Structural determinants of A3 adenosine receptor activation:: Nucleoside ligands at the agonist/antagonist boundary
Structural determinants of A3 adenosine receptor activation:: Nucleoside ligands at the agonist/antagonist boundary
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DOI：
10.1021/jm020211
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发表时间：
2002-09-26
影响因子：
7.3
通讯作者：
Jacobson, KA
中科院分区：
文献类型：
--
作者：
Gao, ZG;Kim, SK;Jacobson, KA
人A（3）腺苷受体（AR）的突变表明，某些氨基酸残基对配体结合和激活过程的贡献不同。在这里，我们证明了各种腺苷修饰，包括腺嘌呤取代和核糖环限制，也有助于这些过程的差异。在表达A（3）AR的完整CHO细胞中，比较了配体对环AMP产生的影响以及对受体结合的影响。值得注意的是，单独的简单2-氟基团或2-氯与N-6-取代的组合显著降低了腺苷衍生物的功效，甚至将激动剂转化为拮抗剂。其他增加亲和力的取代，包括N-6-（3-碘苄基）4和（北方）-甲烷卡巴15，也降低了效力，除了与柔性5 '-氟酰胺组合。2-Cl-N-6-（3-碘苄基）衍生物，均为（N）-甲烷卡巴（即，的北方构象）和核苷系列18和5分别是具有很少残余激动作用的有效拮抗剂。合成了核苷和（AT）-甲烷卡巴系列13和21中的环约束2 '，3'-环氧化物衍生物，以及环化（螺旋）4 '，5'-糖醛酰胺衍生物14，发现它们是人A（3）AR拮抗剂。14与人（26 nM）和大鼠（49 nM）A（3）AR均有效结合。基于视紫红质的A（3）AR模型，包含除C-末端区域外的所有结构域，表明这些腺苷类似物对受体结合和激活的不同结构要求。考虑到突变体A（3）AR处所选衍生物的结合，配体对接以TM3（His95）与腺嘌呤部分以及TM6和7与核糖5 '区的相互作用为特征。拮抗剂N-6-（3-碘苄基）-2-氯腺苷5的5 ′-OH基团与N274形成氢键，但不与S271形成氢键。核苷拮抗剂的5 ′-取代基向TM7移动，远离TM6。保守的Trp243（6.48）侧链，参与识别经典的（非核苷）A（3）AR拮抗剂，但不参与腺苷衍生配体，在激动剂对接时显示出特有的运动。因此，A（3）AR的激活似乎需要在5 ′-和3 ′-位的柔性，这在（AT）-甲烷卡巴、螺环和环氧化物类似物中减少，并且是TM6和TM7处核糖相互作用的特征。
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'-fluronamide. 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 (AT)-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 (AT)-methanocarba, spiro, and epoxide analogues, and was characteristic of ribose interactions at TM6 and TM7.