Series of (([1,1'-Biphenyl]-2-yl)methyl)sulfinylalkyl Alicyclic Amines as Novel and High Affinity Atypical Dopamine Transporter Inhibitors with Reduced hERG Activity.

Currently, there are no FDA-approved medications for the treatment of psychostimulant use disorders (PSUD). We have previously discovered "atypical" dopamine transporter (DAT) inhibitors that do not display psychostimulant-like behaviors and may be useful as medications to treat PSUD. Lead candidates (e.g., JJC8-091, 1) have shown promising in vivo profiles in rodents; however, reducing hERG (human ether-à-go-go-related gene) activity, a predictor of cardiotoxicity, has remained a challenge. Herein, a series of 30 (([1,1'-biphenyl]-2-yl)methyl)sulfinylalkyl alicyclic amines was synthesized and evaluated for DAT and serotonin transporter (SERT) binding affinities. A subset of analogues was tested for hERG activity, and the IC50 values were compared to those predicted by our hERG QSAR models, which showed robust predictive power. Multiparameter optimization scores (MPO > 3) indicated central nervous system (CNS) penetrability. Finally, comparison of affinities in human DAT and its Y156F and Y335A mutants suggested that several compounds prefer an inward facing conformation indicating an atypical DAT inhibitor profile.

Rational Approach to Improve Detergent Efficacy for Membrane Protein Stabilization.

Membrane protein structures are essential for the molecular understanding of diverse cellular processes and drug discovery. Detergents are not only widely used to extract membrane proteins from membranes but also utilized to preserve native protein structures in aqueous solution. However, micelles formed by conventional detergents are suboptimal for membrane protein stabilization, necessitating the development of novel amphiphilic molecules with enhanced protein stabilization efficacy. In this study, we prepared two sets of tandem malonate-derived glucoside (TMG) variants, both of which were designed to increase the alkyl chain density in micelle interiors. The alkyl chain density was modulated either by reducing the spacer length (TMG-Ms) or by introducing an additional alkyl chain between the two alkyl chains of the original TMGs (TMG-Ps). When evaluated with a few membrane proteins including a G protein-coupled receptor, TMG-P10,8 was found to be substantially more efficient at extracting membrane proteins and also effective at preserving protein integrity in the long term compared to the previously described TMG-A13. This result reveals that inserting an additional alkyl chain between the two existing alkyl chains is an effective way to optimize detergent properties for membrane protein study. This new biochemical tool and the design principle described have the potential to facilitate membrane protein structure determination.

Exploring the K+ binding site and its coupling to transport in the neurotransmitter:sodium symporter LeuT.

The neurotransmitter:sodium symporters (NSSs) are secondary active transporters that couple the reuptake of substrate to the symport of one or two sodium ions. One bound Na+ (Na1) contributes to the substrate binding, while the other Na+ (Na2) is thought to be involved in the conformational transition of the NSS. Two NSS members, the serotonin transporter (SERT) and the Drosophila dopamine transporter (dDAT), also couple substrate uptake to the antiport of K+ by a largely undefined mechanism. We have previously shown that the bacterial NSS homologue, LeuT, also binds K+, and could therefore serve as a model protein for the exploration of K+ binding in NSS proteins. Here, we characterize the impact of K+ on substrate affinity and transport as well as on LeuT conformational equilibrium states. Both radioligand binding assays and transition metal ion FRET (tmFRET) yielded similar K+ affinities for LeuT. K+ binding was specific and saturable. LeuT reconstituted into proteoliposomes showed that intra-vesicular K+ dose-dependently increased the transport velocity of [3H]alanine, whereas extra-vesicular K+ had no apparent effect. K+ binding induced a LeuT conformation distinct from the Na+- and substrate-bound conformation. Conservative mutations of the Na1 site residues affected the binding of Na+ and K+ to different degrees. The Na1 site mutation N27Q caused a >10-fold decrease in K+ affinity but at the same time a ~3-fold increase in Na+ affinity. Together, the results suggest that K+ binding to LeuT modulates substrate transport and that the K+ affinity and selectivity for LeuT is sensitive to mutations in the Na1 site, pointing toward the Na1 site as a candidate site for facilitating the interaction with K+ in some NSSs.

