Maria Letizia Trincavelli

The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinyl-leukotrienes receptor

Nucleotides and cysteinyl‐leukotrienes (CysLTs) are unrelated signaling molecules inducing multiple effects through separate G‐protein‐coupled receptors: the P2Y and the CysLT receptors. Here we show that GPR17, a Gi‐coupled orphan receptor at intermediate phylogenetic position between P2Y and CysLT receptors, is specifically activated by both families of endogenous ligands, leading to both adenylyl cyclase inhibition and intracellular calcium increases. Agonist‐response profile, as determined by [35S]GTPγS binding, was different from that of already known CysLT and P2Y receptors, with EC50 values in the nanomolar and micromolar range, for CysLTs and uracil nucleotides, respectively. Both rat and human receptors are highly expressed in the organs typically undergoing ischemic damage, that is, brain, heart and kidney. In vivo inhibition of GPR17 by either CysLT/P2Y receptor antagonists or antisense technology dramatically reduced ischemic damage in a rat focal ischemia model, suggesting GPR17 as the common molecular target mediating brain damage by nucleotides and CysLTs. In conclusion, the deorphanization of GPR17 revealed a dualistic receptor for two endogenous unrelated ligand families. These findings may lead to dualistic drugs of previously unexplored therapeutic potential.

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.

The oxysterol–CXCR2 axis plays a key role in the recruitment of tumor-promoting neutrophils

Tumor-derived oxysterols recruit protumor neutrophils in an LXR-independent, CXCR2-dependent manner, thus favoring tumor growth by promoting neoangiogenesis and immunosuppression.

Structure-based optimization of pyrazolo[3,4-d]pyrimidines as Abl inhibitors and antiproliferative agents toward human leukemia cell lines.

Results from molecular docking calculations and Grid mapping laid the foundations for a structure-based optimization approach to improve the biological properties of pyrazolo-pyrimidine derivatives in terms of inhibition of Abl enzymatic activity and antiproliferative properties toward human leukemia cells. Insertion of halogen substituents with various substitution patterns, suggested by simulations, led to a significant improvement of leukemia cell growth inhibition and to an increase up to 1 order of magnitude of the affinity toward Abl.

Combining Galantamine and Memantine in Multitargeted, New Chemical Entities Potentially Useful in Alzheimer’s Disease

Herein we report on a novel series of multitargeted compounds obtained by linking together galantamine and memantine. The compounds were designed by taking advantage of the crystal structures of acetylcholinesterase (AChE) in complex with galantamine derivatives. Sixteen novel derivatives were synthesized, using spacers of different lengths and chemical composition. The molecules were then tested as inhibitors of AChE and as binders of the N-methyl-d-aspartate (NMDA) receptor (NMDAR). Some of the new compounds were nanomolar inhibitors of AChE and showed micromolar affinities for NMDAR. All compounds were also tested for selectivity toward NMDAR containing the 2B subunit (NR2B). Some of the new derivatives showed a micromolar affinity for NR2B. Finally, selected compounds were tested using a cell-based assay to measure their neuroprotective activity. Three of them showed a remarkable neuroprotective profile, inhibiting the NMDA-induced neurotoxicity at subnanomolar concentrations (e.g., 5, named memagal, IC(50) = 0.28 nM).

Regulation of A2B adenosine receptor functioning by tumour necrosis factor a in human astroglial cells

Low‐affinity A2B adenosine receptors (A2B ARs), which are expressed in astrocytes, are mainly activated during brain hypoxia and ischaemia, when large amounts of adenosine are released. Cytokines, which are also produced at high levels under these conditions, may regulate receptor responsiveness. In the present study, we detected A2B AR in human astrocytoma cells (ADF) by both immunoblotting and real‐time PCR. Functional studies showed that the receptor stimulated adenylyl cyclase through Gs proteins. Moreover, A2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF‐α) treatment (24‐ h) increased A2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF‐α markedly reduced agonist‐dependent receptor phosphorylation on threonine residues and attenuated agonist‐mediated A2B ARs desensitization. In the presence of TNF‐α, A2B AR stimulation in vitro induced the elongation of astrocytic processes, a typical morphological hallmark of in vivo reactive astrogliosis. This event was completely prevented by the selective A2B AR antagonist MRS 1706 and required the presence of TNF‐α. These results suggest that, in ADF cells, TNF‐α selectively modulates A2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A2B AR in response to brain damage.

