| 1. |
- Betari, Nibal, et al.
(author)
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Discovery and biological characterization of a novel scaffold for potent inhibitors of peripheral serotonin synthesis
- 2020
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In: Future Medicinal Chemistry. - : Future Science. - 1756-8919 .- 1756-8927. ; 12:16, s. 1461-1474
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Journal article (peer-reviewed)abstract
- Aim: Tryptophan hydroxylase 1 (TPH1) catalyzes serotonin synthesis in peripheral tissues. Selective TPH1 inhibitors may be useful for treating disorders related to serotonin dysregulation.Results & methodology: Screening using a thermal shift assay for TPH1 binders yielded Compound1(2-(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one), which showed high potency (50% inhibition at 98 +/- 30 nM) and selectivity for inhibiting TPH over related aromatic amino acid hydroxylases in enzyme activity assays. Structure-activity relationships studies revealed several analogs of1showing comparable potency. Kinetic studies suggested a noncompetitive mode of action of1, with regards to tryptophan and tetrahydrobiopterin. Computational docking studies and live cell assays were also performed.Conclusion: This TPH1 inhibitor scaffold may be useful for developing new therapeutics for treating elevated peripheral serotonin.
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| 2. |
- Betari, Nibal, et al.
(author)
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Inhibition of Tryptophan Hydroxylases and Monoamine Oxidase-A by the Proton Pump Inhibitor, Omeprazole-In Vitro and In Vivo Investigations
- 2020
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In: Frontiers in Pharmacology. - : Frontiers Media S.A.. - 1663-9812. ; 11
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Journal article (peer-reviewed)abstract
- Serotonin (5-HT) is a hormone and neurotransmitter that modulates neural activity as well as a wide range of other physiological processes including cardiovascular function, bowel motility, and platelet aggregation. 5-HT synthesis is catalyzed by tryptophan hydroxylase (TPH) which exists as two distinct isoforms; TPH1 and TPH2, which are responsible for peripheral and central 5-HT, respectively. Due to the implication of 5-HT in a number of pathologies, including depression, anxiety, autism, sexual dysfunction, irritable bowel syndrome, inflammatory bowel disease, and carcinoid syndrome, there has been a growing interest in finding modulators of these enzymes in recent years. We thus performed high-throughput screening (HTS) using a fluorescence-based thermal shift assay (DSF) to search the Prestwick Chemical Library containing 1,280 compounds, mostly FDA-approved drugs, for TPH1 binders. We here report the identification of omeprazole, a proton pump inhibitor, as an inhibitor of TPH1 and TPH2 with low micromolar potency and high selectivity over the other aromatic amino acid hydroxylases. The S-enantiomer of omeprazole, esomeprazole, has recently also been described as an inhibitor of monoamine oxidase-A (MAO-A), the main enzyme responsible for 5-HT degradation, albeit with lower potency compared to the effect on TPH1 and TPH2. In order to investigate the net effect of simultaneous inhibition of TPH and MAO-A in vivo, we administered high-dose (100 mg/kg) omeprazole to CD-1 mice for 4 days, after which the animals were subjected to the tail suspension test. Finally, central (whole brain) and peripheral (serum) 5-HT content was measured using liquid chromatography-mass spectrometry (LC-MS). Omeprazole treatment significantly increased 5-HT concentrations, both in brain and in serum, and reduced the time spent immobile in the tail suspension test relative to vehicle control. Thus, the MAO-A inhibition afforded by high-dose omeprazole appears to overcome the opposing effect on 5-HT produced by inhibition of TPH1 and TPH2. Further modification of proton pump inhibitor scaffolds may yield more selective modulators of 5-HT metabolism.
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| 3. |
- Betari, Nibal, et al.
(author)
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Synthetic corticosteroids as tryptophan hydroxylase stabilizers
- 2021
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In: Future Medicinal Chemistry. - : Future Science Ltd.. - 1756-8919 .- 1756-8927. ; 13:17, s. 1465-1474
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Journal article (peer-reviewed)abstract
- Background: Clinically, corticosteroids are used mainly for their immune-modulatory properties but are also known to influence mood. Despite evidence of a role in regulating tryptophan hydroxylases (TPH), key enzymes in serotonin biosynthesis, a direct action of corticosteroids on these enzymes has not been systematically investigated.Methodology & results: Corticosteroid effects on TPHs were tested using an in vitro assay. The compound with the strongest modulatory effect, beclomethasone dipropionate, activated TPH1 and TPH2 with low micromolar potency. Thermostability assays suggested a stabilizing mechanism, and computational docking indicated that beclomethasone dipropionate interacts with the TPH active site.Conclusion: Beclomethasone dipropionate is a stabilizer of TPHs, acting as a pharmacological chaperone. Our findings may inspire further development of steroid scaffolds as putative antidepressant drugs.
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| 4. |
- Burström, Viktor, et al.