Modifications to 1-(4-(2-Bis(4-fluorophenyl)methyl)sulfinyl)alkyl Alicyclic Amines That Improve Metabolic Stability and Retain an Atypical DAT Inhibitor Profile.

Atypical dopamine transporter (DAT) inhibitors have shown therapeutic potential in the preclinical models of psychostimulant use disorders (PSUD). In rats, 1-(4-(2-((bis(4-fluorophenyl)methyl)sulfinyl)ethyl)-piperazin-1-yl)-propan-2-ol (JJC8-091, 3b) was effective in reducing the reinforcing effects of both cocaine and methamphetamine but did not exhibit psychostimulant behaviors itself. Improvements in DAT affinity and metabolic stability were desirable for discovering pipeline drug candidates. Thus, a series of 1-(4-(2-bis(4-fluorophenyl)methyl)sulfinyl)alkyl alicyclic amines were synthesized and evaluated for binding affinities at DAT and the serotonin transporter (SERT). Replacement of the piperazine with either a homopiperazine or a piperidine ring system was well tolerated at DAT (Ki range = 3-382 nM). However, only the piperidine analogues (20a-d) showed improved metabolic stability in rat liver microsomes as compared to the previously reported analogues. Compounds 12b and 20a appeared to retain an atypical DAT inhibitor profile, based on negligible locomotor activity in mice and molecular modeling that predicts binding to an inward-facing conformation of DAT.

Melamine-cored glucosides for membrane protein solubilization and stabilization: importance of water-mediated intermolecular hydrogen bonding in detergent performance.

Membrane proteins play essential roles in a number of biological processes, and their structures are important in elucidating such processes at the molecular level and also for rational drug design and development. Membrane protein structure determination is notoriously challenging compared to that of soluble proteins, due largely to the inherent instability of their structures in non-lipid environments. Micelles formed by conventional detergents have been widely used for membrane protein manipulation, but they are suboptimal for long-term stability of membrane proteins, making downstream characterization difficult. Hence, there is an unmet need for the development of new amphipathic agents with enhanced efficacy for membrane protein stabilization. In this study, we designed and synthesized a set of glucoside amphiphiles with a melamine core, denoted melamine-cored glucosides (MGs). When evaluated with four membrane proteins (two transporters and two G protein-coupled receptors), MG-C11 conferred notably enhanced stability compared to the commonly used detergents, DDM and LMNG. These promising findings are mainly attributed to a unique feature of the MGs, i.e., the ability to form dynamic water-mediated hydrogen-bond networks between detergent molecules, as supported by molecular dynamics simulations. Thus, MG-C11 is the first example of a non-peptide amphiphile capable of forming intermolecular hydrogen bonds within a protein-detergent complex environment. Detergent micelles formed via a hydrogen-bond network could represent the next generation of highly effective membrane-mimetic systems useful for membrane protein structural studies.

Dynamic extracellular vestibule of human SERT: Unveiling druggable potential with high-affinity allosteric inhibitors.

The serotonin transporter (SERT) tightly regulates synaptic serotonin levels and has been the primary target of antidepressants. Binding of inhibitors to the allosteric site of human SERT (hSERT) impedes the dissociation of antidepressants bound at the central site and may enhance the efficacy of such antidepressants to potentially reduce their dosage and side effects. Here, we report the identification of a series of high-affinity allosteric inhibitors of hSERT in a unique scaffold, with the lead compound, Lu AF88273 (3-(1-(2-(1H-indol-3-yl)ethyl)piperidin-4-yl)-6-chloro-1H-indole), having 2.1 nM allosteric potency in inhibiting imipramine dissociation. In addition, we find that Lu AF88273 also inhibits serotonin transport in a noncompetitive manner. The binding pose of Lu AF88273 in the allosteric site of hSERT is determined with extensive molecular dynamics simulations and rigorous absolute binding free energy perturbation (FEP) calculations, which show that a part of the compound occupies a dynamically formed small cavity. The predicted binding location and pose are validated by site-directed mutagenesis and can explain much of the structure-activity relationship of these inhibitors using the relative binding FEP calculations. Together, our findings provide a promising lead compound and the structural basis for the development of allosteric drugs targeting hSERT. Further, they demonstrate that the divergent allosteric sites of neurotransmitter transporters can be selectively targeted.

