Synaptic events are important to define treatment strategies for brain disorders. In the present ... more Synaptic events are important to define treatment strategies for brain disorders. In the present paper, freshly obtained rat brain striatal minces were incubated under different times and conditions to determine dopamine biosynthesis, storage, and tyrosine hydroxylase phosphorylation. Remarkably, we found that endogenous dopamine spontaneously accumulated during tissue incubation at 37 °C ex vivo while dopamine synthesis simultaneously decreased. We analyzed whether these changes in brain dopamine biosynthesis and storage were linked to dopamine feedback inhibition of its synthesis-limiting enzyme tyrosine hydroxylase. The aromatic-l-amino-acid decarboxylase inhibitor NSD-1015 prevented both effects. As expected, dopamine accumulation was increased with l-DOPA addition or VMAT2-overexpression, and dopamine synthesis decreased further with added dopamine, the VMAT2 inhibitor tetrabenazine or D2 auto-receptor activation with quinpirole, accordingly to the known synaptic effects of these treatments. Phosphorylation activation and inhibition of tyrosine hydroxylase on Ser31 and Ser40 with okadaic acid, Sp-cAMP and PD98059 also exerted the expected effects. However, no clear-cut association was found between dopamine feedback inhibition of its own biosynthesis and changes of tyrosine hydroxylase phosphorylation, assessed by western blot and mass spectrometry. The later technique also revealed a new Thr30 phosphorylation in rat tyrosine hydroxylase. Our methodological assessment of brain dopamine synthesis and storage dynamics ex vivo could be applied to predict the in vivo effects of pharmacological interventions in animal models of dopamine-related disorders.
Background: Suicide is the leading cause of violent death. In 2014, the Catalan Ministry of Healt... more Background: Suicide is the leading cause of violent death. In 2014, the Catalan Ministry of Health launched a Suicide Risk Protocol, an intervention programme targeting individuals at high risk of suicide. Since 2016 the protocol has covered the whole of Catalonia (Spain). Objectives: To identify clinical, demographic, neuropsychological and biological factors and stress responses (Psychoneuroendocrine, inflammatory, genetic and neuroimaging markers) associated with suicide attempts. Method: Analysis of clinical sub-cohorts from a sample of patients registered under the Suicide Risk Protocol (n=900). All participants will undergo clinical assessment (neuropsychological and biomarkers) and be followed up over 18 months. Patients who repeat a suicide attempt will be compared with two groups: patients with a single suicide attempt and healthy controls with no history of attempted suicide. Discussion and conclusion: The CODIRISC project will also carry out a functional neuroimaging stud...
Employing two different alkyne-modified dopamine agonists to construct bivalent compounds via cli... more Employing two different alkyne-modified dopamine agonists to construct bivalent compounds via click chemistry resulted in the identification of a bivalent ligand (11c) for dopamine D2 receptor homodimer, which, compared to its parent monomeric alkyne, showed a 16-fold higher binding affinity for the dopamine D2 receptor and a 5-fold higher potency in a cAMP assay in HEK 293T cells stably expressing D2R. Molecular modeling revealed that 11c can indeed bridge the orthosteric binding sites of a D2R homodimer in a relaxed conformation via the TM5-TM6 interface and allows to largely rationalize the results of the receptor assays.
A collection of crystal structures of rhodopsin, β2-adrenergic and adenosine A2A receptors in act... more A collection of crystal structures of rhodopsin, β2-adrenergic and adenosine A2A receptors in active, intermediate and inactive states were selected for structural and energetic analyses to identify the changes involved in the activation/deactivation of Class A GPCRs. A set of helix interactions exclusive to either inactive or active/intermediate states were identified. The analysis of these interactions distinguished some local conformational changes involved in receptor activation, in particular, a packing between the intracellular domains of transmembrane helices H3 and H7 and a separation between those of H2 and H6. Also, differential movements of the extracellular and intracellular domains of these helices are apparent. Moreover, a segment of residues in helix H3, including residues L/I3.40 to L3.43, is identified as a key component of the activation mechanism, acting as a conformational hinge between extracellular and intracellular regions. Remarkably, the influence on the act...
Rhodopsin, the visual pigment in the retina, is a Class A G protein-coupled receptor (GPCR) coval... more Rhodopsin, the visual pigment in the retina, is a Class A G protein-coupled receptor (GPCR) covalently bound to retinal chromophore. In dark conditions, retinal is in the cis-isomeric state, stabilizing the rhodopsin inactive state as an inverse agonist. After light absorption, retinal undergoes an isomerization photoreaction to trans-retinal, which includes a conformational change of the receptor to its active state. In absence of retinal, the apoprotein opsin presents a low level of constitutive activity, which depends on pH and with higher propensity of activation at acidic pH. To examine the effect and the underlying mechanism that protonation may have on opsin activation, a number of MD simulations were run varying the number and identity of acidic residues selected for protonation. Results show that the combined protonation of D83, E113 and E247 is of special relevance for the induction of receptor activation. Subsequent conformational analysis of the MD trajectories provides ...