(author)
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Dopamine-induced arrestin recruitment and desensitization of the dopamine D4 receptor is regulated by G protein-coupled receptor kinase-2
- 2023
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In: Frontiers in Pharmacology. - : Frontiers Media S.A.. - 1663-9812. ; 14
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Journal article (peer-reviewed)abstract
- The dopamine D4 receptor (D4R) is expressed in the retina, prefrontal cortex, and autonomic nervous system and has been implicated in attention deficit hyperactivity disorder (ADHD), substance use disorders, and erectile dysfunction. D4R has also been investigated as a target for antipsychotics due to its high affinity for clozapine. As opposed to the closely related dopamine D2 receptor (D2R), dopamine-induced arrestin recruitment and desensitization at the D4R have not been studied in detail. Indeed, some earlier investigations could not detect arrestin recruitment and desensitization of this receptor upon its activation by agonist. Here, we used a novel nanoluciferase complementation assay to study dopamine-induced recruitment of β-arrestin2 (βarr2; also known as arrestin3) and G protein-coupled receptor kinase-2 (GRK2) to the D4R in HEK293T cells. We also studied desensitization of D4R-evoked G protein-coupled inward rectifier potassium (GIRK; also known as Kir3) current responses in Xenopus oocytes. Furthermore, the effect of coexpression of GRK2 on βarr2 recruitment and GIRK response desensitization was examined. The results suggest that coexpression of GRK2 enhanced the potency of dopamine to induce βarr2 recruitment to the D4R and accelerated the rate of desensitization of D4R-evoked GIRK responses. The present study reveals new details about the regulation of arrestin recruitment to the D4R and thus increases our understanding of the signaling and desensitization of this receptor.
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| 5. |
- Hsieh, Chia-Ju, et al.
(author)
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Interaction of ligands for pet with the dopamine d3 receptor : in silico and in vitro methods
- 2021
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In: Biomolecules. - : MDPI. - 2218-273X. ; 11:4
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Journal article (peer-reviewed)abstract
- [18F]Fallypride and [18F]Fluortriopride (FTP) are two different PET radiotracers that bind with sub-nanomolar affinity to the dopamine D3 receptor (D3R). In spite of their similar D3 affinities, the two PET ligands display very different properties for labeling the D3R in vivo: [18F]Fallypride is capable of binding to D3R under "baseline" conditions, whereas [18F]FTP requires the depletion of synaptic dopamine in order to image the receptor in vivo. These data suggest that [18F]Fallypride is able to compete with synaptic dopamine for binding to the D3R, whereas [18F]FTP is not. The goal of this study was to conduct a series of docking and molecular dynamic simulation studies to identify differences in the ability of each molecule to interact with the D3R that could explain these differences with respect to competition with synaptic dopamine. Competition studies measuring the ability of each ligand to compete with dopamine in the β-arrestin assay were also conducted. The results of the in silico studies indicate that FTP has a weaker interaction with the orthosteric binding site of the D3R versus that of Fallypride. The results of the in silico studies were also consistent with the IC50 values of each compound in the dopamine β-arrestin competition assays. The results of this study indicate that in silico methods may be able to predict the ability of a small molecule to compete with synaptic dopamine for binding to the D3R.
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| 6. |
- López-Cano, Marc, et al.
(author)
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Remote local photoactivation of morphine produces analgesia without opioid-related adverse effects
- 2023
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In: British Journal of Pharmacology. - : John Wiley & Sons. - 0007-1188 .- 1476-5381. ; 180:7, s. 958-974
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Journal article (peer-reviewed)abstract
- Background and Purpose: Opioid-based drugs are the gold standard medicines for pain relief. However, tolerance and several side effects (i.e. constipation and dependence) may occur upon chronic opioid administration. Photopharmacology is a promising approach to improve the benefit/risk profiles of these drugs. Thus, opioids can be locally activated with high spatiotemporal resolution, potentially minimizing systemic-mediated adverse effects. Here, we aimed at developing a morphine photo-derivative (photocaged morphine), which can be activated upon light irradiation both in vitro and in vivo.Experimental Approach: Light-dependent activity of pc-morphine was assessed in cell-based assays (intracellular calcium accumulation and electrophysiology) and in mice (formalin animal model of pain). In addition, tolerance, constipation and dependence were investigated in vivo using experimental paradigms.Key results: In mice, pc-morphine was able to elicit antinociceptive effects, both using external light-irradiation (hind paw) and spinal cord implanted fibre-optics. In addition, remote morphine photoactivation was devoid of common systemic opioid-related undesired effects, namely, constipation, tolerance to the analgesic effects, rewarding effects and naloxone-induced withdrawal.Conclusion and Implications: Light-dependent opioid-based drugs may allow effective analgesia without the occurrence of tolerance or the associated and severe opioid-related undesired effects.
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| 7. |
- Paucar, Martin, et al.