Tris(hydroxymethyl)aminomethane Linker-Bearing Triazine-Based Triglucosides for Solubilization and Stabilization of Membrane Proteins.

High-resolution membrane protein structures are essential for a fundamental understanding of the molecular basis of diverse cellular processes and for drug discovery. Detergents are widely used to extract membrane-spanning proteins from membranes and maintain them in a functional state for downstream characterization. Due to limited long-term stability of membrane proteins encapsulated in conventional detergents, development of novel agents is required to facilitate membrane protein structural study. In the current study, we designed and synthesized tris(hydroxymethyl)aminomethane linker-bearing triazine-based triglucosides (TTGs) for solubilization and stabilization of membrane proteins. When these glucoside detergents were evaluated for four membrane proteins including two G protein-coupled receptors, a few TTGs including TTG-C10 and TTG-C11 displayed markedly enhanced behaviors toward membrane protein stability relative to two maltoside detergents [DDM (n-dodecyl-ß-d-maltoside) and LMNG (lauryl maltose neopentyl glycol)]. This is a notable feature of the TTGs as glucoside detergents tend to be inferior to maltoside detergents at stabilizing membrane proteins. The favorable behavior of the TTGs for membrane protein stability is likely due to the high hydrophobicity of the lipophilic groups, an optimal range of hydrophilic-lipophilic balance, and the absence of cis-trans isomerism.

Persistent binding at dopamine transporters determines sustained psychostimulant 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., 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.

3,4-Bis(hydroxymethyl)hexane-1,6-diol-based Maltosides (HDMs) for Membrane-Protein Study: Importance of Detergent Rigidity-Flexibility Balance in Protein Stability.

Detergents have been major contributors to membrane-protein structural study for decades. However, membrane proteins solubilized in conventional detergents tend to aggregate or denature over time. Stability of large eukaryotic membrane proteins with complex structures tends to be particularly poor, necessitating development of novel detergents with improved properties. Here, we prepared a novel class of detergents, designated 3,4-bis(hydroxymethyl)hexane-1,6-diol-based maltosides (HDMs). When tested on three membrane proteins, including two G-protein-coupled receptors (GPCRs), the new detergents displayed significantly better behaviors compared with DDM. Moreover, the HDMs were superior or comparable to LMNG, an amphiphile widely used for GPCR structural study. An optimal balance of detergent rigidity vs. flexibility of the HDMs is likely responsible for their favorable behaviors toward membrane-protein stability. Thus, the current study not only introduces the HDMs, with significant potential for membrane-protein structural study, but also suggests a useful guideline for designing novel detergents for membrane-protein research.

Development of 1,3-acetonedicarboxylate-derived glucoside amphiphiles (ACAs) for membrane protein study.

Detergents are extensively used for membrane protein manipulation. Membrane proteins solubilized in conventional detergents are prone to denaturation and aggregation, rendering downstream characterization of these bio-macromolecules difficult. Although many amphiphiles have been developed to overcome the limited efficacy of conventional detergents for protein stabilization, only a handful of novel detergents have so far proved useful for membrane protein structural studies. Here, we introduce 1,3-acetonedicarboxylate-derived amphiphiles (ACAs) containing three glucose units and two alkyl chains as head and tail groups, respectively. The ACAs incorporate two different patterns of alkyl chain attachment to the core detergent unit, generating two sets of amphiphiles: ACA-As (asymmetrically alkylated) and ACA-Ss (symmetrically alkylated). The difference in the attachment pattern of the detergent alkyl chains resulted in minor variation in detergent properties such as micelle size, critical micelle concentration, and detergent behaviors toward membrane protein extraction and stabilization. In contrast, the impact of the detergent alkyl chain length on protein stability was marked. The two C11 variants (ACA-AC11 and ACA-SC11) were most effective at stabilizing the tested membrane proteins. The current study not only introduces new glucosides as tools for membrane protein study, but also provides detergent structure-property relationships important for future design of novel amphiphiles.

