Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda MD 20892, USA.
Molecular Signaling Section, Laboratory of Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda MD 20892, USA.
Sci Rep. 2017 Mar 20;7:44816. doi: 10.1038/srep44816.
Agonists that target the A, A, A and A adenosine receptors have potential to be potent treatment options for a number of diseases, including autoimmune diseases, cardiovascular disease and cancer. Because each of these adenosine receptors plays a distinct role throughout the body, obtaining highly specific receptor agonists is essential. Of these receptors, the adenosine AR and AR share many sequence and structural similarities but highly differ in their responses to inflammatory stimuli. Our laboratory, using a combination of specially developed cell lines and calcium release analysis hardware, has created a new and faster method for determining specificity of synthetic adenosine agonist compounds for the A and A receptors in human cells. A receptor expression was effectively removed from K562 cells, resulting in the development of a distinct null line. Using HIV-lentivector and plasmid DNA transfection, we also developed A and A receptor over-expressing lines. As adenosine is known to cause changes in intracellular calcium levels upon addition to cell culture, calcium release can be determined in these cell lines upon compound addition, providing a functional readout of receptor activation and allowing us to isolate the most specific adenosine agonist compounds.
针对 A₁、A₂A、A₂B 和 A₃ 腺苷受体的激动剂有可能成为许多疾病的有效治疗选择，包括自身免疫性疾病、心血管疾病和癌症。由于这些腺苷受体在体内的作用各不相同，因此获得高度特异性的受体激动剂至关重要。在这些受体中，腺苷 A₁R 和 A₂A R 具有许多序列和结构相似性，但对炎症刺激的反应却大不相同。我们的实验室使用专门开发的细胞系和钙释放分析硬件的组合，为确定合成腺苷激动剂化合物对人细胞中 A₁ 和 A₂A 受体的特异性创建了一种新的、更快的方法。通过 K562 细胞的有效敲除，我们得到了一个独特的缺失株。通过 HIV 慢病毒载体和质粒 DNA 转染，我们还开发了 A₁ 和 A₂A 受体过表达系。由于腺苷在加入细胞培养物后会引起细胞内钙离子水平的变化，因此可以在这些细胞系中通过化合物的加入来确定钙释放，从而提供受体激活的功能读数，并使我们能够分离出最特异的腺苷激动剂化合物。