(author)
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V374A KCND3 Pathogenic Variant Associated With Paroxysmal Ataxia Exacerbations
- 2021
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In: Neurology Genetics. - : American Academy of Neurology. - 2376-7839. ; 7:1
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Journal article (peer-reviewed)abstract
- Objective: Ataxia channelopathies share common features such as slow motor progression and variable degrees of cognitive dysfunction. Mutations in potassium voltage-gated channel subfamily D member 3 (KCND3), encoding the K+ channel, Kv4.3, are associated with spinocerebellar ataxia (SCA) 19, allelic with SCA22. Mutations in potassium voltage-gated channel subfamily C member 3 (KCNC3), encoding another K+ channel, Kv3.3, cause SCA13. First, a comprehensive phenotype assessment was carried out in a family with autosomal dominant ataxia harboring 2 genetic variants in KCNC3 and KCND3. To evaluate the physiological impact of these variants on channel currents, in vitro studies were performed.Methods: Clinical and psychometric evaluations, neuroimaging, and genotyping of a family (mother and son) affected by ataxia were carried out. Heterozygous and homozygous Kv3.3 A671V and Kv4.3 V374A variants were evaluated in Xenopus laevis oocytes using 2-electrode voltage-clamp. The influence of Kv4 conductance on neuronal activity was investigated computationally using a Purkinje neuron model.Results: The main clinical findings were consistent with adult-onset ataxia with cognitive dysfunction and acetazolamide-responsive paroxysmal motor exacerbations in the index case. Despite cognitive deficits, fluorodeoxyglucose (FDG)-PET displayed hypometabolism mainly in the severely atrophic cerebellum. Genetic analyses revealed the new variant c.1121T>C (V374A) in KCND3 and c.2012T>C (A671V) in KCNC3. In vitro electrophysiology experiments on Xenopus oocytes demonstrated that the V374A mutant was nonfunctional when expressed on its own. Upon equal co-expression of wild-type (WT) and V374A channel subunits, Kv4.3 currents were significantly reduced in a dominant negative manner, without alterations of the gating properties of the channel. By contrast, Kv3.3 A671V, when expressed alone, exhibited moderately reduced currents compared with WT, with no effects on channel activation or inactivation. Immunohistochemistry demonstrated adequate cell membrane translocation of the Kv4.3 V374A variant, thus suggesting an impairment of channel function, rather than of expression. Computational modeling predicted an increased Purkinje neuron firing frequency upon reduced Kv4.3 conductance.Conclusions: Our findings suggest that Kv4.3 V374A is likely pathogenic and associated with paroxysmal ataxia exacerbations, a new trait for SCA19/22. The present FDG PET findings contrast with a previous study demonstrating widespread brain hypometabolism in SCA19/22.
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| 8. |
- Saarinen, Marcus, et al.
(author)
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TAAR1 dependent and independent actions of the potential antipsychotic and dual TAAR1/5-HT1A receptor agonist SEP-383856
- 2022
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In: Neuropsychopharmacology. - : Springer Nature. - 0893-133X .- 1740-634X. ; 47, s. 2319-2329
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Journal article (peer-reviewed)abstract
- SEP-383856 (SEP-856) is a novel antipsychotic under clinical development. It displays a unique pattern of receptor interaction, with only weak (partial agonist) activity at dopamine D2 receptors, yet more potent agonist activity at the trace amine associated receptor (TAAR1) and 5-hydroxytryptamine 1 A receptor (5-HT1A). Nonetheless, these observations await independent confirmation and more detailed characterization of the in vitro and in vivo actions of SEP-856 at TAAR1 and 5-HT1A receptors would be instructive. Herein, we employed luminescence complementation technology in heterologous live cell systems, confocal microscopy, voltage clamp electrophysiology, behavioral readouts and TAAR1 knockout (KO) mice to study SEP-856 in further detail. We provide evidence for the ability of SEP-856 to activate TAAR1 at the surface plasma membrane, and show that this interaction results in Gαs recruitment (pEC50: 6.08 ± 0.22 EMAX: 96.41% ± 15.26) and by extension, to G-protein inwardly rectifying potassium (GIRK) channel activation. Using TAAR1-KO mice, we find TAAR1 to be indispensable for SEP-856 control of body temperature, baseline locomotion reduction and for “antipsychotic-like” efficacy as characterized by a reversal of dizocilipine (MK-801) mediated disruption of pre-pulse inhibition. Conversely, the inhibition by SEP-856 of MK-801 induced locomotion was unaffected in TAAR1 KO mice. SEP-856 behaved as a low-potency, partial agonist at the 5-HT1A receptor, while it partially inhibited recruitment of D2 receptor-coupled Gα and GIRK by DA and acted as a weak partial agonist with low potency at the same receptor when applied alone. Our findings corroborate and extend previous observations on the molecular substrates engaged by this unique, dual TAAR1/5-HT1A receptor agonist and potential antipsychotic that could prove to have major advantages in the treatment of schizophrenia and other psychotic disorders.