Biodiscoveries within the Australian plant genus Eremophila based on international and interdisciplinary collaboration: results and perspectives on outstanding ethical dilemmas.

In a cross-continental research initiative, including researchers working in Australia and Denmark, and based on joint external funding by a 3-year grant from the Novo Nordisk Foundation, we have used DNA sequencing, extensive chemical profiling and molecular networking analyses across the entire Eremophila genus to provide new knowledge on the presence of natural products and their bioactivities using polypharmocological screens. Sesquiterpenoids, diterpenoids and dimers of branched-chain fatty acids with previously unknown chemical structures were identified. The collection of plant material from the Eremophila genus was carried out according to a 'bioprospecting agreement' with the Government of Western Australia. We recognize that several Eremophila species hold immense cultural significance to Australia's First Peoples. In spite of our best intentions to ensure that new knowledge gained about the genus Eremophila and any potential future benefits are shared in an equitable manner, in accordance with the Nagoya Protocol, we encounter serious dilemmas and potential conflicts in making benefit sharing with Australia's First Peoples a reality.

The dopamine transporter antiports potassium to increase the uptake of dopamine.

The dopamine transporter facilitates dopamine reuptake from the extracellular space to terminate neurotransmission. The transporter belongs to the neurotransmitter:sodium symporter family, which includes transporters for serotonin, norepinephrine, and GABA that utilize the Na+ gradient to drive the uptake of substrate. Decades ago, it was shown that the serotonin transporter also antiports K+, but investigations of K+-coupled transport in other neurotransmitter:sodium symporters have been inconclusive. Here, we show that ligand binding to the Drosophila- and human dopamine transporters are inhibited by K+, and the conformational dynamics of the Drosophila dopamine transporter in K+ are divergent from the apo- and Na+-states. Furthermore, we find that K+ increases dopamine uptake by the Drosophila dopamine transporter in liposomes, and visualize Na+ and K+ fluxes in single proteoliposomes using fluorescent ion indicators. Our results expand on the fundamentals of dopamine transport and prompt a reevaluation of the impact of K+ on other transporters in this pharmacologically important family.

Foldable Detergents for Membrane Protein Study: Importance of Detergent Core Flexibility in Protein Stabilization.

Membrane proteins are of biological and pharmaceutical significance. However, their structural study is extremely challenging mainly due to the fact that only a small number of chemical tools are suitable for stabilizing membrane proteins in solution. Detergents are widely used in membrane protein study, but conventional detergents are generally poor at stabilizing challenging membrane proteins such as G protein-coupled receptors and protein complexes. In the current study, we prepared tandem triazine-based maltosides (TZMs) with two amphiphilic triazine units connected by different diamine linkers, hydrazine (TZM-Hs) and 1,2-ethylenediamine (TZM-Es). These TZMs were consistently superior to a gold standard detergent (DDM) in terms of stabilizing a few membrane proteins. In addition, the TZM-Es containing a long linker showed more general protein stabilization efficacy with multiple membrane proteins than the TZM-Hs containing a short linker. This result indicates that introduction of the flexible1,2-ethylenediamine linker between two rigid triazine rings enables the TZM-Es to fold into favourable conformations in order to promote membrane protein stability. The novel concept of detergent foldability introduced in the current study has potential in rational detergent design and membrane protein applications.

Glyco-Steroidal Amphiphiles (GSAs) for Membrane Protein Structural Study.

