Mechanistic Insights into the Stimulant Properties of Novel Psychoactive Substances (NPS) and Their Discrimination by the Dopamine Transporter-In Silico and In Vitro Exploration of Dissociative Diarylethylamines

Novel psychoactive substances (NPS) may have unsuspected addiction potential through possessing stimulant properties. Stimulants normally act at the dopamine transporter (DAT) and thus increase dopamine (DA) availability in the brain, including nucleus accumbens, within the reward and addiction pathway. This paper aims to assess DAT responses to dissociative diarylethylamine NPS by means of in vitro and in silico approaches. We compared diphenidine (DPH) and 2-methoxydiphenidine (methoxphenidine, 2-MXP/MXP) for their binding to rat DAT, using autoradiography assessment of [ I]RTI-121 displacement in rat striatal sections. The drugs' effects on electrically-evoked DA efflux were measured by means of fast cyclic voltammetry in rat accumbens slices. Computational modeling, molecular dynamics and alchemical free energy simulations were used to analyse the atomistic changes within DAT in response to each of the five dissociatives: DPH, 2-MXP, 3-MXP, 4-MXP and 2-Cl-DPH, and to calculate their relative binding free energy. DPH increased DA efflux as a result of its binding to DAT, whereas MXP had no significant effect on either DAT binding or evoked DA efflux. Our computational findings corroborate the above and explain the conformational responses and atomistic processes within DAT during its interactions with the dissociative NPS. We suggest DPH can have addictive liability, unlike MXP, despite the chemical similarities of these two NPS

Identification of a nucleoside analog active against adenosine kinase-expressing plasma cell malignancies

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase–inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI–sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers

Combined in vitro and in silico approaches to the assessment of stimulant properties of novel psychoactive substances – The case of the benzofuran 5-MAPB

Novel psychoactive substances (NPS) are increasingly prevalent world-wide although their pharmacological characteristics are largely unknown; those with stimulant properties, due to interactions with the dopamine transporter (DAT), have addictive potential which their users may not realise. We evaluated the binding of 1-(1-benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB) to rat striatal DAT by means of quantitative autoradiography with [125I]RTI-121, and the effects of 5-MAPB on electrically-evoked dopamine efflux by fast-cyclic voltammetry in rat brain slices. 5-MAPB displaced [125I]RTI-121 in a concentration-dependent manner, with significant effects at 10 and 30 μM. The voltammetry data suggest that 5-MAPB reduces the rate of dopamine reuptake; while the peak dopamine efflux was not increased, the area under the curve was augmented. 5-MAPB can also cause reverse dopamine transport consistent with stimulant properties, more similar to amphetamine than cocaine. Molecular modelling and docking studies compared the binding site of DAT in complex with 5-MAPB to dopamine, amphetamine, 5-APB, MDMA, cocaine and RTI-121. This structural comparison reveals a binding mode for 5-MAPB found in the primary binding (S1) site, central to transmembrane domains 1, 3, 6 and 8, which overlaps with the binding modes of dopamine, cocaine and its analogues. Atomistic molecular dynamics simulations further show that, when in complex with 5-MAPB, DAT can exhibit conformational transitions that spontaneously isomerize the transporter into inward-facing state, similarly to that observed in dopamine-bound DAT. These novel insights, offered by the combination of computational methods of biophysics with neurobiological procedures, provide structural context for NPS at DAT and relate them with their functional properties at DAT as the molecular target of stimulants

R-Ras Regulates Migration through an Interaction with Filamin A in Melanoma Cells

