Aminorex, a metabolite of the cocaine adulterant levamisole, exerts amphetamine like actions at monoamine transporters

AbstractPsychostimulants such as amphetamine and cocaine are illicitly used drugs that act on neurotransmitter transporters for dopamine, serotonin or norepinephrine. These drugs can by themselves already cause severe neurotoxicity. However, an additional health threat arises from adulterant substances which are added to the illicit compound without declaration. One of the most frequently added adulterants in street drugs sold as cocaine is the anthelmintic drug levamisole. We tested the effects of levamisole on neurotransmitter transporters heterologously expressed in HEK293 cells. Levamisole was 100 and 300-fold less potent than cocaine in blocking norepinephrine and dopamine uptake, and had only very low affinity for the serotonin transporter. In addition, levamisole did not trigger any appreciable substrate efflux. Because levamisole and cocaine are frequently co-administered, we searched for possible allosteric effects; at 30μM, a concentration at which levamisole displayed already mild effects on norepinephrine transport it did not enhance the inhibitory action of cocaine. Levamisole is metabolized to aminorex, a formerly marketed anorectic drug, which is classified as an amphetamine-like substance. We examined the uptake-inhibitory and efflux-eliciting properties of aminorex and found it to exert strong effects on all three neurotransmitter transporters in a manner similar to amphetamine. We therefore conclude that while the adulterant levamisole itself has only moderate effects on neurotransmitter transporters, its metabolite aminorex may exert distinct psychostimulant effects by itself. Given that the half-time of levamisole and aminorex exceeds that of cocaine, it may be safe to conclude that after the cocaine effect “fades out” the levamisole/aminorex effect “kicks in”

Substrate selectivity profiling of the human monoamine transporters

Die Serotonin, Dopamin und Noradrenalin Transporter (bzw. SERT, DAT, NET) werden zusammen als die Monoamin Transporter (MAT) bezeichnet und sind an einer Vielzahl von psychiatrischen Störungen wie Depression, Sucht und Aufmerksamkeitsdefizit-/Hyperaktivitätsstörung (ADHS) beteiligt. Daher haben sie sich zu einem zentralen Thema in der Forschung der Lebenswissenschaften der letzten Jahrzehnte gemacht. Die Selektivität der MAT Substrate wurde im Rahmen dieser Arbeit mittels Computer-Kalkulationen und biochemischen Methoden gründlich studiert. Die Bindungsmodi und Selektivität von exogenen Verbindungen wurden vor dieser Arbeit noch nicht extensiv erklärt. Kathinone sind Verbindungen mit steigender Popularität in der Party-Szene, die auch durch MAT transportiert werden. Ihre aufnahmehemmende Aktivität auf die MAT wurde genutzt, um das Selektivitätsphänomen genauer zu erklären. Das molekulare Docken von einem Set dieser Kathinone in einem Homologie-Model des SERTs, hat ihren Bindungsmodus in der Substrat Bindungstasche bestätigt und lässt darauf schließen, dass ihre chemischen Gerüste überlappen. Zusätzlich wurde die Bindungsaktivität mit Hilfe der Interaktionen zwischen den chemischen Substituenten und den Aminosäuren im Protein, die sie umgeben, erläutert. Eine weitere Validierung wurde durch Hansch-Analyse erhalten. Dieser Ansatz deutet darauf hin, dass ein polarisierbarer oder lipophiler para-Substituent für SERT Affinität wichtig ist. Datensätze zeigten, dass das Fehlen von Substituenten am aromatischen Ring die Selektivität der Verbindungen zugunsten von DAT im Vergleich zu SERT erhöht. Daher wurden Moleküldynamik (MD) Simulationen und Studien zur Thermodynamischen Integration (TI) durchgeführt, die darauf hindeuteten, dass die Substrat-Bindungstasche der für die Affinität meist bestimmende Bindungsplatz dieser Verbindungen ist. Die höhere Selektivität von DAT verglichen mit SERT wurden durch die folgenden Punkte erklärt: 1. eine günstigere aromatische Stapelung, vermutlich aufgrund eines weniger sperrigen Val152, im Vergleich zu Ile172 in SERT, das eine solche Interaktion verhindern würde. 2. attraktivere elektrostatische Interaktionen wegen einer leicht strafferen Bindungstasche mit einer im Durchschnitt geringeren Anzahl an Wassermolekülen, im Vergleich zu SERT. Die Hypothesen wurden mittels Aufnahmehemmungsassays an Mutanten der Transporterproteinen validiert.The serotonin, dopamine and norepinephrine transporter proteins (SERT, DAT, NET, respectively) are collectively named as the monoamine transporters (MATs) and are involved in a variety of psychiatric disorders such as depression, addiction and attention-deficit hyperactivity disorder (ADHD). This has made them a central topic in life sciences research during the last decades. The selectivity of MAT substrates has been thoroughly studied during this thesis, using computational and biochemical methods. The binding modes and selectivity of exogenous compounds have not been studied extensively before this study. Cathinones are compounds gaining increased popularity in the party scene and are also transported by the MATs. Their uptake inhibitory activity on the MATs was exploited in order to better understand the selectivity phenomenon. Molecular docking of a set of these cathinones into a homology model of SERT validated their binding mode in the substrate binding site and indicated that their chemical scaffold overlap. Additionally, their binding activity was rationalized based on the interaction between the chemical substituents and protein residues that surround them. Further validation was obtained by Hansch analysis. This approach indicated that a polarizable or lipophilic para-substituent increases SERT affinity, whereas a bulky nitrogen substituent would be unfavorable. Different datasets indicated that a lack of substituents on the aromatic ring rendered the compounds DAT-over-SERT selective. Therefore, a molecular dynamics (MD) and thermodynamic integration (TI) study was conducted, which indicated the substrate binding site to be the major recognition site for these compounds. The DAT-over-SERT selectivity was ascribed to: 1. more favorable aromatic stacking interactions which were more favorable in DAT presumably, due to a less bulky Val152 compared to Ile172 in SERT that would disrupt such an interaction. 2. More attractive electrostatic interactions caused by a slightly tighter DAT binding pocket with a smaller number of waters on average entering the binding site, as compared to SERT. The hypotheses were validated by uptake inhibitory assays on mutants of the transporters