Integral membrane proteins pose considerable challenges to high resolution structural analysis. Maintaining membrane proteins in their native state during protein isolation is essential for structural study of these bio-macromolecules. Detergents are the most commonly used amphiphilic compounds for stabilizing membrane proteins in solution outside a lipid bilayer. We previously introduced a glyco-diosgenin (GDN) detergent that was shown to be highly effective at stabilizing a wide range of membrane proteins. This steroidal detergent has additionally gained attention due to its compatibility with membrane protein structure study via cryo-EM. However, synthetic inconvenience limits widespread use of GDN in membrane protein study. To improve its synthetic accessibility and to further enhance detergent efficacy for protein stabilization, we designed a new class of glyco-steroid-based detergents using three steroid units: cholestanol, cholesterol and diosgenin. These new detergents were efficiently prepared and showed marked efficacy for protein stabilization in evaluation with a few model membrane proteins including two G protein-coupled receptors. Some new agents were not only superior to a gold standard detergent, DDM (n-dodecyl-ß-d-maltoside), but were also more effective than the original GDN at preserving protein integrity long term. These agents represent valuable alternatives to GDN, and are likely to facilitate structural determination of challenging membrane proteins.

Elucidating the Mechanism Behind Sodium-Coupled Neurotransmitter Transporters by Reconstitution.

Sodium-coupled neurotransmitter transporters play a fundamental role in the termination of synaptic neurotransmission, which makes them a major drug target. The reconstitution of these secondary active transporters into liposomes has shed light on their molecular transport mechanisms. From the earliest days of the reconstitution technique up to today's single-molecule studies, insights from live functioning transporters have been indispensable for our understanding of their physiological impact. The two classes of sodium-coupled neurotransmitter transporters, the neurotransmitter: sodium symporters and the excitatory amino acid transporters, have vastly different molecular structures, but complementary proteoliposome studies have sought to unravel their ion-dependence and transport kinetics. Furthermore, reconstitution experiments have been used on both protein classes to investigate the role of e.g. the lipid environment, of posttranslational modifications, and of specific amino acid residues in transport. Techniques that allow the detection of transport at a single-vesicle resolution have been developed, and single-molecule studies have started to reveal single transporter kinetics, which will expand our understanding of how transport across the membrane is facilitated at protein level. Here, we review a selection of the results and applications where the reconstitution of the two classes of neurotransmitter transporters has been instrumental.

Probing the Impact of Temperature and Substrates on the Conformational Dynamics of the Neurotransmitter:Sodium symporter LeuT.

The crucial function of neurotransmitter:sodium symporters (NSS) in facilitating the reuptake of neurotransmitters into neuronal cells makes them attractive drug targets for treating multiple mental diseases. Due to the challenges in working with eukaryotic NSS proteins, LeuT, a prokaryotic amino acid transporter, has served as a model protein for studying structure-function relationships of NSS family proteins. With hydrogen-deuterium exchange mass spectrometry (HDX-MS), slow unfolding/refolding kinetics were identified in multiple regions of LeuT, suggesting that substrate translocation involves cooperative fluctuations of helical stretches. Earlier work has solely been performed at non-native temperatures (25 °C) for LeuT, which is evolutionarily adapted to function at high temperatures (85 - 95 °C). To address the effect of temperature on LeuT dynamics, we have performed HDX-MS experiments at elevated temperatures (45 °C and 60 °C). At these elevated temperatures, multiple regions in LeuT exhibited increased dynamics compared to 25 °C. Interestingly, coordinated slow unfolding/refolding of key regions could still be observed, though considerably faster. We have further investigated the conformational impact of binding the efficiently transported substrate alanine (Ala) relative to the much slower transported substrate leucine (Leu). Comparing the HDX of the Ala-bound versus Leu-bound state of LeuT, we observe distinct differences that could explain the faster transport rate (kcat) of Ala relative to Leu. Importantly, slow unfolding/refolding dynamics could still be observed in regions of Ala-bound LeuT . Overall, our work brings new insights into the conformational dynamics of LeuT and provides a better understanding of the transport mechanism of LeuT and possibly other transporters bearing the LeuT fold.

