Tritium-labeled agonists as tools for studying adenosine A2B receptors

Purinergic Signalling

Wessam M. Alnouri¹  Sonja Hinz² 

[1] Bayer Pharma AG;Pharmaceutical Chemistry I

关键词: Adenosine A2B receptor;    Agonist radioligand;    [3H]BAY 60-6583;    [3H]NECA;    Kinetics;    Radioligand binding;   

DOI  :  10.1007/s11302-018-9608-5

学科分类：分子生物学，细胞生物学和基因

来源: Springer

【 摘 要 】

A selective agonist radioligand for A2B adenosine receptors (A2BARs) is currently not available. Such a tool would be useful for labeling the active conformation of the receptors. Therefore, we prepared BAY 60-6583, a potent and functionally selective A2BAR (partial) agonist, in a tritium-labeled form. Despite extensive efforts, however, we have not been able to establish a radioligand binding assay using [3H]BAY 60-6583. This is probably due to its high non-specific binding and its moderate affinity, which had previously been overestimated based on functional data. As an alternative, we evaluated the non-selective A2BAR agonist [3H]NECA for its potential to label A2BARs. [3H]NECA showed specific, saturable, and reversible binding to membrane preparations of Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells stably expressing human, rat, or mouse A2BARs. In competition binding experiments, the AR agonists 2-chloroadenosine (CADO) and NECA displayed significantly higher affinity when tested versus [3H]NECA than versus the A2B-antagonist radioligand [3H]PSB-603 while structurally diverse AR antagonists showed the opposite effects. Although BAY 60-6583 is an A2BAR agonist, it displayed higher affinity versus [3H]PSB-603 than versus [3H]NECA. These results indicate that nucleoside and non-nucleoside agonists are binding to very different conformations of the A2BAR. In conclusion, [3H]NECA is currently the only useful radioligand for determining the affinity of ligands for an active A2BAR conformation.

【 授权许可】

CC BY