Agonist‐Induced Internalization and Recycling of the Human A3 Adenosine Receptors

Abstract: A3 adenosine receptors have been proposed to play an important role in the pathophysiology of cerebral ischemia with a regimen‐dependent nature of the therapeutic effects probably related to receptor desensitization and down‐regulation. Here we studied the agonist‐induced internalization of human A3 adenosine receptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internalization and signal desensitization and resensitization. Binding of N6‐(4‐amino‐3‐[125I]iodobenzyl)adenosine‐5′‐N‐methyluronamide to membranes from Chinese hamster ovary cells stably transfected with the human A3 adenosine receptor showed a profile typical of these receptors in other cell lines (KD = 1.3 ± 0.08 nM; Bmax = 400 ± 28 fmol/mg of proteins). The iodinated agonist, bound at 4°C to whole transfected cells, was internalized by increasing the temperature to 37°C with a rate constant of 0.04 ± 0.034 min‐1. Agonist‐induced internalization of A3 adenosine receptors was directly demonstrated by immunogold electron microscopy, which revealed the localization of these receptors in plasma membranes and intracellular vesicles. Moreover, short‐term exposure of these cells to the agonist caused rapid desensitization as tested in adenylyl cyclase assays. Subsequent removal of the agonist led to restoration of the receptor function and recycling of the receptors to the cell surface. The rate constant of receptor recycling was 0.02 ± 0.0017 min‐1. Blockade of internalization and recycling demonstrated that internalization did not affect signal desensitization, whereas recycling of internalized receptors was implicated in the signal resensitization.

Co-localization and functional cross-talk between A1 and P2Y1 purine receptors in rat hippocampus.

Adenosine and ATP, via their specific P1 and P2 receptors, modulate a wide variety of cellular and tissue functions, playing a neuroprotective or neurodegenerative role in brain damage conditions. Although, in general, adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, recent data suggest the existence of a heterodimerization and a functional interaction between P1 and P2 receptors in the brain. In particular, interactions of adenosine A1 and P2Y1 receptors may play important roles in the purinergic signalling cascade. In the present work, we investigated the subcellular localization/co‐localization of the receptors and their functional cross‐talk at the membrane level in Wistar rat hippocampus. This is a particularly vulnerable brain area, which is sensitive to adenosine‐ and ATP‐mediated control of glutamatergic transmission. The postembedding immunogold electron microscopy technique showed that the two receptors are co‐localized at the synaptic membranes and surrounding astroglial membranes of glutamatergic synapses. To investigate the functional cross‐talk between the two types of purinergic receptors, we evaluated the reciprocal effects of their activation on their G protein coupling. P2Y1 receptor stimulation impaired the potency of A1 receptor coupling to G protein, whereas the stimulation of A1 receptors increased the functional responsiveness of P2Y1 receptors. The results demonstrated an A1–P2Y1 receptor co‐localization at glutamatergic synapses and surrounding astrocytes and a functional interaction between these receptors in hippocampus, suggesting ATP and adenosine can interact in purine‐mediated signalling. This may be particularly important during pathological conditions, when large amounts of these mediators are released.

New 1,2,3-triazolo[1,5-a]quinoxalines: synthesis and binding to benzodiazepine and adenosine receptors. II.

On pursuing research about 1,2,3-triazolo[1,5-a]quinoxalines, in this paper we report synthesis and binding assays toward the benzodiazepine and A(1) and A(2A) adenosine receptors, of a new series of derivatives, bearing some structural changes (introduction of fluorine and trifluoromethyl in the seventh position, amino substituents in the fourth position, benzyl group in the fifth position and aroyl substituents in the third position). The biological tests have shown that only the 7-fluorosubstituted compounds 3a and 4a and the N-benzyl derivative 7 have a good affinity toward the benzodiazepine receptors, while only the 7-trifluoromethyl substituted compound 3b presents a moderate affinity with low selectivity toward the A(1) adenosine receptors. The other structural modifications strongly decreased biological activity.

Adenosine receptors: what we know and what we are learning.

Adenosine, beside its role in the intermediate metabolism, mediates its physiological functions by interacting with four receptor subtypes named A(1), A(2A), A(2B) and A(3). All these receptors belong to the superfamily of G protein-coupled receptors that represent the most widely targeted pharmacological protein class. Since adenosine receptors are widespread throughout the body, they are involved in a variety of physiological processes and pathology including neurological, cardiovascular, inflammatory diseases and cancer. At now, it is ascertained that the biological responses evoked by the activation of a single receptor are the result of complex and integrated signalling pathways targeted by different receptor proteins, interacting each other. These pathways may in turn control receptor responsiveness over time through fine regulatory mechanisms including desensitization-internalization processes. The knowledge of adenosine receptor structure as well as the molecular mechanisms underlying the regulation of receptor functioning and of receptor-receptor interactions during physio and pathological conditions represent a pivotal starting point to the development of new drugs with high efficacy and selectivity for each adenosine receptor subtype. The goal of this review is to summarize what we now and what we are learning about adenosine receptor structure, signalling and regulatory mechanisms. In addition, to dissect the potential therapeutic application of adenosine receptor ligands, the pathophysiological role of the receptor subtypes in different tissues are discussed.