Edited by: Aviv M. Weinstein, Ariel University, Israel
Reviewed by: Dino Luethi, Medical University of Vienna, Austria; Michael H. Baumann, National Institute on Drug Abuse (NIDA), United States
*Correspondence: Jolanta Opacka-Juffry,
This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology
†These authors share first authorship
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Stimulant drugs, including novel psychoactive substances (NPS, formerly “legal highs”) have addictive potential which their users may not realize. Stimulants increase extracellular dopamine levels in the brain, including the reward and addiction pathways, through interacting with dopamine transporter (DAT). This work aimed to assess the molecular and atomistic mechanisms of stimulant NPS actions at DAT, which translate into biological outcomes such as dopamine release in the brain’s reward pathway. We applied combined
Typical drugs of addiction, stimulants such as cocaine or amphetamine, have been known to share among them the ability to activate the brain’s reward system and increase extracellular levels of dopamine (DA) in the mesolimbic pathway, and preferentially in the nucleus accumbens (NAc) (
Stimulants can share similar structural moieties, such as phenylethylamine which is a common structural feature found embedded in many stimulants like amphetamine and methylamphetamine and is also found in the naturally occurring neurotransmitter dopamine (
Molecular structures of various substrates and stimulants: dopamine, amphetamine, cocaine, 2-diphenylmethylpiperidine (2-DPMP), pipradrol, methylphenidate, and methylamphetamine (which structurally belongs to the substituted amphetamine class of compounds). These different compounds, although occupying the same central binding site in the dopamine transporter, trigger different downstream effects and prefer different conformational states of the protein. Desoxypipradrol (2-DPMP), shares similar structural and pharmacological characteristics with pipradrol and methylphenidate. They have a hydrophobic diphenylmethyl group attached to the α-carbon atom of a cyclic amine (
Stimulants can be found among novel/new psychoactive substances (NPS) (
In the present paper, we chose desoxypipradrol, also known as 2-diphenylmethylpiperidine (2-DPMP) (
In the present study, we assessed the stimulant profile of 2-DPMP by means of
As the pharmacology of 2-DPMP has been reasonably described (
For the
For the
All chemicals, including desoxypipradrol hydrochloride solution D-082, were purchased from Sigma Chemicals (Poole, UK). The radioligand for the dopamine transporter, [125I]RTI-121 (specific activity 81.4TBq/mmol) was purchased from Perkin Elmer.
2-DPMP was purchased from Sigma-Aldrich (Ref. Nr. D-082) as 1 ml/ml solution in methanol. For microdialysis studies, the methanol was evaporated by N2 stream to dryness and the resulting residue was dissolved in a vehicle containing 2% ethanol, 2% Tween 80 and saline. The drug was administered intravenously at the volume of 1 ml/kg. Control rats received the vehicle alone.
Brains were rapidly removed and frozen at −40°C, then stored at −80°C. Frozen brains were cut into 20-µm coronal sections to harvest the ventral and dorsal striatum areas at +1.7mm to −0.3mm against bregma (
Rats were anesthetised with isoflurane gas (4%–5%), and maintained under anesthesia using a breathing tube under a scavenging system while placed in a stereotaxic apparatus, and implanted with a catheter consisting of a polyethylene tubing (Dow Corning Corporation, Michigan, USA) in the right jugular vein and stable fixed in the mid-scapular region of the back. During the same surgical session, rats were implanted with vertical dialysis probes aimed at the NAc shell and CPu. The following coordinates were used according to
At the end of the experiment, all animals were sacrificed under the anesthesia, the probes were gently removed, and the brains harvested and cut in coronal sections with a vibratome to check the correctness of a microdialysis probe location in every brain. Fiber placement was determined as consistent with the coordinates by
Autoradiography data were analyzed using two-way ANOVAs followed by post-hoc Tukey’s test. Data were expressed as mean percentage ± standard error of mean (SEM) against the control value. Binding of [125 I]RTI-121 was analyzed in the presence of increasing concentrations of the drugs in both CPu and NAc shell.
