title: Structural determinants of A(3) adenosine receptor activation: nucleoside ligands at the agonist/antagonist boundary.
abstract: 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.
authors.affiliation: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Zhan-Guo
authors.initials: ZG
authors.lastname: Gao
authors.forename: Soo-Kyung
authors.initials: SK
authors.lastname: Kim
authors.forename: Thibaud
authors.initials: T
authors.lastname: Biadatti
authors.forename: Wangzhong
authors.initials: W
authors.lastname: Chen
authors.forename: Kyeong
authors.initials: K
authors.lastname: Lee
authors.forename: Dov
authors.initials: D
authors.lastname: Barak
authors.forename: Seong Gon
authors.initials: SG
authors.lastname: Kim
authors.forename: Carl R
authors.initials: CR
authors.lastname: Johnson
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Jacobson
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D004852:Epoxy Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D011869:Radioligand Assay; D051381:Rats; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1 / Q000235:genetics; D012266:Ribose / Q000737:chemistry*; D013141:Spiro Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
chemical_list: D004852:Epoxy Compounds; D008024:Ligands; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1; D013141:Spiro Compounds; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012266:Ribose; D000241:Adenosine