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| 9. |
- Sahlholm, Kristoffer
(author)
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Voltage sensitivity of dopamine D2-like receptors
- 2011
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Doctoral thesis (other academic/artistic)abstract
- G protein coupled receptors (GPCRs) mediate a multitude of responses serving hormonal, neurotransmitter, and sensory functions. These receptors are important drug targets; in fact, about 27 % of prescribed drugs are GPCR ligands. The dopamine D2 receptor is prominently expressed within the CNS as two distinct isoforms; D2L (long isoform) and D2S (short isoform). The former is mainly expressed postsynaptically, whereas the latter functions primarily as an inhibitory auto- and heteroreceptor. The D2 receptor is of considerable pharmacological interest, as it constitutes the main target for most antiparkinsonian and antipsychotic drugs in clinical use. While many ion channels have long been known to be voltage sensitive, this property has not been attributed to GPCRs until quite recently. As a notable example, the muscarinic M2 receptor was shown to display depolarization-induced decreases in agonist binding and functional potency. M2 receptor voltage sensitivity has been implicated in the autoreceptor function of this GPCR, by permitting rapid control of neurotransmitter release kinetics by membrane voltage. The present work investigated the voltage sensitivities of the three D2-like dopamine receptors; D2, D3, and D4. The bulk of the experiments were carried out in Xenopus oocytes heterologously expressing D2-like receptors with G protein-coupled inwardly rectifying potassium channel (GIRK) subunits. GIRK channels are activated by Gβγ subunits from inhibitory G proteins and were used as readout of receptor activation. It was found that dopamine potency was reduced by depolarization to a similar extent at both isoforms of the D2 receptor. However, at the dopamine D3 receptor dopamine potency was not significantly affected, while a weak, albeit significant potency decrease was observed at the dopamine D4 receptor. Moreover, in mammalian cells expressing fluorescent G protein subunits, changes in inter-subunit Förster Resonance Energy Transfer (FRET) were used as readout of D2S receptor activation. Determination of dopamine concentration-response relationships in single cells under simultaneous patch clamp revealed similar depolarization-induced potency shifts as when studying GIRK channel activation in oocytes. Furthermore, radioligand binding experiments carried out on oocytes in hyperpolarizing vs. depolarizing buffer established that dopamine binding is reduced by depolarization. Interestingly, the effect of voltage was different for different agonists at the D2S receptor, including efficacious, high-affinity antiparkinsonian agonists. This agonist-specificity did not reflect selective signalling via distinct G protein subtypes. However, contacts between agonist hydroxyl groups and receptor serine residues, as well as between the agonist amine group and a conserved aspartate residue, were found to be important for the voltage induced potency shift of phenethylamine agonists, such as dopamine. In conclusion, the findings presented in this thesis suggest that the dopamine D2-like receptors are differentially affected by voltage. At the D2S receptor, specific agonist- receptor interactions determine the effect of the receptor’s voltage sensitivity on agonist potency and efficacy. This information demonstrates the relevance of GPCR voltage sensitivity to dopaminergic signalling, reveals new details about the mechanism of voltage sensitive agonism, and points to the possibility of using differentially voltage- modulated agonists to investigate the relevance of this phenomenon in native tissue.
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| 10. |
- Stan, Tiberiu Loredan, et al.
(author)
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Neurophysiological treatment effects of mesdopetam, pimavanserin and clozapine in a rodent model of Parkinson's disease psychosis
- 2024
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In: Neurotherapeutics. - : Elsevier. - 1878-7479 .- 1933-7213. ; 21:2, s. 1-12
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Journal article (peer-reviewed)abstract
- Psychosis in Parkinson's disease is a common phenomenon associated with poor outcomes. To clarify the pathophysiology of this condition and the mechanisms of antipsychotic treatments, we have here characterized the neurophysiological brain states induced by clozapine, pimavanserin, and the novel prospective antipsychotic mesdopetam in a rodent model of Parkinson's disease psychosis, based on chronic dopaminergic denervation by 6-OHDA lesions, levodopa priming, and the acute administration of an NMDA antagonist. Parallel recordings of local field potentials from eleven cortical and sub-cortical regions revealed shared neurophysiological treatment effects for the three compounds, despite their different pharmacological profiles, involving reversal of features associated with the psychotomimetic state, such as a reduction of aberrant high-frequency oscillations in prefrontal structures together with a decrease of abnormal synchronization between different brain regions. Other drug-induced neurophysiological features were more specific to each treatment, affecting network oscillation frequencies and entropy, pointing to discrete differences in mechanisms of action. These findings indicate that neurophysiological characterization of brain states is particularly informative when evaluating therapeutic mechanisms in conditions involving symptoms that are difficult to assess in rodents such as psychosis, and that mesdopetam should be further explored as a potential novel antipsychotic treatment option for Parkinson psychosis.