Dopamine D2 receptors (D2R) are known to form transient homodimer complexes, of which the increas... more Dopamine D2 receptors (D2R) are known to form transient homodimer complexes, of which the increased formation has already been associated with development of schizophrenia. Pharmacological targeting and modulation of the equilibrium of these receptor homodimers might lead to a better understanding of the critical role played by these complexes in physiological and pathological conditions. Whereas agonist addition has shown to prolong the D2R dimer lifetime and increase the level of dimer formation, the possible influence of D2R antagonists on dimerization has remained rather unexplored. Here, using a live-cell reporter assay based on the functional complementation of a split Nanoluciferase, a panel of six D2R antagonists were screened for their ability to modulate the level of D2LR dimer formation. Incubation with the D2R antagonist spiperone decreased the level of D2LR dimer formation significantly by 40–60% in real-time and after long-term (≥16 h) incubations. The fact that dimer ...
Correction for ‘Rational design of a peptide capture agent for CXCL8 based on a model of the CXCL... more Correction for ‘Rational design of a peptide capture agent for CXCL8 based on a model of the CXCL8:CXCR1 complex’ by Dorothea Helmeret al.,RSC Adv., 2015,5, 25657–25668.
Light-operated drugs constitute a major target in drug discovery, since they may provide spatiote... more Light-operated drugs constitute a major target in drug discovery, since they may provide spatiotemporal resolution for the treatment of complex diseases (i.e. chronic pain). JF-NP-26 is an inactive photocaged derivative of the metabotropic glutamate type 5 (mGlu5) receptor negative allosteric modulator raseglurant. Violet light illumination of JF-NP-26 induces a photochemical reaction prompting the active-drug's release, which effectively controls mGlu5 receptor activity both in ectopic expressing systems and in striatal primary neurons. Systemic administration in mice followed by local light-emitting diode (LED)-based illumination, either of the thalamus or the peripheral tissues, induced JF-NP-26-mediated light-dependent analgesia both in neuropathic and in acute/tonic inflammatory pain models. These data offer the first example of optical control of analgesia in vivo using a photocaged mGlu5 receptor negative allosteric modulator. This approach shows potential for precisely t...
Synaptic events are important to define treatment strategies for brain disorders. In the present ... more Synaptic events are important to define treatment strategies for brain disorders. In the present paper, freshly obtained rat brain striatal minces were incubated under different times and conditions to determine dopamine biosynthesis, storage, and tyrosine hydroxylase phosphorylation. Remarkably, we found that endogenous dopamine spontaneously accumulated during tissue incubation at 37 °C ex vivo while dopamine synthesis simultaneously decreased. We analyzed whether these changes in brain dopamine biosynthesis and storage were linked to dopamine feedback inhibition of its synthesis-limiting enzyme tyrosine hydroxylase. The aromatic-l-amino-acid decarboxylase inhibitor NSD-1015 prevented both effects. As expected, dopamine accumulation was increased with l-DOPA addition or VMAT2-overexpression, and dopamine synthesis decreased further with added dopamine, the VMAT2 inhibitor tetrabenazine or D2 auto-receptor activation with quinpirole, accordingly to the known synaptic effects of these treatments. Phosphorylation activation and inhibition of tyrosine hydroxylase on Ser31 and Ser40 with okadaic acid, Sp-cAMP and PD98059 also exerted the expected effects. However, no clear-cut association was found between dopamine feedback inhibition of its own biosynthesis and changes of tyrosine hydroxylase phosphorylation, assessed by western blot and mass spectrometry. The later technique also revealed a new Thr30 phosphorylation in rat tyrosine hydroxylase. Our methodological assessment of brain dopamine synthesis and storage dynamics ex vivo could be applied to predict the in vivo effects of pharmacological interventions in animal models of dopamine-related disorders.
Background: Suicide is the leading cause of violent death. In 2014, the Catalan Ministry of Healt... more Background: Suicide is the leading cause of violent death. In 2014, the Catalan Ministry of Health launched a Suicide Risk Protocol, an intervention programme targeting individuals at high risk of suicide. Since 2016 the protocol has covered the whole of Catalonia (Spain). Objectives: To identify clinical, demographic, neuropsychological and biological factors and stress responses (Psychoneuroendocrine, inflammatory, genetic and neuroimaging markers) associated with suicide attempts. Method: Analysis of clinical sub-cohorts from a sample of patients registered under the Suicide Risk Protocol (n=900). All participants will undergo clinical assessment (neuropsychological and biomarkers) and be followed up over 18 months. Patients who repeat a suicide attempt will be compared with two groups: patients with a single suicide attempt and healthy controls with no history of attempted suicide. Discussion and conclusion: The CODIRISC project will also carry out a functional neuroimaging stud...