Changes in cell adhesion and migration in the tumor microenvironment are key in the initiation and progression of metastasis. R-Ras is one of several small GTPases that regulate cell adhesion and migration on the extracellular matrix, however the mechanism has not been completely elucidated. Using a yeast two-hybrid approach we sought to identify novel R-Ras binding proteins that might mediate its effects on integrins.We identified Filamin A (FLNa) as a candidate interacting protein. FLNa is an actin-binding scaffold protein that also binds to integrin β1, β2 and β7 tails and is associated with diverse cell processes including cell migration. Indeed, M2 melanoma cells require FLNa for motility. We further show that R-Ras and FLNa interact in co-immunoprecipitations and pull-down assays. Deletion of FLNa repeat 3 (FLNaΔ3) abrogated this interaction. In M2 melanoma cells active R-Ras co-localized with FLNa but did not co-localize with FLNa lacking repeat 3. Thus, activated R-Ras binds repeat 3 of FLNa. The functional consequence of this interaction was that active R-Ras and FLNa coordinately increased cell migration. In contrast, co-expression of R-Ras and FLNaΔ3 had a significantly reduced effect on migration. While there was enhancement of integrin activation and fibronectin matrix assembly, cell adhesion was not altered. Finally, siRNA knockdown of endogenous R-Ras impaired FLNa-dependent fibronectin matrix assembly.These data support a model in which R-Ras functionally associates with FLNa and thereby regulates integrin-dependent migration. Thus in melanoma cells R-Ras and FLNa may cooperatively promote metastasis by enhancing cell migration

Quantifying Water-Mediated Protein–Ligand Interactions in a Glutamate Receptor: A DFT Study

It is becoming increasingly clear that careful treatment of water molecules in ligand–protein interactions is required in many cases if the correct binding pose is to be identified in molecular docking. Water can form complex bridging networks and can play a critical role in dictating the binding mode of ligands. A particularly striking example of this can be found in the ionotropic glutamate receptors. Despite possessing similar chemical moieties, crystal structures of glutamate and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) in complex with the ligand-binding core of the GluA2 ionotropic glutamate receptor revealed, contrary to all expectation, two distinct modes of binding. The difference appears to be related to the position of water molecules within the binding pocket. However, it is unclear exactly what governs the preference for water molecules to occupy a particular site in any one binding mode. In this work we use density functional theory (DFT) calculations to investigate the interaction energies and polarization effects of the various components of the binding pocket. Our results show (i) the energetics of a key water molecule are more favorable for the site found in the glutamate-bound mode compared to the alternative site observed in the AMPA-bound mode, (ii) polarization effects are important for glutamate but less so for AMPA, (iii) ligand–system interaction energies alone can predict the correct binding mode for glutamate, but for AMPA alternative modes of binding have similar interaction energies, and (iv) the internal energy is a significant factor for AMPA but not for glutamate. We discuss the results within the broader context of rational drug-design

Case study: Biochemistry without borders: a case study utilising infographics

Brief reportThe present paper addresses a case study on the implementation of an online learning exercise utilising infographics in undergraduate Biochemistry and General Chemistry courses at the University of Roehampton (UoR) and Hostos Community College (HCC) of the City University of New York (CUNY). Students at UoR were asked to create infographics on topics related to the four major classes of biomolecules: carbohydrates, lipids, proteins and nucleic acids, and these infographics were shared with HCC students in an active learning exercise which incorporated peer evaluation and feedback. We highlight the various teaching and learning strategies, as well as the challenges related to the implementation of digital tools, in the educational process during the COVID-19 pandemic to maintain student engagement and active learning. Student feedback revealed positive learning gains on biochemistry concepts related to the four biomolecules. The exercise was viewed favourably by students, with learners indicating the acquisition of digital skills to effectively represent and visualise their understanding of biochemical concepts and explain these processes to peers.The authors received no financial support for conducting the case study, manuscript preparation, and/or publication of this article

Molecular mechanisms of action of stimulant novel psychoactive substances that target the high-affinity transporter for dopamine