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| 11. |
- Stepniewski, Tomasz Maciej, et al.
(author)
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Mechanistic insights into dopaminergic and serotonergic neurotransmission : concerted interactions with helices 5 and 6 drive the functional outcome
- 2021
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In: Chemical Science. - : Royal Society of Chemistry. - 2041-6520 .- 2041-6539. ; 12:33, s. 10990-11003
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Journal article (peer-reviewed)abstract
- Brain functions rely on neurotransmitters that mediate communication between billions of neurons. Disruption of this communication can result in a plethora of psychiatric and neurological disorders. In this work, we combine molecular dynamics simulations, live-cell biosensor and electrophysiological assays to investigate the action of the neurotransmitter dopamine at the dopaminergic D2 receptor (D2R). The study of dopamine and closely related chemical probes reveals how neurotransmitter binding translates into the activation of distinct subsets of D2R effectors (i.e.: Gi2, GoB, Gz and β-arrestin 2). Ligand interactions with key residues in TM5 (S5.42) and TM6 (H6.55) in the D2R binding pocket yield a dopamine-like coupling signature, whereas exclusive TM5 interaction is typically linked to preferential G protein coupling (in particular GoB) over β-arrestin. Further experiments for serotonin receptors indicate that the reported molecular mechanism is shared by other monoaminergic neurotransmitter receptors. Ultimately, our study highlights how sequence variation in position 6.55 is used by nature to fine-tune β-arrestin recruitment and in turn receptor signaling and internalization of neurotransmitter receptors.
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| 12. |
- Valle-Leon, Marta, et al.
(author)
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Decreased striatal adenosine A2A-dopamine D2 receptor heteromerization in schizophrenia
- 2021
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In: Neuropsychopharmacology. - : Springer Nature. - 0893-133X .- 1740-634X. ; 46, s. 665-672
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Journal article (peer-reviewed)abstract
- According to the adenosine hypothesis of schizophrenia, the classically associated hyperdopaminergic state may be secondary to a loss of function of the adenosinergic system. Such a hypoadenosinergic state might either be due to a reduction of the extracellular levels of adenosine or alterations in the density of adenosine A(2A)receptors (A(2A)Rs) or their degree of functional heteromerization with dopamine D(2)receptors (D2R). In the present study, we provide preclinical and clinical evidences for this latter mechanism. Two animal models for the study of schizophrenia endophenotypes, namely the phencyclidine (PCP) mouse model and the A(2A)R knockout mice, were used to establish correlations between behavioural and molecular studies. In addition, a new AlphaLISA-based method was implemented to detect native A(2A)R-D2R heteromers in mouse and human brain. First, we observed a reduction of prepulse inhibition in A(2A)R knockout mice, similar to that observed in the PCP animal model of sensory gating impairment of schizophrenia, as well as a significant upregulation of striatal D2R without changes in A(2A)R expression in PCP-treated animals. In addition, PCP-treated animals showed a significant reduction of striatal A(2A)R-D2R heteromers, as demonstrated by the AlphaLISA-based method. A significant and pronounced reduction of A(2A)R-D2R heteromers was next demonstrated in postmortem caudate nucleus from schizophrenic subjects, even though both D2R and A(2A)R were upregulated. Finally, in PCP-treated animals, sub-chronic administration of haloperidol or clozapine counteracted the reduction of striatal A(2A)R-D2R heteromers. The degree of A(2A)R-D2R heteromer formation in schizophrenia might constitute a hallmark of the illness, which indeed should be further studied to establish possible correlations with chronic antipsychotic treatments.
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| 13. |
- Valle-León, Marta, et al.
(author)
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Unique effect of clozapine on adenosine A2A-dopamine D2 receptor heteromerization
- 2023
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In: Biomedicine and Pharmacotherapy. - : Elsevier. - 0753-3322 .- 1950-6007. ; 160
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Journal article (peer-reviewed)abstract
- The striatal dopamine D2 receptor (D2R) is generally accepted to be involved in positive symptoms of schizophrenia and is a main target for clinically used antipsychotics. D2R are highly expressed in the striatum, where they form heteromers with the adenosine A2A receptor (A2AR). Changes in the density of A2AR-D2R heteromers have been reported in postmortem tissue from patients with schizophrenia, but the degree to which A2R are involved in schizophrenia and the effect of antipsychotic drugs is unknown. Here, we examine the effect of exposure to three prototypical antipsychotic drugs on A2AR-D2R heteromerization in mammalian cells using a NanoBiT assay. After 16 h of exposure, a significant increase in the density of A2AR-D2R heteromers was found with haloperidol and aripiprazole, but not with clozapine. On the other hand, clozapine, but not haloperidol or aripiprazole, was associated with a significant decrease in A2AR-D2R heteromerization after 2 h of treatment. Computational binding models of these compounds revealed distinctive molecular signatures that explain their different influence on heteromerization. The bulky tricyclic moiety of clozapine displaces TM 5 of D2R, inducing a clash with A2AR, while the extended binding mode of haloperidol and aripiprazole stabilizes a specific conformation of the second extracellular loop of D2R that enhances the interaction with A2AR. It is proposed that an increase in A2AR-D2R heteromerization is involved in the extrapyramidal side effects (EPS) of antipsychotics and that the specific clozapine-mediated destabilization of A2AR-D2R heteromerization can explain its low EPS liability.
