Development of the first in vivo GPR17 ligand through an iterative drug discovery pipeline: A novel disease-modifying strategy for multiple sclerosis

The GPR17 receptor, expressed on oligodendroglial precursors (OPCs, the myelin producing cells), has emerged as an attractive target for a pro-myelinating strategy in multiple sclerosis (MS). However, the proof-of-concept that selective GPR17 ligands actually exert protective activity in vivo is still missing. Here, we exploited an iterative drug discovery pipeline to prioritize novel and selective GPR17 pro-myelinating agents out of more than 1,000,000 compounds. We first performed an in silico high-throughput screening on GPR17 structural model to identify three chemically-diverse ligand families that were then combinatorially exploded and refined. Top-scoring compounds were sequentially tested on reference pharmacological in vitro assays with increasing complexity, ending with myelinating OPC-neuron co-cultures. Successful ligands were filtered through in silico simulations of metabolism and pharmacokinetics, to select the most promising hits, whose dose and ability to target the central nervous system were then determined in vivo. Finally, we show that, when administered according to a preventive protocol, one of them (named by us as galinex) is able to significantly delay the onset of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. This outcome validates the predictivity of our pipeline to identify novel MS-modifying agents

Surface Plasmon Resonance as a Tool for Ligand Binding Investigation of Engineered GPR17 Receptor, a G Protein Coupled Receptor Involved in Myelination

The aim of this study was to investigate the potential of surface plasmon resonance (SPR) spectroscopy for the measurement of real-time ligand-binding affinities and kinetic parameters for GPR17, a G protein-coupled receptor (GPCR) of major interest in medicinal chemistry as potential target in demyelinating diseases. The receptor was directly captured, in a single-step, from solubilized membrane extracts on the sensor chip through a covalently bound anti-6x-His-antibody and retained its ligand binding activity for over 24h. Furthermore, our experimental setup made possible, after a mild regeneration step, to remove the bound receptor without damaging the antibody, and thus to reuse many times the same chip. Two engineered variants of GPR17, designed for crystallographic studies, were expressed in insect cells, extracted from crude membranes and analyzed for their binding with two high affinity ligands: the antagonist Cangrelor and the agonist Asinex 1. The calculated kinetic parameters and binding constants of ligands were in good agreement with those reported from activity assays and highlighted a possible functional role of the N-terminal residues of the receptor in ligand recognition and binding. Validation of SPR results was obtained by docking and molecular dynamics of GPR17-ligands interactions and by functional in vitro studies. The latter allowed us to confirm that Asinex 1 behaves as GPR17 receptor agonist, inhibits forskolin-stimulated adenylyl cyclase pathway and promotes oligodendrocyte precursor cell maturation and myelinating ability

GPR17 molecular modelling: interactions with non-conventional pro-inflammatory ligands

GPR17 is a class A-GPCRs operated by di erent classes of ligands, such as uracil nucleotides, cysteinyl-leukotrienes and oxysterols. Similar to other receptors of the same class, GPR17 can associate into homo- and hetero-dimers. Recent ndings suggest its promiscuous behavior namely the possibility to be operated by ligands able to transversely interact with more than one GPCRs. In fact, both GPR17 and CXCR2 are operated by oxysterols, and both GPR17 and CXCRn ligands have demonstrated roles in orchestrating in ammatory responses and oligodendrocyte precursor cell (OPC) di erentiation to myelinating cells in acute and chronic diseases of the CNS. Here we demonstrate that GPR17 can be activated by the chemokine stromal-derived factor-1 (SDF-1), a ligand of CXCR4 and CXCR7, and investigate the underlying molecular recognition mechanism, by combining in silico modelling data with in vitro validation in (i) a classical reference pharmacological assay for GPCR activity and (ii) a model of maturation of primary OPCs. We also demonstrate that cangrelor, a GPR17 orthosteric antagonist, can block the SDF-1-mediated activation of GPR17 in a concentration-dependent manner. The ability of GPR17 to respond to di erent classes of GPCR ligands suggests that this receptor modi es its function depending on changes occurring in the extracellular mileu changes occurring under speci c pathophysiological conditions and advocates it as a strategic target for neurodegenerative diseases with an in ammatory/immune component

Adenosine: old passion, new perspectives

In this review is summarized my research in the adenosine field from the beginning of my carrier to day. My research began preparing the degree thesis with Prof. Carlo Alfonso Rossi, about the purification and characterization of adenosine deaminase. My scientific interest for adenosine has spread during the years and I have been interested in the study of ARs and their related transduction and in the study of molecular mechanisms involved in homologous and heterologous AR receptor regulatio

Desensitization as A3 adenosine receptor regulation: physiopathological implications

A3 adenosine receptors are subjected to fast desensitisation, internalisation and down regulation after agonist exposure. As almost all G-protein coupled receptors, these regulatory mechanisms are mediated by receptor phosphorylation in a process that results in the recruitment of arrestins. Receptor phosphorylation is a flexible and dynamic process in which the receptors are regulated in a unique manner depending on the cell type in which the receptors are expressed. This rapid regulatory mechanism has an important pathophysiological role: in fact the impairment of receptor responses obtained following agonist exposure may be therapeutically equivalent to antagonist occupancy and suggest the use of agonists in different pathological conditions in which the activity of A3 receptors should be regulated, such as neurodegenerative and cancer diseases. In this scenario, phosphorylation offers a mechanism of regulating the signalling outcome of G-protein coupled receptors that can be tailored to meet specific physiological role

