Azidobupramine, an Antidepressant-Derived Bifunctional Neurotransmitter Transporter Ligand Allowing Covalent Labeling and Attachment of Fluorophores

The aim of this study was to design, synthesize and validate a multifunctional antidepressant probe that is modified at two distinct positions. The purpose of these modifications was to allow covalent linkage of the probe to interaction partners, and decoration of probe-target complexes with fluorescent reporter molecules. The strategy for the design of such a probe (i.e., azidobupramine) was guided by the need for the introduction of additional functional groups, conveying the required properties while keeping the additional moieties as small as possible. This should minimize the risk of changing antidepressant-like properties of the new probe azidobupramine. To control for this, we evaluated the binding parameters of azidobupramine to known target sites such as the transporters for serotonin (SERT), norepinephrine (NET), and dopamine (DAT). The binding affinities of azidobupramine to SERT, NET, and DAT were in the range of structurally related and clinically active antidepressants. Furthermore, we successfully visualized azidobupramine-SERT complexes not only in SERT-enriched protein material but also in living cells stably overexpressing SERT. To our knowledge, azidobupramine is the first structural analogue of a tricyclic antidepressant that can be covalently linked to target structures and further attached to reporter molecules while preserving antidepressant-like properties and avoiding radioactive isotopes

The Recently Identified P2Y-Like Receptor GPR17 Is a Sensor of Brain Damage and a New Target for Brain Repair

Deciphering the mechanisms regulating the generation of new neurons and new oligodendrocytes, the myelinating cells of the central nervous system, is of paramount importance to address new strategies to replace endogenous damaged cells in the adult brain and foster repair in neurodegenerative diseases. Upon brain injury, the extracellular concentrations of nucleotides and cysteinyl-leukotrienes (cysLTs), two families of endogenous signaling molecules, are markedly increased at the site of damage, suggesting that they may act as “danger signals” to alert responses to tissue damage and start repair. Here we show that, in brain telencephalon, GPR17, a recently deorphanized receptor for both uracil nucleotides and cysLTs (e.g., UDP-glucose and LTD4), is normally present on neurons and on a subset of parenchymal quiescent oligodendrocyte precursor cells. We also show that induction of brain injury using an established focal ischemia model in the rodent induces profound spatiotemporal-dependent changes of GPR17. In the lesioned area, we observed an early and transient up-regulation of GPR17 in neurons expressing the cellular stress marker heat shock protein 70. Magnetic Resonance Imaging in living mice showed that the in vivo pharmacological or biotechnological knock down of GPR17 markedly prevents brain infarct evolution, suggesting GPR17 as a mediator of neuronal death at this early ischemic stage. At later times after ischemia, GPR17 immuno-labeling appeared on microglia/macrophages infiltrating the lesioned area to indicate that GPR17 may also acts as a player in the remodeling of brain circuitries by microglia. At this later stage, parenchymal GPR17+ oligodendrocyte progenitors started proliferating in the peri-injured area, suggesting initiation of remyelination. To confirm a specific role for GPR17 in oligodendrocyte differentiation, the in vitro exposure of cortical pre-oligodendrocytes to the GPR17 endogenous ligands UDP-glucose and LTD4 promoted the expression of myelin basic protein, confirming progression toward mature oligodendrocytes. Thus, GPR17 may act as a “sensor” that is activated upon brain injury on several embryonically distinct cell types, and may play a key role in both inducing neuronal death inside the ischemic core and in orchestrating the local remodeling/repair response. Specifically, we suggest GPR17 as a novel target for therapeutic manipulation to foster repair of demyelinating wounds, the types of lesions that also occur in patients with multiple sclerosis

Is "option B+" also being adopted in pregnant women in high-income countries? Temporal trends from a national study in Italy

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A2A-D2 Receptor Heterodimerization and Psychosis