Evaluating the potential cancer chemopreventive efficacy of two different solvent extracts of Seriphidium herba-alba in vitro

Cancer is the second leading cause of death world-wide. One of the most important medical practices of the 21st century is the chemoprevention of cancer. For a long history, it has been accepted that plants could prevent and exert suitable anti-carcinogenic effects for multiple types of cancers. Seriphidium herba-alba family Asteraceae has been used in the folk medicine by many cultures for treatment of various ailments since ancient times. In the current research we were aimed to evaluate the cancer chemopreventive activity of two crude extracts of S. herba-alba, methylene chloride extract and methanol extract on two cell lines: Human breast cancer cells (MCF-7) and human hepatocellular carcinoma cells (Hep-G2). Assessment of cytotoxicity using methyl thiazole tetrazolium (MTT assay) indicated that both extracts exhibit poor cytotoxicity with half maximal inhibitory concentration (IC50) >20 µg/mL. Assessment of glutathione-S-transferases (GSTs) activity (spectrophotometrically) showed statistically significant enhancement of enzyme activity after treatment with three different doses of methylene chloride extract and glutathione (GSH) concentrations were decreased. Analysis of cell mode of death by Ethidium bromide/Acridine orange (EB/AO) staining revealed that the dominant mode of death in MCF-7 cells was apoptosis. Assessment of vascular endothelial growth factor (VEGF) and platelets derived growth factor (PDGFBB) using ELISA showed that VEGF and PDGFBB levels were statistically significant decreased. In Conclusion: both extracts may be cancer chemopreventive agents since they had tumor anti-initiating, and anti-promoting activity

Synthesis and in Silico Evaluation of Novel Compounds for PET-Based Investigations of the Norepinephrine Transporter

Since the norepinephrine transporter (NET) is involved in a variety of diseases, the investigation of underlying dysregulation-mechanisms of the norepinephrine (NE) system is of major interest. Based on the previously described highly potent and selective NET ligand 1-(3-(methylamino)-1-phenylpropyl)-3-phenyl-1,3-dihydro-2H-benzimidaz- ol-2-one (Me@APPI), this paper aims at the development of several fluorinated methylamine-based analogs of this compound. The newly synthesized compounds were computationally evaluated for their interactions with the monoamine transporters and represent reference compounds for PET-based investigation of the NET

Synthesis and in Silico Evaluation of Novel Compounds for PET-Based Investigations of the Norepinephrine Transporter

Since the norepinephrine transporter (NET) is involved in a variety of diseases, the investigation of underlying dysregulation-mechanisms of the norepinephrine (NE) system is of major interest. Based on the previously described highly potent and selective NET ligand 1-(3-(methylamino)-1-phenylpropyl)-3-phenyl-1,3-dihydro-2H-benzimidaz- ol- 2-one (Me@APPI), this paper aims at the development of several fluorinated methylaminebased analogs of this compound. The newly synthesized compounds were computationally evaluated for their interactions with the monoamine transporters and represent reference compounds for PET-based investigation of the NET