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Summary: Extracellular nucleotides, including ATP, are essential regulators of liver function and serve as danger signals that trigger inflammation upon injury. Ectonucleotidases, which are expressed by liver-resident cells and recruited immune cells sequentially hydrolyse nucleotides to adenosine. The nucleotide/nucleoside balance orchestrates liver homeostasis, tissue repair, and functional restoration by regulating the crosstalk between liver-resident cells and recruited immune cells. In this review, we discuss our current knowledge on the role of purinergic signals in liver homeostasis, restriction of inflammation, stimulation of liver regeneration, modulation of fibrogenesis, and regulation of carcinogenesis. Moreover, we discuss potential targeted therapeutic strategies for liver diseases based on purinergic signals involving blockade of nucleotide receptors, enhancement of ectonucleoside triphosphate diphosphohydrolase activity, and activation of adenosine receptors.

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS) affecting more than 2.5 million individuals worldwide. In the present study, myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) mice were treated with adenosine receptor A2A antagonist SCH58261 at different periods of EAE development. The administration of SCH58261 at 11–28 days post-immunization (d.p.i.) with MOG improved the neurological deficits. This time window corresponds to the therapeutic time window for MS treatment. SCH58261 significantly reduced the CNS neuroinflammation including reduced local infiltration of inflammatory cells, demyelination, and the numbers of macrophage/microglia in the spinal cord. Importantly, SCH58261 ameliorated the EAE-induced neurobehavioral deficits. By contrast, the SCH58261 treatment was ineffective when administered at the beginning of the onset of EAE (i.e., 1–10 d.p.i). The identification of the effective therapeutic window of A2A receptor antagonist provide insight into the role of A2A receptor signaling in EAE, and support SCH58261 as a candidate for the treatment of MS in human.

In high throughput screening of our file compounds, a novel structure 1 was identified as a potent A(2A) receptor antagonist with no selectivity over the A1 adenosine receptor. The structure-activity relationship investigation using 1 as a template lead to identification of a novel class of compounds as potent and selective antagonists of A(2A) adenosine receptor. Compound 26 was identified to be the most potent A(2A) receptor antagonist (Ki = 0.8 nM) with 100-fold selectivity over the A1 adenosine receptor.

G protein-coupled receptors (GPCRs) represent the largest family of integral membrane receptors and are involved in many intracellular communications in response to diverse external stimuli. Despite advances in GPCR biophysical characterization, a major challenge continues to be the isolation of large quantities of membrane protein in functional form. Here we report the purification of a GPCR, namely the human adenosine receptor A2A, using solely the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After purification, the receptor remains folded and capable of binding to ligand. Interestingly, no cholesterol addition was required to retain receptor activity and no detergent removal step was necessary. Additionally, this approach will enable the addition of longer chain lipids create biomimetic membrane systems (e.g. bilayered micelles) that can be oriented in the presence of a magnetic field, which are widely used in NMR studies.

Predicting accurate ligand poses and ligand selective receptor conformations are imperative in designing efficacious, functionally specific drugs for G-protein coupled receptors. Comparison of the crystal structures of carazolol (inverse agonist), formoterol(agonist), and agonist with G-protein mimic bound β2 Adrenergic Receptor (β2AR) shows that the agonist stabilizes a slightly different conformation when there is no G-protein bound. The G-protein bound receptor state exhibits a high affinity conformation for the agonist. Using the computational method LITiCon, we have calculated the activation pathways for full, partial and inverse agonists of β2AR, which are in agreement with fluorescence intensity lifetime measurements. MD simulations starting from various conformations along the activation pathway (total ∼1.5 μs) show that in the absence of G protein, norepinephrine (agonist) stabilizes an intermediate receptor state that is similar to the formoterol bound intermediate state without the G-protein, in agreement with the recent crystal structure of formoterol bound β2AR. Thus coupling to G protein may be needed for stabilizing the fully active state.Using Liticon method we have calculated the activation pathway of agonist bound adenosine receptor A2A starting from its inactive state. We found that water plays a important role in the docking of the antagonist as well as the agonist.We recently predicted the structures of D3 dopamine receptor and CXCR4 chemokine receptor as part of the competition for the assessment of computational methods in GPCR modeling (GPCR Dock 2010). The predicted docked poses of both the ligands are in close agreement with the crystal structures. The details of the methods and comparison with the crystal structures will be presented.