Employing two different alkyne-modified dopamine agonists to construct bivalent compounds via cli... more Employing two different alkyne-modified dopamine agonists to construct bivalent compounds via click chemistry resulted in the identification of a bivalent ligand (11c) for dopamine D2 receptor homodimer, which, compared to its parent monomeric alkyne, showed a 16-fold higher binding affinity for the dopamine D2 receptor and a 5-fold higher potency in a cAMP assay in HEK 293T cells stably expressing D2R. Molecular modeling revealed that 11c can indeed bridge the orthosteric binding sites of a D2R homodimer in a relaxed conformation via the TM5-TM6 interface and allows to largely rationalize the results of the receptor assays.
A collection of crystal structures of rhodopsin, β2-adrenergic and adenosine A2A receptors in act... more A collection of crystal structures of rhodopsin, β2-adrenergic and adenosine A2A receptors in active, intermediate and inactive states were selected for structural and energetic analyses to identify the changes involved in the activation/deactivation of Class A GPCRs. A set of helix interactions exclusive to either inactive or active/intermediate states were identified. The analysis of these interactions distinguished some local conformational changes involved in receptor activation, in particular, a packing between the intracellular domains of transmembrane helices H3 and H7 and a separation between those of H2 and H6. Also, differential movements of the extracellular and intracellular domains of these helices are apparent. Moreover, a segment of residues in helix H3, including residues L/I3.40 to L3.43, is identified as a key component of the activation mechanism, acting as a conformational hinge between extracellular and intracellular regions. Remarkably, the influence on the act...
Rhodopsin, the visual pigment in the retina, is a Class A G protein-coupled receptor (GPCR) coval... more Rhodopsin, the visual pigment in the retina, is a Class A G protein-coupled receptor (GPCR) covalently bound to retinal chromophore. In dark conditions, retinal is in the cis-isomeric state, stabilizing the rhodopsin inactive state as an inverse agonist. After light absorption, retinal undergoes an isomerization photoreaction to trans-retinal, which includes a conformational change of the receptor to its active state. In absence of retinal, the apoprotein opsin presents a low level of constitutive activity, which depends on pH and with higher propensity of activation at acidic pH. To examine the effect and the underlying mechanism that protonation may have on opsin activation, a number of MD simulations were run varying the number and identity of acidic residues selected for protonation. Results show that the combined protonation of D83, E113 and E247 is of special relevance for the induction of receptor activation. Subsequent conformational analysis of the MD trajectories provides ...
Dopamine D2 receptors (D2R) are known to form transient homodimer complexes, of which the increas... more Dopamine D2 receptors (D2R) are known to form transient homodimer complexes, of which the increased formation has already been associated with development of schizophrenia. Pharmacological targeting and modulation of the equilibrium of these receptor homodimers might lead to a better understanding of the critical role played by these complexes in physiological and pathological conditions. Whereas agonist addition has shown to prolong the D2R dimer lifetime and increase the level of dimer formation, the possible influence of D2R antagonists on dimerization has remained rather unexplored. Here, using a live-cell reporter assay based on the functional complementation of a split Nanoluciferase, a panel of six D2R antagonists were screened for their ability to modulate the level of D2LR dimer formation. Incubation with the D2R antagonist spiperone decreased the level of D2LR dimer formation significantly by 40–60% in real-time and after long-term (≥16 h) incubations. The fact that dimer ...
Correction for ‘Rational design of a peptide capture agent for CXCL8 based on a model of the CXCL... more Correction for ‘Rational design of a peptide capture agent for CXCL8 based on a model of the CXCL8:CXCR1 complex’ by Dorothea Helmeret al.,RSC Adv., 2015,5, 25657–25668.
Light-operated drugs constitute a major target in drug discovery, since they may provide spatiote... more Light-operated drugs constitute a major target in drug discovery, since they may provide spatiotemporal resolution for the treatment of complex diseases (i.e. chronic pain). JF-NP-26 is an inactive photocaged derivative of the metabotropic glutamate type 5 (mGlu5) receptor negative allosteric modulator raseglurant. Violet light illumination of JF-NP-26 induces a photochemical reaction prompting the active-drug's release, which effectively controls mGlu5 receptor activity both in ectopic expressing systems and in striatal primary neurons. Systemic administration in mice followed by local light-emitting diode (LED)-based illumination, either of the thalamus or the peripheral tissues, induced JF-NP-26-mediated light-dependent analgesia both in neuropathic and in acute/tonic inflammatory pain models. These data offer the first example of optical control of analgesia in vivo using a photocaged mGlu5 receptor negative allosteric modulator. This approach shows potential for precisely t...
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