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| 14. |
- Zeberg, Hugo, et al.
(author)
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A Neanderthal Sodium Channel Increases Pain Sensitivity in Present-Day Humans
- 2020
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In: Current Biology. - : Elsevier. - 0960-9822 .- 1879-0445. ; 30:17, s. 3465-3469
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Journal article (peer-reviewed)abstract
- The sodium channel Nav1.7 is crucial for impulse generation and conduction in peripheral pain pathways [1]. In Neanderthals, the Nav1.7 protein carried three amino acid substitutions (M932L, V991L, and D1908G) relative to modern humans. We expressed Nav1.7 proteins carrying all combinations of these substitutions and studied their electrophysiological effects. Whereas the single amino acid substitutions do not affect the function of the ion channel, the full Neanderthal variant carrying all three substitutions, as well as the combination of V991L with D1908G, shows reduced inactivation, suggesting that peripheral nerves were more sensitive to painful stimuli in Neanderthals than in modern humans. We show that, due to gene flow from Neanderthals, the three Neanderthal substitutions are found in ∼0.4% of present-day Britons, where they are associated with heightened pain sensitivity.
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| 15. |
- Ågren, Richard, et al.
(author)
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An E280K Missense Variant in KCND3/Kv4.3—Case Report and Functional Characterization
- 2023
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In: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 24:13
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Journal article (peer-reviewed)abstract
- A five-year-old girl presented with headache attacks, clumsiness, and a history of transient gait disturbances. She and her father, mother, twin sister, and brother underwent neurological evaluation, neuroimaging, and exome sequencing covering 357 genes associated with movement disorders. Sequencing revealed the new variant KCND3 c.838G>A, p.E280K in the father and sisters, but not in the mother and brother. KCND3 encodes voltage-gated potassium channel D3 (Kv4.3) and mutations have been associated with spinocerebellar ataxia type 19/22 (SCA19/22) and cardiac arrhythmias. SCA19/22 is characterized by ataxia, Parkinsonism, peripheral neuropathy, and sometimes, intellectual disability. Neuroimaging, EEG, and ECG were unremarkable. Mild developmental delay with impaired fluid reasoning was observed in both sisters, but not in the brother. None of the family members demonstrated ataxia or parkinsonism. In Xenopus oocyte electrophysiology experiments, E280K was associated with a rightward shift in the Kv4.3 voltage-activation relationship of 11 mV for WT/E280K and +17 mV for E280K/E280K relative to WT/WT. Steady-state inactivation was similarly right-shifted. Maximal peak current amplitudes were similar for WT/WT, WT/E280K, and E280K/E280K. Our data indicate that Kv4.3 E280K affects channel activation and inactivation and is associated with developmental delay. However, E280K appears to be relatively benign considering it does not result in overt ataxia.
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| 16. |
- Ågren, Richard, et al.
(author)
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Dopamine d2 receptor agonist binding kinetics : role of a conserved serine residue
- 2021
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In: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:8
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Journal article (peer-reviewed)abstract
- The forward (kon) and reverse (koff ) rate constants of drug–target interactions have important implications for therapeutic efficacy. Hence, time-resolved assays capable of measuring these binding rate constants may be informative to drug discovery efforts. Here, we used an ion channel activation assay to estimate the kons and koff s of four dopamine D2 receptor (D2R) ago-nists; dopamine (DA), p-tyramine, (R)-and (S)-5-OH-dipropylaminotetralin (DPAT). We further probed the role of the conserved serine S1935.42 by mutagenesis, taking advantage of the preferential interaction of (S)-, but not (R)-5-OH-DPAT with this residue. Results suggested similar koff s for the two 5-OH-DPAT enantiomers at wild-type (WT) D2R, both being slower than the koff s of DA and p-tyramine. Conversely, the kon of (S)-5-OH-DPAT was estimated to be higher than that of (R)-5-OH-DPAT, in agreement with the higher potency of the (S)-enantiomer. Furthermore, S1935.42A mutation lowered the kon of (S)-5-OH-DPAT and reduced the potency difference between the two 5-OH-DPAT enantiomers. Kinetic Kds derived from the koff and kon estimates correlated well with EC50 values for all four compounds across four orders of magnitude, strengthening the notion that our assay captured meaningful information about binding kinetics. The approach presented here may thus prove valuable for characterizing D2R agonist candidate drugs.
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| 17. |
- Ågren, Richard, et al.
