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Comparative Study
. 2015 Sep;11(3):389-407.
doi: 10.1007/s11302-015-9460-9. Epub 2015 Jul 1.

Selectivity is species-dependent: Characterization of standard agonists and antagonists at human, rat, and mouse adenosine receptors

Mohamad Wessam Alnouri  1 , Stephan Jepards, Alessandro Casari, Anke C Schiedel, Sonja Hinz, Christa E Müller
Affiliations
Comparative Study

Selectivity is species-dependent: Characterization of standard agonists and antagonists at human, rat, and mouse adenosine receptors

Mohamad Wessam Alnouri et al. Purinergic Signal. 2015 Sep.

Abstract

Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species. However, preclinical studies are mostly performed in mouse or rat, and standard AR agonists and antagonists are frequently used for studies in rodents without knowing their selectivity in the investigated species. In the present study, we selected a set of frequently used standard AR ligands, 8 agonists and 16 antagonists, and investigated them in radioligand binding studies at all four AR subtypes, A1, A2A, A2B, and A3, of three species, human, rat, and mouse. Recommended, selective agonists include CCPA (for A1AR of rat and mouse), CGS-21680 (for A2A AR of rat), and Cl-IB-MECA (for A3AR of all three species). The functionally selective partial A2B agonist BAY60-6583 was found to additionally bind to A1 and A3AR and act as an antagonist at both receptor subtypes. The antagonists PSB-36 (A1), preladenant (A2A), and PSB-603 (A2B) displayed high selectivity in all three investigated species. MRS-1523 acts as a selective A3AR antagonist in human and rat, but is only moderately selective in mouse. The comprehensive data presented herein provide a solid basis for selecting suitable AR ligands for biological studies.

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Figures

Fig. 1
Fig. 1
Saturation binding assays at ARs stably expressed in recombinant CHO cells: a mA1AR using [3H]CCPA; b mA1AR using [3H]DPCPX; c mA2AAR using [3H]CGS-21680; d mA2AAR using [3H]MSX-2. Data are means of three independent saturation assays each performed in duplicates. Curves represent specific binding and gray lines represent non-specific binding. Curves and lines were obtained by plotting the counts per minute against increasing concentrations of the radioligand
Fig. 2
Fig. 2
Saturation binding assays at ARs stably expressed in recombinant CHO cells: a rA2BAR using [3H]PSB-603; b mA2BAR using [3H]PSB-603; c rA3AR using [3H]NECA; d mA3AR using [3H]NECA. Data are means of three independent saturation assays each performed in duplicates. Curves represent the specific binding and were obtained by plotting the counts per minute against increasing concentrations of the radioligand
Fig. 3
Fig. 3
Structures and selectivity of the investigated adenosine receptor agonists
Fig. 4
Fig. 4
Structures and selectivity of the investigated adenosine receptor antagonists
Fig. 5
Fig. 5
Concentration-dependent antagonistic effect of BAY60-6583 at a hA1AR (IC50 = 7.40 ± 2.19 μM) and b hA3AR (IC50 = 6.70 ± 0.83 μM). The hA1AR was activated by CCPA (100 nM), the hA3AR was activated by Cl-IB-MECA (30 nM). The employed agonist concentrations corresponded to their EC80 values. Receptor-induced ß-arrestin translocation was measured by using a ß-galactosidase complementation assay. Values represent means of three independent experiments. K i value BAY60-6583 at hA1 is 3190 ± 780 nM, K i value BAY60-6583 at hA3 is 5630 ± 700 nM
Fig. 6
Fig. 6
Antagonistic effects of BAY60-6583 at the Gi-coupled hA1 and hA3 ARs determined in cAMP accumulation assays; a at the hA1AR a rightward shift of the curve for the agonist NECA was observed; the EC50 was shifted from 56.5 ± 6.7 nM in the absence of BAY60-6583 to 6060 ± 780 nM in the presence of 10 μM BAY60-6583; b at the hA3AR, a rightward shift of the curve for NECA was also observed from an EC50 of 56.4 ± 7.3 nM in the absence of BAY60-6583 to an EC50 of 348 ± 25 nM in the presence of 10 μM BAY 60–6583. Cells were stimulated with forskolin at a final concentration of 10 μM. Calculated K b values were 0.347 ± 0.032 μM (hA1) and 0.652 ± 0.058 μM (hA3)
Fig. 7
Fig. 7
pKi values of the A1 agonist CCPA and the A1 antagonist PSB-36 at human, rat, and mouse AR subtypes; number sign indicates the highest tested concentration was 10 μM and K i value is >10 μM
Fig. 8
Fig. 8
pK i values of the A2A agonist CGS-21680 and the A2A antagonist preladenant; (number sign) highest tested concentration was 10 μM and K i value is >10 μM, (section sign) highest tested concentration was 1 μM and K i value is >1 μM
Fig. 9
Fig. 9
pK ivalues of the A2B partial agonist BAY60-6583 and the A2B antagonist PSB-603 at the four adenosine receptor subtypes; (number sign) highest tested concentration was 10 μM and K i value is >10 μM
Fig. 10
Fig. 10
pK i values of the A3 agonist Cl-IB-MECA and the A3 antagonist MRS-1523 at the four adenosine receptor subtypes; (number sign) highest tested concentration was 10 μM and K i value is >10 μM
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