Neuroleptic drugs, classified in typical and atypical, mainly act as D2 dopamine receptor antagonists. D2 and A2A receptors are co-expressed in the striatum where they structurally interact to form functional heterodimers. It has been demonstrated these receptors show a positive or negative functional cross-talk in dependence on their relative expression levels. To clarify the reciprocal interactions between adenosine and dopaminergic system in physiological conditions, we investigated the regulation of A2A adenosine receptor induced by D2 receptor antagonists in PC12 cells co-expressing both receptors in native conditions and in CHO cells co-expressing both receptors at levels comparable to those reported in striatum. In PC12 we were able to detect both receptors in the monomeric and dimeric form. Moreover, after chronic treatment with the typical antipsychotic haloperidol our results showed an increase of affinity and density of A2A receptor compared with the control, while treatment with atypical antipsychotics risperidone and clozapine did not show any significant difference. Treatment with haloperidol and risperidone showed an uncoupling of A2A receptor to G proteins and a significant decrease of cAMP levels. Chronic treatment with clozapine did not show any difference also in the G protein coupling and cAMP levels. In CHO cells our data showed that acute and chronic treatment with haloperidol increased the A2A AR affinity and responsiveness whereas no effects were detected with the atypical drug, clozapine. Otherwise, risperidone, an atypical antipsychotic but with high affinity for D2 DR, after long treatment induced a significant decrease of A2A AR affinity and responsiveness. Our results ascertain that A2A receptors are modulated by typical antipsychotics and by risperidone which profile does not fully fit to the atypical antipsychotic profile and these effects depend on the receptor levels. Our data suggest adenosine system as an alternative target for pathologies characterized by dopamine system dysfunction. In the same way, since A2A receptors are involved in brain damage, individual control of antipsychotic therapy and the associated regulation of adenosine system, may represent an important goal to control neurodegenerative processes considered as an amplificatory loop in several psychiatric disorders

Regulation of erythropoietin receptor activity in endothelial cells by different erythropoietin (EPO) derivatives: An

Abstract: In endothelial cells, erythropoietin receptors (EPORs) mediate the protective, proliferative and angiogenic effects of EPO and its analogues, which act as EPOR agonists. Because hormonal receptors undergo functional changes upon chronic exposure to agonists and because erythropoiesis-stimulating agents (ESAs) are used for the long-term treatment of anemia, it is critical to determine the mechanism by which EPOR responsiveness is regulated at the vascular level after prolonged exposure to ESAs. Here, we investigated EPOR desensitization/resensitization in human umbilical vein endothelial cells (HUVECs) upon exposure to three ESAs with different pharmacokinetic profiles, epoetin alpha (EPOα), darbepoetin alpha (DarbEPO) and continuous EPOR activator (CERA). These agonists all induced activation of the transcription factor STAT-5, which is a component of the intracellular pathway associated with EPORs. STAT-5 activation occurred with either monophasic or biphasic kinetics for EPOα/DarbEPO and CERA, respectively. ESAs, likely through activation of the STAT-5 pathway, induced endothelial cell proliferation and stimulated angiogenesis in vitro, demonstrating a functional role for epoetins on endothelial cells. All epoetins induced EPOR desensitization with more rapid kinetics forInt. J. Mol. Sci. 2013, 14 225

Loratadine and analogs: Discovery and preliminary SAR of inhibitors of the amino acid transporter B0AT2

B0AT2, encoded by the SLC6A15 gene, is a transporter for neutral amino acids that has recently been implicated in mood and metabolic disorders. It is predominantly expressed in the brain but little is otherwise known about its function. In order to identify inhibitors for this transporter we screened a library of different 3133 bioactive compounds. Loratadine, a clinically used histamine H1 receptor antagonist, was identified as a selective inhibitor of B0AT2 with an IC50 of 4 μM while being less active or inactive against several other members of the SLC6 family. Reversible inhibition of B0AT2 was confirmed by electrophysiology. A series of loratadine analogs was synthesized to get insight into the structure-activity relationships. Our studies provide the first chemical tool for B0AT2

Loratadine and Analogues: Discovery and Preliminary Structure–Activity Relationship of Inhibitors of the Amino Acid Transporter B⁰AT2

B⁰AT2, encoded by the SLC6A15 gene, is a transporter for neutral amino acids that has recently been implicated in mood and metabolic disorders. It is predominantly expressed in the brain, but little is otherwise known about its function. To identify inhibitors for this transporter, we screened a library of 3133 different bioactive compounds. Loratadine, a clinically used histamine H₁ receptor antagonist, was identified as a selective inhibitor of B⁰AT2 with an IC₅₀ of 4 μM while being less active or inactive against several other members of the SLC6 family. Reversible inhibition of B⁰AT2 was confirmed by electrophysiology. A series of loratadine analogues were synthesized to gain insight into the structure–activity relationships. Our studies provide the first chemical tool for B⁰AT2