Use of the triazolotriazine [3H]ZM 241385 as a radioligand at recombinant human A2B adenosine receptors

Abstract

Radiolabeled ZM 241385 (4-(2-[7-amino-2- ¿furyl¿¿1,2,4¿triazolo¿2,3-a¿¿1,3,5¿triazin-5-ylaminoethyl)p henol), has previously been used as a high affinity radioligand for the labeling of A2A adenosine receptors in cell membranes. Another subtype, the A2B receptor, is the least well-defined subtype of adenosine receptors and lacks selective pharmacological probes. In the present study, we have used [3H]ZM 241385 as a radioligand to label recombinant human A2B adenosine receptors in HEK-293 cell membranes, that do not express A2A adenosine receptors, and found that the pharmacological profile is consistent with the SAR of A2B receptors. Saturable, specific binding (Kd 33.6 nM, Bmax 4.48 pmol/mg protein) that was best described by a one-site model was found, and specific binding was approximately 75% of total binding. [3H]ZM 241385 binding was displaceable by a large number of compounds known to interact with A2B receptors; thus, this method has promise as a tool in the search for agonists and antagonists selective for this subtype. Xanthine analogs, which are antagonists, proved to be the most potent displacers. The Ki of XAC, xanthine amine congener, was 12.3 nM, while CPX (8-cyclopentyl-1,3-dipropylxanthine) was less potent. The non-selective triazoloquinazoline antagonist CGS 15943 (Ki 16.4 nM), which is similar in structure to ZM 241385, was slightly less potent than XAC. The non-xanthine A2B-antagonist alloxazine displaced [3H]ZM 241385-binding with a Ki of 462 nM, similar to its affinity in functional assays. Adenosine derivatives known to activate this receptor subtype, such as NECA (5'-N-ethylcarboxamidoadenosine) and R-PIA (N6-phenylisopropyladenosine), were considerably less potent than the 8-substituted xanthines examined.

FIGURE 1

Structures and potencies at A_2B adenosine receptors of agonists and antagonists. Agonist EC₅₀ and antagonist KB values, when given are expressed in μM and are from functional assays at A_2B receptors in fibroblasts (either stimulation of adenylyl cyclase or antagonism of agonist-stimulated adenyly1 cyclase) [8,9,12] or in mammalian tells expressing the recombinant human A_2B adenosine receptor [5,12,13].

FIGURE 2

Dependence of the percent specific binding of [³H]ZM 241385 to the human A_2B receptor on the amount of protein present in each assay tube. [³H]ZM 241385 (30 nM) was incubated with membranes for 60 min at 25 °C. Non-specific binding was delcnnined with 20 μM CPX.

FIGURE 3

Time plot of association of [³H]ZM 241385 to HEK-293 cell membranes expressing the human A_2B receptor. Total binding (○). non-specific binding (▲), and specific binding (●) are shown. [³H]ZM 241385 (30 nM) was incubated wilh 18 μg of membranes at 25 °C. Non-specific binding was determined with 20 μM CPX.

FIGURE 4

(a) Saturation isotherm of specific [³H]ZM 241385 binding 10 human A_2B receptors expressed in HEK-293 cell membranes and (b) Scatchard analysis of the same data. [³H]ZM 241385 (2–225 nM) was incubated with 30 μg of membranes for 60 min. at 25 °C. Non-specific binding was determined with 20 μM CPX. Four experiments were carried out; data from one representative experiment (each data point measured in duplicate) are shown.

FIGURE 5

Effects of the antagonists XAC (●), CPX (○), and enprofylline (▲) and the agonist R-PIA (solid ▲) on radioligand binding to human A_2B receptors expressed in HEK-293 cell membranes. Membranes (30 μg) were incubated for 60 min at 25 °C with 30 nM [³H]ZM 241385. Non-specific binding was determined with 20 μM CPX, Foor experiments were carried out; data from one representative experiment (each data point measured in duplicate) are shown.