(author)
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Evidence for two modes of binding of the negative allosteric modulator SB269,652 to the dopamine D2 receptor
- 2022
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In: Biomedicines. - : MDPI. - 2227-9059. ; 10:1
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Journal article (peer-reviewed)abstract
- SB269,652 has been described as the first negative allosteric modulator (NAM) of the dopamine D2 receptor (D2 R), however, the binding mode and allosteric mechanism of action of this ligand remain incompletely understood. SB269,652 comprises an orthosteric, primary pharmacophore and a secondary (or allosteric) pharmacophore joined by a hydrophilic cyclohexyl linker and is known to form corresponding interactions with the orthosteric binding site (OBS) and the secondary binding pocket (SBP) in the D2 R. Here, we observed a surprisingly low potency of SB269,652 to negatively modulate the D2 R-mediated activation of G protein-coupled inward-rectifier potassium channels (GIRK) and decided to perform a more detailed investigation of the interaction between dopamine and SB269,652. The results indicated that the SB269,652 inhibitory potency is increased 6.6-fold upon ligand pre-incubation, compared to the simultaneous co-application with dopamine. Mutagenesis experiments implicated both S193 in the OBS and E95 in the SBP in the effect of pre-application. The present findings extend previous knowledge about how SB269,652 competes with dopamine at the D2 R and may be useful for the development of novel D2 R ligands, such as antipsychotic drug candidates.
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| 18. |
- Ågren, Richard, et al.
(author)
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G protein-coupled receptor kinase-2 confers isoform-specific calcium sensitivity to dopamine D2 receptor desensitization
- 2021
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In: The FASEB Journal. - : John Wiley & Sons. - 0892-6638 .- 1530-6860. ; 35:11
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Journal article (peer-reviewed)abstract
- The dopamine D2 receptor (D2R) functions as an autoreceptor on dopaminergic cell bodies and terminals and as a postsynaptic receptor on a variety of neurons in the central nervous system. As a result of alternative splicing, the D2R is expressed as two isoforms: long (D2LR) and short (D2SR) differing by a stretch of 29 residues in the third intracellular loop, with D2SR being the predominant presynaptic isoform. Recent reports described a Ca2+ sensitivity of the desensitization time course of potassium currents elicited via D2SR, but not via D2LR, when either isoform was selectively expressed in dopaminergic neurons. Here, we aimed to study the mechanism behind this subtype-specific Ca2+ sensitivity. Thus, we measured the desensitization of potassium channel responses evoked by D2LR and D2SR using two-electrode voltage clamp in Xenopus oocytes in the absence and presence of different amounts of β-arrestin2 and G protein-coupled receptor kinase-2 (GRK2), both of which are known to play important roles in D2R desensitization in native cells. We found that co-expression of both GRK2 and β-arrestin2 was necessary for reconstitution of the Ca2+ sensitivity of D2SR desensitization, while D2LR did not display Ca2+ sensitivity under these conditions. The effect of Ca2+ chelation by BAPTA-AM to slow the rate of D2SR desensitization was mimicked by the GRK2 inhibitor, Cmpd101, and by the kinase-inactivating GRK2 mutation, K220R, but not by the PKC inhibitor, Gö6976, nor by the calmodulin antagonist, KN-93. Thus, Ca2+-sensitive desensitization of D2SR appears to be mediated via a GRK2 phosphorylation-dependent mechanism.
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| 19. |
- Ågren, Richard, et al.
(author)
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In vitro comparison of ulotaront (SEP-363856) and ralmitaront (RO6889450) : two TAAR1 agonist candidate antipsychotics
- 2023
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In: International Journal of Neuropsychopharmacology. - : Oxford University Press. - 1461-1457 .- 1469-5111. ; 26:9, s. 599-606
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Journal article (peer-reviewed)abstract
- BACKGROUND: Trace amine-associated receptor-1 (TAAR1) agonists have been proposed as potential antipsychotics, with ulotaront and ralmitaront having reached clinical trials. While ulotaront demonstrated efficacy in a recent Phase II trial, a corresponding study studies of ralmitaront failed to show efficacy as a monotherapy or as an adjunct to atypical antipsychotics. In addition to TAAR1 agonism, ulotaront is a partial agonist at the serotonin 1A receptor (5-HT1AR). However, little is known about ralmitaront.METHODS: We compared ulotaront and ralmitaront at TAAR1, 5-HT1AR, and dopamine D2 using luciferase complementation-based G protein recruitment, cAMP accumulation, and G protein-coupled inward rectifier potassium channel activation assays.RESULTS: Ralmitaront showed lower efficacy at TAAR1 in G protein recruitment, cAMP accumulation, and GIRK activation assays. Moreover, ralmitaront lacked detectable activity at 5-HT1AR and dopamine D2.CONCLUSIONS: Compared with ulotaront, ralmitaront shows lower efficacy and slower kinetics at TAAR1 and lacks efficacy at 5-HT1AR. These data may be relevant to understanding differences in clinical profiles of these 2 compounds.
