The P2X7 Receptor in Infection and Inflammation.

Adenosine triphosphate (ATP) accumulates at sites of tissue injury and inflammation. Effects of extracellular ATP are mediated by plasma membrane receptors named P2 receptors (P2Rs). The P2R most involved in inflammation and immunity is the P2X7 receptor (P2X7R), expressed by virtually all cells of innate and adaptive immunity. P2X7R mediates NLRP3 inflammasome activation, cytokine and chemokine release, T lymphocyte survival and differentiation, transcription factor activation, and cell death. Ten human P2RX7 gene splice variants and several SNPs that produce complex haplotypes are known. The P2X7R is a potent stimulant of inflammation and immunity and a promoter of cancer cell growth. This makes P2X7R an appealing target for anti-inflammatory and anti-cancer therapy. However, an in-depth knowledge of its structure and of the associated signal transduction mechanisms is needed for an effective therapeutic development.

Machine Learning Scoring Functions for Drug Discovery from Experimental and Computer-Generated Protein-Ligand Structures: Towards Per-Target Scoring Functions.

In recent years, machine learning has been proposed as a promising strategy to build accurate scoring functions for computational docking finalized to numerically empowered drug discovery. However, the latest studies have suggested that over-optimistic results had been reported due to the correlations present in the experimental databases used for training and testing. Here, we investigate the performance of an artificial neural network in binding affinity predictions, comparing results obtained using both experimental protein-ligand structures as well as larger sets of computer-generated structures created using commercial software. Interestingly, similar performances are obtained on both databases. We find a noticeable performance suppression when moving from random horizontal tests to vertical tests performed on target proteins not included in the training data. The possibility to train the network on relatively easily created computer-generated databases leads us to explore per-target scoring functions, trained and tested ad-hoc on complexes including only one target protein. Encouraging results are obtained, depending on the type of protein being addressed.

Dual Anta-Inhibitors Targeting Protein Kinase CK1δ and A2A Adenosine Receptor Useful in Neurodegenerative Disorders.

Currently, the number of patients with neurodegenerative pathologies is estimated at over one million, with consequences also on the economic level. Several factors contribute to their development, including overexpression of A2A adenosine receptors (A2AAR) in microglial cells and up-regulation and post-translational alterations of some casein kinases (CK), among them, CK-1δ. The aim of the work was to study the activity of A2AAR and CK1δ in neurodegeneration using in-house synthesized A2A/CK1δ dual anta-inhibitors and to evaluate their intestinal absorption. Experiments were performed on N13 microglial cells, which were treated with a proinflammatory CK cocktail to simulate an inflammatory state typical of neurodegenerative diseases. Results showed that the dual anta-inhibitors have the ability to counteract the inflammatory state, even if compound 2 is more active than compound 1. In addition, compound 2 displayed an important antioxidant effect similar to the reference compound ZM241385. Since many known kinase inhibitors are very often unable to cross lipid bilayer membranes, the ability of A2A/CK1δ double anta-inhibitors to cross the intestinal barrier was investigated by an everted gut sac assay. HPLC analysis revealed that both compounds are able to cross the intestinal barrier, making them promising candidates for oral therapy.

A2A Adenosine Receptor Antagonists: Are Triazolotriazine and Purine Scaffolds Interchangeable?

The A2A adenosine receptor (A2AAR) is one of the four subtypes activated by nucleoside adenosine, and the molecules able to selectively counteract its action are attractive tools for neurodegenerative disorders. In order to find novel A2AAR ligands, two series of compounds based on purine and triazolotriazine scaffolds were synthesized and tested at ARs. Compound 13 was also tested in an in vitro model of neuroinflammation. Some compounds were found to possess high affinity for A2AAR, and it was observed that compound 13 exerted anti-inflammatory properties in microglial cells. Molecular modeling studies results were in good agreement with the binding affinity data and underlined that triazolotriazine and purine scaffolds are interchangeable only when 5- and 2-positions of the triazolotriazine moiety (corresponding to the purine 2- and 8-positions) are substituted.

New 8-amino-1,2,4-triazolo[4,3-a]pyrazin-3-one derivatives. Evaluation of different moieties on the 6-aryl ring to obtain potent and selective human A2A adenosine receptor antagonists.

In this work, further structural investigations on the 8-amino-2-phenyl-6-aryl-1,2,4-triazolo[4,3-a]pyrazin-3-one series were carried out to achieve potent and selective human A2A adenosine receptor (AR) antagonists. Different ether and amide moieties were attached at the para-position of the 6-phenyl ring, thus leading to compounds 1-9 and 10-18, respectively. Most of these moieties contained terminal basic rings (pyrrolidine, morpholine, piperidine and substituted piperazines) which were thought to confer good physicochemical and drug-like properties. Compounds 11-16, bearing the amide linker, possessed high affinity and selectivity for the hA2A AR (Ki = 3.6-11.8 nM). Also derivatives 1-9, featuring an ether linker, preferentially targeted the hA2A AR but with lower affinity, compared to those of the relative amide compounds. Docking studies, carried out at the hA2A AR binding site, highlighted some crucial ligand-receptor interactions, particularly those provided by the appended substituent whose nature deeply affected hA2A AR affinity.

Neuroprotective potential of adenosine A1 receptor partial agonists in experimental models of cerebral ischemia.

