Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0810, USA.
Biochem Pharmacol. 2013 Apr 15;85(8):1171-81. doi: 10.1016/j.bcp.2013.01.021. Epub 2013 Jan 31.
Various fluorescent nucleoside agonists of the A3 adenosine receptor (AR) were compared as high affinity probes using radioligands and flow cytometry (FCM). They contained a fluorophore linked through the C2 or N(6) position and rigid A3AR-enhancing (N)-methanocarba modification. A hydrophobic C2-(1-pyrenyl) derivative MRS5704 bound nonselectively. C2-Tethered cyanine5-dye labeled MRS5218 bound selectively to hA3AR expressed in whole CHO cells and membranes. By FCM, binding was A3AR-mediated (blocked by A3AR antagonist, at least half through internalization), with t1/2 for association 38min in mA3AR-HEK293 cells; 26.4min in sucrose-treated hA3AR-CHO cells (Kd 31nM). Membrane binding indicated moderate mA3AR affinity, but not selectivity. Specific accumulation of fluorescence (50nM MRS5218) occurred in cells expressing mA3AR, but not other mouse ARs. Evidence was provided suggesting that MRS5218 detects endogenous expression of the A3AR in the human promyelocytic leukemic HL-60 cell line. Therefore, MRS5218 promises to be a useful tool for characterizing the A3AR.
研究人员利用放射性配体和流式细胞术（FCM）比较了几种作为高亲和力探针的 A3 腺苷受体（AR）荧光核苷激动剂。这些探针的荧光团通过 C2 或 N(6)位置连接，并具有刚性的 A3AR 增强（N）-甲叉碳修饰。疏水性 C2-（1-蒽基）衍生物 MRS5704 非选择性结合。C2 连接的菁染料标记的 MRS5218 选择性地结合在整个 CHO 细胞和膜中表达的 hA3AR。通过 FCM，结合是 A3AR 介导的（被 A3AR 拮抗剂阻断，至少一半通过内化），在 mA3AR-HEK293 细胞中缔合的 t1/2 为 38min；在蔗糖处理的 hA3AR-CHO 细胞中为 26.4min（Kd 为 31nM）。膜结合表明 mA3AR 具有中等亲和力，但没有选择性。在表达 mA3AR 的细胞中，荧光的特异性积累（50nM MRS5218）发生，但在其他小鼠 AR 中没有发生。有证据表明，MRS5218 可用于检测人早幼粒细胞白血病 HL-60 细胞系中 A3AR 的内源性表达。因此，MRS5218 有望成为表征 A3AR 的有用工具。
