Tritium-labeled agonists as tools for studying adenosine A2B receptors

Abstract

A selective agonist radioligand for A adenosine receptors (AARs) 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 AAR (partial) agonist, in a tritium-labeled form. Despite extensive efforts, however, we have not been able to establish a radioligand binding assay using [H]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 AAR agonist [H]NECA for its potential to label AARs. [H]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 AARs. In competition binding experiments, the AR agonists 2-chloroadenosine (CADO) and NECA displayed significantly higher affinity when tested versus [H]NECA than versus the A-antagonist radioligand [H]PSB-603 while structurally diverse AR antagonists showed the opposite effects. Although BAY 60-6583 is an AAR agonist, it displayed higher affinity versus [H]PSB-603 than versus [H]NECA. These results indicate that nucleoside and non-nucleoside agonists are binding to very different conformations of the AAR. In conclusion, [H]NECA is currently the only useful radioligand for determining the affinity of ligands for an active AAR conformation.

NanoBRET ligand binding at a GPCR under endogenous promotion facilitated by CRISPR/Cas9 genome editing

“…However, in recent studies the affinity of PSB 603 was reported to be significantly lower (Ki 1.89 nM) when 30 nM [ 3 H] NECA was used as the tracer 48 . Furthermore, the apparent dissociation equilibrium constant (KB) of PSB 603 against a range of agonists was reported to be 3.6 -66 nM 47 .…”

“…pKi values from literature are from the IUPHAR/BPS Guide To PHARMACOLOGY 41 and are against various probes. Values for PSB 603 38,48 and SCH 442416 39…”

Chemical Probes for the Adenosine Receptors

“…This is probably due to its moderate affinity at the human A 2B receptor and high level of non-specific binding. The only results obtained using this radioligand indicate that nucleoside and non-nucleoside agonists most probably bind the receptor in different conformations [25].…”

“…Considering labeled derivatives for binding studies only an agonist for A2B AR named [ 3 H]-BAY60-6583 (1) was recently reported by the group of Prof. C.A. Müller (Figure 1) [25]. This partial agonist in its tritiated form (the position of tritium is not reported) failed to be a good probe for binding studies.…”

“…Considering labeled derivatives for binding studies only an agonist for A 2B AR named [ 3 H]-BAY60-6583 (1) was recently reported by the group of Prof. C.A. Müller (Figure 1) [25]. biology and drug discovery [17].…”