Cerebral ischemia is the second most common cause of death and a major cause of disability worldwide. Available therapies are based only on anticoagulants or recombinant tissue plasminogen activator. Extracellular adenosine increases during ischemia and acts as a neuroprotective endogenous agent mainly by activating adenosine A1 receptors (A1 Rs) which control calcium influx, glutamate release, membrane potential, and metabolism. Accordingly, in many experimental paradigms it has been already demonstrated that the stimulation of A1 R with full agonists is able to reduce ischemia-related structural and functional brain damage; unfortunately, cardiovascular side effects and desensitization of A1 R induced by these compounds have strongly limited their exploitation in stroke therapy so far. Among the newly emerging compounds, A1 R partial agonists could be almost free of side effects and equally effective. Therefore, we decided to evaluate the neuroprotective potential of two A1 R partial agonists, namely 2'-dCCPA and 3'-dCCPA, in in vitro and ex vivo experimental models of cerebral ischemia. Within the experimental paradigm of oxygen-glucose deprivation in vitro in human neuroblastoma (SH-SY5Y) cells both A1 R partial agonists increased cell viability. Considering the high level of expression of A1 Rs in the hippocampus and the susceptibility of CA1 region to hypoxia, we performed electrophysiological experiments in this subfield. The application of 7 min of oxygen-glucose deprivation constantly produces an irreversible synaptic failure in all the C57Bl/6 mice hippocampal slices evaluated; both tested compounds allowed a significant recovery of synaptic transmission. These findings demonstrate that A1 R and its partial agonists are still of interest for cerebral ischemia therapy. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Novel human adenosine receptor antagonists based on the 7-amino-thiazolo[5,4-d]pyrimidine scaffold. Structural investigations at the 2-, 5- and 7-positions to enhance affinity and tune selectivity.

This paper describes the synthesis of novel 7-amino-thiazolo[5,4-d]pyrimidines bearing different substituents at positions 2, 5 and 7 of the thiazolopyrimidine scaffold. The synthesized compounds 2-27 were evaluated in radioligand binding (A1, A2A and A3) and adenylyl cyclase activity (A2B and A2A) assays, in order to evaluate their affinity and potency at human adenosine receptor subtypes. The current study allowed us to support that affinity and selectivity of 7-amino-thiazolo[5,4-d]pyrimidine derivatives towards the adenosine receptor subtypes can be modulated by the nature of the groups attached at positions 2, 5 and 7 of the bicyclic scaffold. To rationalize the hypothetical binding mode of the newly synthesized compounds, we also performed docking calculations in human A2A, A1 and A3 structures.

New sensible method to quantize the intestinal absorption of receptor ligands.

In recent years, special attention has been paid to the A3 adenosine receptor (A3AR) as a possible pharmacological target to treat intestinal inflammation. In this work, it was set up a novel method to quantify the concentration of a promising anti-inflammatory agent inside and outside of intestinal barrier using the everted gut sac technique. The compound chosen for the present study is one of the most potent and selective A3AR agonist reported so far, named AR 170 (N6-methyl-2-phenylethynyl-5'-N-methylcarboxamidoadenosine). In order to evaluate the intestinal absorption of AR 170 the radioligand binding assay in comparison with HPLC-DAD was used. Results showed that the compound is absorbed via passive diffusion by paracellular pathway. The concentrations determined in the serosal (inside the sac) fluid by radioligand binding assay are in good agreement with those obtained through the widely used HPLC/MS protocol, demonstrating the reliability of the method. It is worthwhile to note that the radioligand binding assay allows detecting very low concentrations of analyte, thus offering an excellent tool to measure the intestinal absorption of receptor ligands. Moreover, the AR 170 quantity outside the gut sac and the interaction with A3AR could presuppose good topical anti-inflammatory effects of this compound.

Novel 8-amino-1,2,4-triazolo[4,3-a]pyrazin-3-one derivatives as potent human adenosine A1 and A2A receptor antagonists. Evaluation of their protective effect against ß-amyloid-induced neurotoxicity in SH-SY5Y cells.

In this work, an enlarged series of 1,2,4-triazolo[4,3-a]pyrazin-3-ones was designed to target the human (h) A2A adenosine receptor (AR) or both hA1 and hA2A ARs. The novel 8-amino-1,2,4-triazolopyrazin-3-one derivatives 1-25 featured a phenyl or a benzyl pendant at position 2 while different aryl/heteroaryl substituents were placed at position 6. Two compounds (8 and 10) endowed with high affinity (Ki = 7.2 and 10.6 nM) and a complete selectivity for the hA2A AR were identified. Moreover, several derivatives possessed nanomolar affinity for both hA1 and hA2A ARs (both Ki < 20 nM) and different degrees of selectivity versus the hA3 AR. Two selected compounds (10 and 25) demonstrated ability in preventing ß-amyloid peptide (25-35)-induced neurotoxicity in SH-SY5Y cells. Results of docking studies at the hA2A and hA1 AR crystal structures helped us to rationalize the observed affinity data and to highlight that the steric hindrance of the substituents at the 2- and 6-position of the bicyclic core affects the binding mode in the receptor cavity.

New A2A adenosine receptor antagonists: a structure-based upside-down interaction in the receptor cavity.

Adenosine receptor antagonists are generally based on heterocyclic core structures presenting substituents of various volumes and chemical-physical profiles. Adenine and purine-based adenosine receptor antagonists have been reported in literature. In this work we combined various substituents in the 2, 6, and 8-positions of 9-ethylpurine to depict a structure-affinity relationship analysis at the human adenosine receptors. Compounds were rationally designed trough molecular modeling analysis and then synthesized and evaluated at radioligand binding studies at human adenosine receptors. The new compounds showed affinity for the human adenosine receptors, with some derivatives endowed with low nanomolar Ki data, in particular at the A2AAR subtype. The purine core proves to be a versatile core structure for the development of novel adenosine receptor antagonists with nanomolar affinity for these membrane proteins.