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| 20. |
- Ågren, Richard, et al.
(author)
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Ligand with Two Modes of Interaction with the Dopamine D-2 Receptor-An Induced-Fit Mechanism of Insurmountable Antagonism
- 2020
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In: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 11:19, s. 3130-3143
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Journal article (peer-reviewed)abstract
- A solid understanding of the mechanisms governing ligand binding is crucial for rational design of therapeutics targeting the dopamine D2 receptor (D2R). Here, we use G protein-coupled inward rectifier potassium (GIRK) channel activation in Xenopus oocytes to measure the kinetics of D2R antagonism by a series of aripiprazole analogues, as well as the recovery of dopamine (DA) responsivity upon washout. The aripiprazole analogues comprise an orthosteric and a secondary pharmacophore and differ by the length of the saturated carbon linker joining these two pharmacophores. Two compounds containing 3- and 5-carbon linkers allowed for a similar extent of recovery from antagonism in the presence of 1 or 100 μM DA (>25 and >90% of control, respectively), whereas recovery was less prominent (∼20%) upon washout of the 4-carbon linker compound, SV-III-130, both with 1 and 100 μM DA. Prolonging the coincubation time with SV-III-130 further diminished recovery. Curve-shift experiments were consistent with competition between SV-III-130 and DA. Two mutations in the secondary binding pocket (V91A and E95A) of D2R decreased antagonistic potency and increased recovery from SV-III-130 antagonism, whereas a third mutation (L94A) only increased recovery. Our results suggest that the secondary binding pocket influences recovery from inhibition by the studied aripiprazole analogues. We propose a mechanism, supported by in silico modeling, whereby SV-III-130 initially binds reversibly to the D2R, after which the drug-receptor complex undergoes a slow transition to a second ligand-bound state, which is dependent on secondary binding pocket integrity and irreversible during the time frame of our experiments.
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| 21. |
- Ågren, Richard, et al.
(author)
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Major genetic risk factors for Dupuytren's disease are inherited from neandertals
- 2023
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In: Molecular biology and evolution. - : Oxford University Press. - 0737-4038 .- 1537-1719. ; 40:6
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Journal article (peer-reviewed)abstract
- Dupuytren's disease is characterized by fingers becoming permanently bent in a flexed position. Whereas people of African ancestry are rarely afflicted by Dupuytren's disease, up to ∼30% of men over 60 years suffer from this condition in northern Europe. Here, we meta-analyze 3 biobanks comprising 7,871 cases and 645,880 controls and find 61 genome-wide significant variants associated with Dupuytren's disease. We show that 3 of the 61 loci harbor alleles of Neandertal origin, including the second and third most strongly associated ones (P = 6.4 × 10-132 and P = 9.2 × 10-69, respectively). For the most strongly associated Neandertal variant, we identify EPDR1 as the causal gene. Dupuytren's disease is an example of how admixture with Neandertals has shaped regional differences in disease prevalence.
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| 22. |
- Ågren, Richard, et al.
(author)
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Voltage-Dependent Dopamine Potency at D-1-Like Dopamine Receptors
- 2020
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In: Frontiers in Pharmacology. - : Frontiers Media S.A.. - 1663-9812. ; 11
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Journal article (peer-reviewed)abstract
- In recent years, transmembrane voltage has been found to modify agonist potencies at several G protein-coupled receptors (GPCRs). Whereas the voltage sensitivities of the Gαi/o-coupled dopamine D2-like receptors (D2R, D3R, D4R) have previously been investigated, the putative impact of transmembrane voltage on agonist potency at the mainly Gαs/olf-coupled dopamine D1-like receptors (D1R, D5R) has hitherto not been reported. Here, we assayed the potency of dopamine in activating G protein-coupled inward rectifier potassium (GIRK) channels co-expressed with D1R and D5R in Xenopus oocytes, at -80 mV and at 0 mV. Furthermore, GIRK response deactivation rates upon dopamine washout were measured to estimate dopamine dissociation rate (koff) constants. Depolarization from -80 to 0 mV was found to reduce dopamine potency by about 7-fold at both D1R and D5R. This potency reduction was accompanied by an increase in estimated dopamine koffs at both receptors. While the GIRK response elicited via D1R was insensitive to pertussis toxin (PTX), the response evoked via D5R was reduced by 64% (-80 mV) and 71% (0 mV) in the presence of PTX. Injection of oocytes with Gαs antisense oligonucleotide inhibited the D1R-mediated response by 62% (-80 mV) and 76% (0 mV) and abolished the D5R response when combined with PTX. Our results suggest that depolarization decreases dopamine affinity at D1R and D5R. The voltage-dependent affinities of dopamine at D1R and D5R may be relevant to the functions of these receptors in learning and memory.
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