Microdialysis DA data were analyzed by ANOVA for repeated measures followed by the post-hoc Tukey’s test; data were presented as mean ± SEM. Statistica for Windows (Version 10) software was used throughout; significance was set at p < 0.05.
The Schrödinger Release 2019-1 (Schrödinger Release 2019-1: Maestro, Schrödinger, LLC, New York, NY) with the OPLS3e force field was used to prepare the compounds and dock them into a homology model of rDAT, the construction and validation of which has been previously described (
A standard protocol was used to study these docked homology models of rat DAT (rDAT) based on the crystal structure of the
Unbiased atomistic molecular dynamics (MD) simulation trajectories (totaling 4.5 μs) were analyzed and images were created with VMD (
The displacement of [125I]RTI-121 was examined in NAc and CPu.
Concentration-dependent displacement of [125I]RTI-121 by 2-diphenylmethylpiperidine (2-DPMP) in rat nucleus accumbens (NAc) and caudate-putamen (CPu) sections.
In detail, two-way ANOVA revealed a significant effect of the drug concentrations (F5,60 = 705.4; P < 0.0001), a significant effect of the area (F1,60 = 23.49; P < 0.0001), and a significant concentration x area interaction (F5,60 = 3.18; P < 0.05) (see
The acute i.v. administration of 2-DPMP elicited a dose-dependent increase in extracellular DA in the NAc shell and in the CPu of freely moving animals, with an onset of action within the first hour after the treatment (
Three-way ANOVA revealed a significant effect of the brain area [F(1,22)=8.78; p < 0.001], dose [F(3,22)=13.10; p < 0.0001], time [F(9,198)=46.24; p < 0.000001] and a significant brain area × time [F(9,198)=1.95; p < 0.05], dose × time [F(27,198)=10.57; p < 0.000001], and brain area × dose × time interaction [F(27,198)=1.69; p < 0.05]. In the NAc shell,
The
Docking studies were performed by the Induced Fit Protocol in Schrödinger to identify the potential binding sites for AMPH, cocaine and 2-DPMP. The protocol performed in the presence of the internal ions 2Na+ and Cl- obtained positions for all three drugs near a common high affinity binding site that is near the primary substrate-binding S1 site (
The binding poses of AMPH and cocaine were consistent with previous studies (
Cross-sectional illustration of the dopamine transporter (DAT) and molecular models of DAT/ligand complexes.
In our study, unbiased MD simulations show a preferential substrate translocation for AMPH, whereby the DAT adopts an inward-facing conformation indicated by the structural dynamics of the salt-bridge forming pairs of the intracellular (IC) vestibule (R60-D436, K66-D345 and E428–R445) (
Conformational changes of dopamine transporter (DAT) when bound to the three different compounds.
Cocaine, on the other hand, adopts a position in the S1 site where there is minor destabilization of the EC gate Y156–F320 bond at these time scales. As the extracellular salt-bridge R85-D476 remains closed, we interpret it as showing that the DAT is maintained in the original outward-facing conformation (
With the understanding that 2-DPMP binds in the S1 site, our simulations provide evidence whereby the structural arrangement leaves DAT in the outward-facing conformation, similarly to cocaine (
To sum up, our
We employed
As hypothesized, 2-DPMP behaved like a highly potent DAT ligand. The effects of 2-DPMP were more pronounced than those of cocaine, which, when tested under the same conditions, was able to significantly displace [125I]RTI-121, starting at higher concentrations than 2-DPMP in the rat NAc (
The present profile of the “on-and-off” of extracellular DA in response to i.v. 2-DPMP, with a decisive descending phase, resembles that of DA responses to cocaine as observed in a comparable experimental design applied previously in the same research lab of Di Chiara (
The present findings of the potent direct effects of 2-DPMP on DAT and DA availability in the reward pathway help explain why, according to users’ reports, physical and psychoactive effects of 2-DPMP, such as pleasure, euphoria, and increased energy and sociability may start as early as after 15 min of the dose (and last for hours), depending on the dose and route of administration (
In the present study, we elaborated the molecular features that may determine the mode of drug binding at DAT by modeling two structurally distinct inhibitors as well as the releaser amphetamine at the binding site of DAT. MD simulations provide a suitable computational method to reveal structural changes within DAT in response to stimulant effects. Amphetamine, transported