Maltose-bis(hydroxymethyl)phenol (MBPs) and Maltose-tris(hydroxymethyl)phenol (MTPs) Amphiphiles for Membrane Protein Stability.

Membrane protein structures provide a fundamental understanding of their molecular actions and are of importance for drug development. Detergents are widely used to solubilize, stabilize, and crystallize membrane proteins, but membrane proteins solubilized in conventional detergents are prone to denaturation and aggregation. Thus, developing novel detergents with enhanced efficacy for protein stabilization remains important. We report herein the design and synthesis of a class of phenol-derived maltoside detergents. Using two different linkers, we prepared two sets of new detergents, designated maltose-bis(hydroxymethyl)phenol (MBPs) and maltose-tris(hydroxymethyl)phenol (MTPs). The evaluation of these detergents with three transporters and two G-protein coupled receptors allowed us to identify a couple of new detergents (MBP-C9 and MTP-C12) that consistently conferred enhanced stability to all tested proteins compared to a gold standard detergent (DDM). Furthermore, the data analysis based on the detergent structures provides key detergent features responsible for membrane protein stabilization that together will facilitate the future design of novel detergents.

The antidepressant drug vilazodone is an allosteric inhibitor of the serotonin transporter.

Depression is a common mental disorder. The standard medical treatment is the selective serotonin reuptake inhibitors (SSRIs). All characterized SSRIs are competitive inhibitors of the serotonin transporter (SERT). A non-competitive inhibitor may produce a more favorable therapeutic profile. Vilazodone is an antidepressant with limited information on its molecular interactions with SERT. Here we use molecular pharmacology and cryo-EM structural elucidation to characterize vilazodone binding to SERT. We find that it exhibits non-competitive inhibition of serotonin uptake and impedes dissociation of [3H]imipramine at low nanomolar concentrations. Our SERT structure with bound imipramine and vilazodone reveals a unique binding pocket for vilazodone, expanding the boundaries of the extracellular vestibule. Characterization of the binding site is substantiated with molecular dynamics simulations and systematic mutagenesis of interacting residues resulting in decreased vilazodone binding to the allosteric site. Our findings underline the versatility of SERT allosteric ligands and describe the unique binding characteristics of vilazodone.

Type I and type II positive allosteric modulators of α7 nicotinic acetylcholine receptors induce antidepressant-like activity in mice by a mechanism involving receptor potentiation but not neurotransmitter reuptake inhibition. Correlation with mTOR intracellular pathway activation.

The aim of this study is to determine whether type I and type II positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (nAChRs) induce antidepressant-like activity in mice after acute, subchronic, and chronic treatments, and to assess whether α7-PAMs inhibit neurotransmitter transporters and activate mTOR (mammalian target of rapamycin) and/or ERK (extracellular signal-regulated protein kinases) signaling. The forced swim (FST) and tail suspension (TST) test results indicated that NS-1738 (type I PAM), PNU-120596 and PAM-2 (type II PAMs) induce antidepressant-like activity after subchronic treatment, whereas PAM-2 was also active after chronic treatment. Methyllycaconitine (α7-antagonist) inhibited the observed effects, highlighting the involvement of α7 nAChRs in this process. Drug interaction studies showed synergism between PAM-2 and bupropion (antidepressant), but not between PAM-2 and DMXBA (α7-agonist). The studied PAMs showed no high affinity (< 1 µM) for the human dopamine, serotonin, and noradrenaline transporters, suggesting that transporter inhibition is not the underlying mechanism for the observed activity. To assess whether mTOR and ERK signaling pathways are involved in the activity of α7-PAMs, the phosphorylation status of key signaling nodes was determined in prefrontal cortex and hippocampus from mice chronically treated with PAM-2. In conclusion, the antidepressant-like activity of type I and type II PAMs is mediated by a mechanism involving α7 potentiation but not α7 desensitization or neurotransmitter transporter blockade, and is correlated with activation of both mTOR and ERK signaling pathways. These results support the view that α7-PAMs might be clinically used to ameliorate depression disorders .