Table 6. NanoBRET Competition-Binding Assay at Human Wild-Type and Mutant Nluc-A1Ra.
| A1R | pKia (cmpd) |
|||||
|---|---|---|---|---|---|---|
| BnOCPA | n | 20 | n | 27 | n | |
| WT | 6.24 ± 0.04 | 3 | 6.38 ± 0.17 | 3 | 7.20 ± 0.16 | 3 |
| I692.64A | 5.03 ± 0.02* | 3 | 5.06 ± 0.04* | 3 | 6.37 ± 0.10* | 3 |
| N702.65A | 6.01 ± 0.05 | 3 | 5.48 ± 0.15* | 3 | 7.00 ± 0.24 | 3 |
| T2576.58A | 6.93 ± 0.10* | 3 | 6.23 ± 0.02 | 3 | 8.09 ± 0.18* | 3 |
| Y2717.36A | 5.40 ± 0.23* | 3 | 5.19 ± 0.13* | 3 | 6.33 ± 0.10* | 3 |
a
Compound affinity (pKi) determined through NanoBRET competition-binding assays with CA200645 in wild-type (WT) or mutant Nluc-A1R stably expressing HEK293 cells. The resulting concentration-dependent decrease in BRET ratio at 10 min was used to calculate pKi. Data are expressed as mean ± SEM obtained in n separate experiments. All individual experiments were conducted in duplicate. Statistical significance (*p < 0.05) compared to WT was determined by one-way ANOVA with Dunnett’s post-test and presented according to ref (31).