Drug misuse is a significant social and public health problem worldwide. Misused substances exert their neurobehavioural effects through changing neural signalling within the brain, many of them leading to substance dependence and addiction in the longer term. Among drugs with addictive liability, there are illicit classical stimulants such as cocaine and amphetamine, and their more recently available counterparts known as novel psychoactive substances (NPS). Stimulants normally increase dopamine availability in the brain, including the pathway implicated in reward-related behaviour. This pattern is observed in both animal and human brain. The main biological target of stimulants, both classical and NPS, is the dopamine transporter (DAT) implicated in the dopamine-enhancing effects of these drugs. This article aims at reviewing research on the molecular mechanisms underpinning the interactions between stimulant NPS, such as benzofurans, cathinones or piperidine derivatives and DAT, to achieve a greater understanding of the core phenomena that decide about the addictive potential of stimulant NPS. As the methodology is essential in the process of experimental research in this area, we review the applications of in vitro, in vivo and in silico approaches. The latter, including molecular dynamics, attracts the focus of the present review as the method of choice in molecular and atomistic investigations of the mechanisms of addiction of stimulant NPS. Research of this kind is of interest to not only scientists but also health professionals as updated knowledge of NPS, their modes of action and health risks, is needed to tackle the challenges posed by NPS misuse

Quantifying Water-Mediated Protein–Ligand Interactions in a Glutamate Receptor: A DFT Study

It is becoming increasingly clear that careful treatment of water molecules in ligand–protein interactions is required in many cases if the correct binding pose is to be identified in molecular docking. Water can form complex bridging networks and can play a critical role in dictating the binding mode of ligands. A particularly striking example of this can be found in the ionotropic glutamate receptors. Despite possessing similar chemical moieties, crystal structures of glutamate and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) in complex with the ligand-binding core of the GluA2 ionotropic glutamate receptor revealed, contrary to all expectation, two distinct modes of binding. The difference appears to be related to the position of water molecules within the binding pocket. However, it is unclear exactly what governs the preference for water molecules to occupy a particular site in any one binding mode. In this work we use density functional theory (DFT) calculations to investigate the interaction energies and polarization effects of the various components of the binding pocket. Our results show (i) the energetics of a key water molecule are more favorable for the site found in the glutamate-bound mode compared to the alternative site observed in the AMPA-bound mode, (ii) polarization effects are important for glutamate but less so for AMPA, (iii) ligand–system interaction energies alone can predict the correct binding mode for glutamate, but for AMPA alternative modes of binding have similar interaction energies, and (iv) the internal energy is a significant factor for AMPA but not for glutamate. We discuss the results within the broader context of rational drug-design

Computational modeling of the N-terminus of the human dopamine transporter and its interaction with PIP2 -containing membranes

The dopamine transporter (DAT) is a transmembrane protein belonging to the family of Neurotransmitter:Sodium Symporters (NSS). Members of the NSS are responsible for the clearance of neurotransmitters from the synaptic cleft, and for their translocation back into the presynaptic nerve terminal. The DAT contains long intracellular N- and C-terminal domains that are strongly implicated in the transporter function. The N-terminus (N-term), in particular, regulates the reverse transport (efflux) of the substrate through DAT. Currently, the molecular mechanisms of the efflux remain elusive in large part due to lack of structural information on the N-terminal segment. Here we report a computational model of the N-term of the human DAT (hDAT), obtained through an ab initio structure prediction, in combination with extensive atomistic molecular dynamics (MD) simulations in the context of a lipid membrane. Our analysis reveals that whereas the N-term is a highly dynamic domain, it contains secondary structure elements that remain stable in the long MD trajectories of interactions with the bilayer (totaling >2.2 µs). Combining MD simulations with continuum mean-field modeling we found that the N-term engages with lipid membranes through electrostatic interactions with the charged lipids PIP(2) (phosphatidylinositol 4,5-Biphosphate) or PS (phosphatidylserine) that are present in these bilayers. We identify specific motifs along the N-term implicated in such interactions and show that differential modes of N-term/membrane association result in differential positioning of the structured segments on the membrane surface. These results will inform future structure-based studies that will elucidate the mechanistic role of the N-term in DAT function