To date, there is no structural information available about the binding of 2-DPMP to DAT. We know from the docking study that 2-DPMP can bind to the S1 site, which also supports the autoradiography findings where 2-DPMP competed with the radioligand. Previously mentioned
Overall, amphetamine and cocaine behaved structurally as we expected at the atomistic level, with structural rearrangements of DAT toward the inward-facing conformation and outward-facing conformation respectively. Several residues in both the transmembrane regions TM1 and TM6, including D79 directly interacts with the cationic amine of typical drugs of addiction, including cocaine-like molecules that occupy the primary binding site (
2-DPMP in the simulated time-frame shows characteristics toward an outward-facing conformation and 2-DPMP binding to DAT is a plausible reason for an increase in extracellular dopamine observed
Although DAT features as the main molecular target for typical stimulants, responsible for their DA-enhancing effects, and dopamine is involved in the acute effects of stimulants, the complex and long-term process of addiction involves as well other neurotransmitter systems in addition to that of dopamine. Thus, the noradrenaline/norepinephrine transporter (NET) and serotonin transporter (SERT) contribute to the stimulant characteristics to a varying degree across the range of stimulant drugs (
The present study is not free from limitations. Thus, quantitative behavioral observations of rat mobility, freezing, piloerection, grooming, sniffing, gnawing, rearing and scratching during the
One outcome of this research is the development of a framework for studying stimulant abuse by utilizing both
To conclude: our combination of the
The datasets generated for this study are available on request to the corresponding author.
The animal study was reviewed and approved by: All animal experiments were carried out in accordance with the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioural Research according to Italian (D.L. 116/92 and 152/06) and European Council directives (609/86 and 63/2010) and in compliance with the approved animal policies by the Ethical Committee for Animal Experiments (CESA, University of Cagliari) and the Italian Ministry of Health (Aut. N.162/2016-PR).
BL, MS, and JO-J were responsible for the study concept and design. BL and JO-J conducted the ligand binding experiments and analyzed the data. ML facilitated and designed the microdialysis study. BL collected and interpreted the microdialysis data with ML’s input. HS parameterized the ligands and performed the docking studies. MS performed the molecular simulations and interpreted the findings. JO-J, BL, and MS drafted the manuscript. All authors critically reviewed and edited the content and approved the final version.
This project was supported in part by grants of the European Commission (Drug Prevention and Information Programme 2014–2016; contract JUST/2013/DPIP/AG/4823; EUMADNESS project). Further financial support was provided by the EU Commission‐targeted call on cross-border law enforcement cooperation in the field of drug trafficking—DG Justice/DG Migrations and Home Affairs (JUST/2013/ISEC/ DRUGS/AG/6429) Project EPS/NPS (Enhancing Police Skills concerning Novel Psychoactive Substances; NPS), and by the Drug Policies Department, Presidency of the Council of Ministers, Italy (project: “Effects of NPS: development of a multicentre research for the information enhancement of the Early Warning System” to ML), and RAS-FSC 2014-2020 to ML (Codice intervento: RC_CRP_034; CUP RASSR03071).
The following computational resources are gratefully acknowledged: ARCHER granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim (
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.
We thank Simon Gibbons from the UCL School of Pharmacy, London for stimulating discussions and invaluable suggestions. A special acknowledgement is due for the Ph.D. studentship support from the University of Hertfordshire granted to Barbara Loi and to her supervisors Professors Fabrizio Schifano, John Corkery, Colin Davidson and Mire Zloh. We also thank Shana Bergman, Department of Physiology, Biophysics and Systems Biology, Weill Cornell Medicine, for useful conversations.