Nonalcoholic fatty liver disease is closely associated with metabolic disorders, even in nonobese, nondiabetic subjects. Nonalcoholic fatty liver disease can be considered an early predictor of metabolic disorders, particularly in the normal-weight population.

Mutagenesis of the human A(3) adenosine receptor (AR) suggested that certain amino acid residues contributed differently to ligand binding and activation processes. Here we demonstrated that various adenosine modifications, including adenine substitution and ribose ring constraints, also contributed differentially to these processes. The ligand effects on cyclic AMP production in intact CHO cells expressing the A(3)AR and in receptor binding were compared. Notably, the simple 2-fluoro group alone or 2-chloro in combination with N(6)-substitution dramatically diminished the efficacy of adenosine derivatives, even converting agonist into antagonist. Other affinity-increasing substitutions, including N(6)-(3-iodobenzyl) 4 and the (Northern)-methanocarba 15, also reduced efficacy, except in combination with a flexible 5'-uronamide. 2-Cl-N(6)-(3-iodobenzyl) derivatives, both in the (N)-methanocarba (i.e., of the Northern conformation) and riboside series 18 and 5, respectively, were potent antagonists with little residual agonism. Ring-constrained 2',3'-epoxide derivatives in both riboside and (N)-methanocarba series 13 and 21, respectively, and a cyclized (spiral) 4',5'-uronamide derivative 14 were synthesized and found to be human A(3)AR antagonists. 14 bound potently at both human (26 nM) and rat (49 nM) A(3)ARs. A rhodopsin-based A(3)AR model, containing all domains except the C-terminal region, indicated separate structural requirements for receptor binding and activation for these adenosine analogues. Ligand docking, taking into account binding of selected derivatives at mutant A(3)ARs, featured interactions of TM3 (His95) with the adenine moiety and TMs 6 and 7 with the ribose 5'-region. The 5'-OH group of antagonist N(6)-(3-iodobenzyl)-2-chloroadenosine 5 formed a H-bond with N274 but not with S271. The 5'-substituent of nucleoside antagonists moved toward TM7 and away from TM6. The conserved Trp243 (6.48) side chain, involved in recognition of the classical (nonnucleoside) A(3)AR antagonists but not adenosine-derived ligands, displayed a characteristic movement exclusively upon docking of agonists. Thus, A(3)AR activation appeared to require flexibility at the 5'- and 3'-positions, which was diminished in (N)-methanocarba, spiro, and epoxide analogues, and was characteristic of ribose interactions at TM6 and TM7.

Ligand recognition has been extensively explored in G protein-coupled A 1 , A 2A , and A 2B adenosine receptors but not in the A 3 receptor, which is cerebroprotective and cardioprotective. We mutated several residues of the human A 3 adenosine receptor within transmembrane domains 3 and 6 and the second extracellular loop, which have been predicted by previous molecular modeling to be involved in the ligand recognition, including His 95 , Trp 243 , Leu 244 , Ser 247 , Asn 250 , and Lys 152 . The N250A mutant receptor lost the ability to bind both radiolabeled agonist and antagonist. The H95A mutation significantly reduced affinity of both agonists and antagonists. In contrast, the K152A (EL2), W243A (6.48), and W243F (6.48) mutations did not significantly affect the agonist binding but decreased antagonist affinity by ϳ3-38-fold, suggesting that these residues were critical for the high affinity of A 3 adenosine receptor antagonists. Activation of phospholipase C by wild type (WT) and mutant receptors was measured. The A 3 agonist 2-chloro-N 6 -(3-iodobenzyl)-5-N-methylcarbamoyladenosine stimulated phosphoinositide turnover in the WT but failed to evoke a response in cells expressing W243A and W243F mutant receptors, in which agonist binding was less sensitive to guanosine 5-␥-thiotriphosphate than in WT. Thus, although not important for agonist binding, Trp 243 was critical for receptor activation. The results were interpreted using a rhodopsin-based model of ligand-A 3 receptor interactions.