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

Quinolinedione nucleus as a novel scaffold for A1 and A 2A adenosine receptor antagonists

In the last few years, much effort has been directed towards the synthesis of selective adenosine receptor (AR) antagonists since they are attractive tools for pharmacological intervention in many pathophysiological conditions. During our studies aimed at obtaining new nonclassical adenosine antagonists devoid of phosphodiesterase (PDE) inhibition, a series of 2-pyridones and 2,5-quinolinediones (3a-f, 5a-f, 6a,c-f) has been synthesized as potential AR ligands. Binding affinities of the new compounds were determined for bovine and human adenosine A1, A2A, and A3 receptors. Compound 5f showed good affinity (Ki = 7.8 μM) towards human A1AR but no selectivity (Ki = 7.0 μM) towards human A2AAR, whereas compound 6f showed more affinity towards human A 2A (Ki = 16 μM) than A1 receptor (percentage inhibition at 10 μM concentration = 11). In the 1-100 μM range, the new compounds did not inhibit cardiac PDE3 activity at all. Molecular modeling studies carried out on 5f and 6f support the pharmacological results and suggest 6f as a potential lead compound selective towards A2AAR

Short-term TNF-Alpha treatment induced A2B adenosine receptor desensitization in human astroglial cells

Long-term glial cell treatment with the proinflammatory cytokine TNF-alpha has been demonstrated to increase the functional responsiveness of A2B adenosine receptors (A2B ARs), which in turn synergize with the cytokine inducing chronic astrogliosis. In the present study, we investigated the short-term effects of TNF-alpha on A2B AR functional responses in human astroglial cells (ADF), thus simulating the acute phase of cerebral damage which is characterized by both cytokine and adenosine high level release. Short-term TNF-alpha cell treatment caused A2B AR phosphorylation inducing, in turn, impairment in A2B AR-G protein coupling and cAMP production. These effects occurred in a time-dependent manner with a maximum following 3-h cell exposure. Moreover, we showed PKC intracellular kinase is mainly involved in the TNF-alpha-mediated regulation of A2B AR functional responses. The results may indicate the A2B AR functional impairment as a cell defense mechanism to counteract the A2B receptor-mediated effects during the acute phase of brain damage, underlying A2B AR as a target to modulate early inflammatory responses

A rapid and efficient immunoenzymatic assay to detect receptor protein interactions : G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors, and are the target of at least one-thofd the current therapeutic drugs on the market. Along their life cycle, GPCRs are accompanied by a range of specialized GPCR-interacting proteins (GIPs), which take part in receptor proper folding, targeting to the appropriate subcellular compartments and in receptor signaling task and also in receptor regulation process,esuch as desensitization and internalizoni. The direction of protein-protein interactions and multi-protein complexes formation is crucial in understanding protein function and their implication in pathological. Although several methods have been already developed to assay protein compl s, some of them are quite laborious, expensive, and, more important, they do not generate fully quantitative results. Herein, we show a rapid immunoenzymatic assay to quantify GPCR interactionswith its signaling proteins. The recently -odrphanized GPCR, GPR17, was chosen as a GPCR prototype to optimize the assay. In a GPR17 transfected cell line and primary oligodendrocyte precursor cells, GPR17 interaction withp roteins involved in the typical GPCR regulation, such as desensitization and internalizationmachinery, was investigated. The obtained results were validated by immunoprecipitation experiments, confirming this new method as a rapid and quantitative assay to study protein-protein interactions

Modulation of A1 and A2B adenosine receptor activity: a new strategy to sensitise glioblastoma stem cells to chemotherapy.

Therapies that target the signal transduction and biological characteristics of cancer stem cells (CSCs) are innovative strategies that are used in combination with conventional chemotherapy and radiotherapy to effectively reduce the recurrence and significantly improve the treatment of glioblastoma multiforme (GBM). The two main strategies that are currently being exploited to eradicate CSCs are (a) chemotherapeutic regimens that specifically drive CSCs toward cell death and (b) those that promote the differentiation of CSCs, thereby depleting the tumour reservoir. Extracellular purines, particularly adenosine triphosphate, have been implicated in the regulation of CSC formation, but currently, no data on the role of adenosine and its receptors in the biological processes of CSCs are available. In this study, we investigated the role of adenosine receptor (AR) subtypes in the survival and differentiation of CSCs isolated from human GBM cells. Stimulation of A1AR and A2BAR had a prominent anti-proliferative/pro-apoptotic effect on the CSCs. Notably, an A1AR agonist also promoted the differentiation of CSCs toward a glial phenotype. The differential effects of the two AR agonists on the survival and/or differentiation of CSCs may be ascribed to their distinct regulation of the kinetics of ERK/AKT phosphorylation and the expression of hypoxia-inducible factors. Most importantly, the AR agonists sensitised CSCs to the genotoxic activity of temozolomide (TMZ) and prolonged its effects, most likely through different mechanisms, are as follows: (i) by A2BAR potentiating the pro-apoptotic effects of TMZ and (ii) by A1AR driving cells toward a differentiated phenotype that is more sensitive to TMZ. Taken together, the results of this study suggested that the purinergic system is a novel target for a stem cell-oriented therapy that could reduce the recurrence of GBM and improve the survival rate of GBM patients