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Solvent reorganization is a major driving force of protein–ligand association, but the contribution of binding site waters to ligand affinity is poorly understood.

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Extracellular adenosine is a danger/injury signal that initiates protective physiology, such as hypothermia. Adenosine has been shown to trigger hypothermia via agonism at A and A adenosine receptors (AAR, AAR). Here, we find that adenosine continues to elicit hypothermia in mice null for AAR and AAR and investigated the effect of agonism at AAR or AAR. The poorly brain penetrant AAR agonists CGS-21680 and PSB-0777 caused hypothermia, which was not seen in mice lacking AAR. MRS7352, a likely non-brain penetrant AAR antagonist, inhibited PSB-0777 hypothermia. While vasodilation is probably a contributory mechanism, AAR agonism also caused hypometabolism, indicating that vasodilation is not the sole mechanism. The AAR agonist BAY60-6583 elicited hypothermia, which was lost in mice null for AAR. Low intracerebroventricular doses of BAY60-6583 also caused hypothermia, indicating a brain site of action, with neuronal activation in the preoptic area and paraventricular nucleus of the hypothalamus. Thus, agonism at any one of the canonical adenosine receptors, AAR, AAR, AAR, or AAR, can cause hypothermia. This four-fold redundancy in adenosine-mediated initiation of hypothermia may reflect the centrality of hypothermia as a protective response.

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Prodrugs (MRS7422, MRS7476) of highly selective A 3 adenosine receptor (AR) agonists Cl-IB-MECA and MRS5698, respectively, were synthesized by succinylation of the 2′ and 3′ hydroxyl groups, and the parent, active drug was shown to be readily liberated upon incubation with liver esterases. The prodrug MRS7476 had greatly increased aqueous solubility compared with parent MRS5698 and was fully efficacious and with a longer duration than MRS7422 in reversing mouse neuropathic pain (chronic constriction injury model, 3 μmol/kg, p.o.), a known A 3 AR effect. MRS7476 (5 mg/kg, p.o., twice daily) was found to protect against non-alcoholic steatohepatitis (NASH) in the STAM mouse model, indicated by the NAFLD activity score. Hepatocyte ballooning, IL-10 production, and liver histology were significantly normalized in the MRS7476-treated mice, but not liver fibrosis (no change in ACTA2 levels) or inflammation. Hepatic expression of ADORA3 in human NAFLD patients was 1.9-fold lower compared to normal controls. Adora3 expression determined by qPCR in primary mouse liver was associated with the stellate cells, and its mouse full body A 3 AR knockout worsened liver markers of inflammation and steatosis. Thus, we have introduced a reversible prodrug strategy that enables water solubility and in vivo activity of masked A 3 AR agonists in models of two disease conditions. KeywordsPurinergic receptors . Nucleosides . Adenosine receptor . Prodrug . Pain . Steatohepatitis Abbreviations ACTA2 Smooth muscle α actin ALT Alanine transaminase AR Adenosine receptor AUC Area under the curve CCI Chronic constriction injury Cl-IB-MECA 2-Chloro-N 6 -(3-iodobenzyl)-adenosine-5′-Nmethyluronamide GPCR G protein-coupled receptor HCC Hepatocellular carcinoma

The A2A adenosine receptor is a protein belonging to a family of four GPCR adenosine receptors. It is involved in the regulation of several pathophysiological conditions in both the central nervous system and periphery. In the brain, its localization at pre- and postsynaptic level in striatum, cortex, hippocampus and its effects on glutamate release, microglia and astrocyte activation account for a crucial role in neurodegenerative diseases, including Alzheimer’s disease (AD). This ailment is considered the main form of dementia and is expected to exponentially increase in coming years. The pathological tracts of AD include amyloid peptide-β extracellular accumulation and tau hyperphosphorylation, causing neuronal cell death, cognitive deficit, and memory loss. Interestingly, in vitro and in vivo studies have demonstrated that A2A adenosine receptor antagonists may counteract each of these clinical signs, representing an important new strategy to fight a disease for which unfortunately only symptomatic drugs are available. This review offers a brief overview of the biological effects mediated by A2A adenosine receptors in AD animal and human studies and reports the state of the art of A2A adenosine receptor antagonists currently in clinical trials. As an original approach, it focuses on the crucial role of pharmacokinetics and ability to pass the blood–brain barrier in the discovery of new agents for treating CNS disorders. Considering that A2A receptor antagonist istradefylline is already commercially available for Parkinson’s disease treatment, if the proof of concept of these ligands in AD is confirmed and reinforced, it will be easier to offer a new hope for AD patients.

The A 3 adenosine receptor (A 3 AR) is a target for pain, ischemia, and inflammatory disease therapy. Among the ligand tools available are selective agonists and antagonists, including radioligands, but most high-affinity non-nucleoside antagonists are limited in selectivity to primate species. We have explored the structure−activity relationship of a previously reported A 3 AR antagonist DPTN 9 (N-[4-(3,5-dimethylphenyl)-5-(4-pyridyl)-1,3-thiazol-2-yl]nicotinamide) for radiolabeling, including 3-halo derivatives (3-iodo, MRS7907), and characterized 9 as a high -affinity radioligand [ 3 H]MRS7799. A 3 AR K d values were (nM): 0.55 (human), 3.74 (mouse), and 2.80 (rat). An extended methyl acrylate (MRS8074, 19) maintained higher affinity (18.9 nM) than a 3-((5-chlorothiophen-2-yl)ethynyl) derivative 20. Compound 9 had an excellent brain distribution in rats (brain/plasma ratio ∼1). Receptor docking predicted its orthosteric site binding by engaging residues that were previously found to be essential for AR binding. Thus the new radioligand promises to be a useful species-general antagonist tracer for receptor characterization and drug discovery.