug targets for neurological and neurodegenerative disorders. We review and discuss data on the binding sites for Na+, Cl-, substrates, and inhibitors by building on the recent cryo-EM structure of GAT1 to highlight specific molecular determinants of transporter functions. The role of the two proteins in GABA homeostasis is investigated by looking at the transport coupling mechanism, as well as structural and kinetic transport models. Furthermore, we review information on selective inhibitors together with the pharmacophore hypothesis of transporter substrates.

Bile acid interactions with neurotransmitter transporters.

Synthesized in the liver from cholesterol, the bile acids (BAs) primary role is emulsifying fats to facilitate their absorption. BAs can cross the blood-brain barrier (BBB) and be synthesized in the brain. Recent evidence suggests a role for BAs in the gut-brain signaling by modulating the activity of various neuronal receptors and transporters, including the dopamine transporter (DAT). In this study, we investigated the effects of BAs and their relationship with substrates in three transporters of the solute carrier 6 family. The exposure to obeticholic acid (OCA), a semi-synthetic BA, elicits an inward current (IBA) in the DAT, the GABA transporter 1 (GAT1), and the glycine transporter 1 (GlyT1b); this current is proportional to the current generated by the substrate, respective to the transporter. Interestingly, a second consecutive OCA application to the transporter fails to elicit a response. The full displacement of BAs from the transporter occurs only after exposure to a saturating concentration of a substrate. In DAT, perfusion of secondary substrates norepinephrine (NE) and serotonin (5-HT) results in a second OCA current, decreased in amplitude and proportional to their affinity. Moreover, co-application of 5-HT or NE with OCA in DAT, and GABA with OCA in GAT1, did not alter the apparent affinity or the Imax, similar to what was previously reported in DAT in the presence of DA and OCA. The findings support the previous molecular model that suggested the ability of BAs to lock the transporter in an occluded conformation. The physiological significance is that it could possibly avoid the accumulation of small depolarizations in the cells expressing the neurotransmitter transporter. This achieves better transport efficiency in the presence of a saturating concentration of the neurotransmitter and enhances the action of the neurotransmitter on their receptors when they are present at reduced concentrations due to decreased availability of transporters.

Betaine-The dark knight of the brain.

The role of betaine in the liver and kidney has been well documented, even from the cellular and molecular point of view. Despite literature reporting positive effects of betaine supplementation in Alzheimer's, Parkinson's and schizophrenia, the role and function of betaine in the brain are little studied and reviewed. Beneficial effects of betaine in neurodegeneration, excitatory and inhibitory imbalance and against oxidative stress in the central nervous system (CNS) have been collected and analysed to understand the main role of betaine in the brain. There are many 'dark' aspects needed to complete the picture. The understanding of how this osmolyte is transported across neuron and glial cells is also controversial, as the expression levels and functioning of the known protein capable to transport betaine expressed in the brain, betaine-GABA transporter 1 (BGT-1), is itself not well clarified. The reported actions of betaine beyond BGT-1 related to neuronal degeneration and memory impairment are the focus of this work. With this review, we underline the scarcity of detailed molecular and cellular information about betaine action. Consequently, the requirement of detailed focus on and study of the interaction of this molecule with CNS components to sustain the therapeutic use of betaine.

The "www" of Xenopus laevis Oocytes: The Why, When, What of Xenopus laevis Oocytes in Membrane Transporters Research.

After 50 years, the heterologous expression of proteins in Xenopus laevis oocytes is still essential in many research fields. New approaches and revised protocols, but also classical methods, such as the two-electrode voltage clamp, are applied in studying membrane transporters. New and old methods for investigating the activity and the expression of Solute Carriers (SLC) are reviewed, and the kinds of experiment that are still useful to perform with this kind of cell are reported. Xenopus laevis oocytes at the full-grown stage have a highly efficient biosynthetic apparatus that correctly targets functional proteins at the defined compartment. This small protein factory can produce, fold, and localize almost any kind of wild-type or recombinant protein; some tricks are required to obtain high expression and to verify the functionality. The methodologies examined here are mainly related to research in the field of membrane transporters. This work is certainly not exhaustive; it has been carried out to be helpful to researchers who want to quickly find suggestions and detailed indications when investigating the functionality and expression of the different members of the solute carrier families.

SSM-based electrophysiology, a label-free real-time method reveals sugar binding & transport events in SGLT1.

Here, we present a solid-supported membrane (SSM)-based electrophysiological approach to study sugar binding and Na+/glucose cotransport by SGLT1 in membrane vesicles. SSM-based electrophysiology delivers a cumulative real-time current readout from numerous SGLT1 proteins simultaneously using a gold-coated sensor chip. In contrast to conventional techniques, which mainly operate with voltage steps, currents are triggered by sugar or sodium addition. Sugar concentration jumps in the presence of sodium lead to transport currents between 5 and 10 nA. Remarkably, in the absence of sodium (i.e. no transport), we observed fast pre-steady-state (PSS) currents with time constants between 3 and 10 ms. These PSS currents mainly originate from sugar binding. Sodium binding does not induce PSS currents. Due to high time resolution, PSS currents were distinguished from transport and eventually correlated with conformational transitions within the sugar translocation pathway. In addition, we analyzed the impact of driving forces on transport and binding currents, showing that membrane voltage and sodium concentration gradients lead to an increased transport rate without affecting sugar binding kinetics. We also compared Na+/sugar efflux with physiologically relevant influx and found similar transport rates, but lower affinity in efflux mode. SSM-based electrophysiology is a powerful technique, which overcomes bottlenecks for transport measurements observed in other techniques such as the requirement of labels or the lack of real-time data. Rapid solution exchange enables the observation of substrate-induced electrogenic events like conformational transitions, opening novel perspectives for in-depth functional studies of SGLT1 and other transporters.