Table 2.
Activity of ligands at H4R and selected mutants
| Histamine | Clobenpropit | Clozapine | VUF6884 | JNJ7777120 | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| hH4R | pKi | Fold (pKi) | pEC50 | Fold (pEC50) | pKi | Fold (pKi) | pEC50 | Fold (pEC50) | pKi | Fold (pKi) | pEC50 | Fold (pEC50) | pKi | Fold (pKi) | pEC50 | Fold (pEC50) | pKi | Fold (pKi) |
| WT | 7.7 ± 0.1 (4) | 8.4 ± 0.2 (4) | 8.0 ± 0.1 (3) | 8.1 ± 0.2 (6) | 6.5 ± 0.0 (4) | 6.6 ± 0.2 (4) | 7.7 ± 0.1 (4) | 7.6 ± 0.1 (4) | 8.5 ± 0.1 (3) | |||||||||
| D943.32A | ND (3) | ND (4) | ND (3) | ND (3) | ND (3) | ND (3) | ND (3) | ND (4) | ND (3) | |||||||||
| D943.32N | ND (3) | ND (4) | ND (3) | ND (3) | ND (3) | ND (3) | ND (3) | ND (4) | ND (3) | |||||||||
| Y953.33A |
6.2 ± 0.1 (3) **** p < 0.0001 |
32 ↓ |
6.1 ± 0.1 (4) **** p < 0.0001 |
200 ↓ |
7.3 ± 0.2 (3) ** p = 0.0013 |
5 ↓ |
ND (3) |
5.7 ± 0.2 (4) **** p < 0.0001 |
6 ↓ |
ND (4) | 6.6 ± 0.2 (4) **** p < 0.0001 |
12 ↓ |
6.6 ± 0.2 (4) ** p = 0.0036 |
10 ↓ |
6.7 ± 0.2 (3) **** p < 0.0001 |
63 ↓ |
||
| E1825.46A |
5.0 ± 0.1 (4) **** p < 0.0001 |
501 ↓ |
5.4 ± 0.5 (4) **** p < 0.0001 |
1000↓ |
6.8 ± 0.0 (3) **** p < 0.0001 |
16 ↓ |
6.1 ± 0.3 (7) inverse agonism |
100 ↓ |
6.2 ± 0.1 (4) * p = 0.0385 |
2 ↓ |
6.4 ± 0.3 (3) ns p = 0.9475 |
2 ↓ |
7.0 ± 0.2 (4) ** p = 0.0086 |
5 ↓ |
7.6 ± 0.3 (4) ns p = 0.9999 |
1 ↓ |
6.0 ± 0.3 (3) **** p < 0.0001 |
316 ↓ |
| E1825.46Q |
5.2 ± 0.1 (4) **** p < 0.0001 |
316↓ |
5.4 ± 0.4 (3) **** p < 0.0001 |
1000↓ |
7.2 ± 0.0 (3) *** p = 0.0004 |
6 ↓ |
8.0 ± 0.3 (3) inverse agonism |
1 ↓ |
5.9 ± 0.0 (4) **** p < 0.0001 |
4 ↓ |
6.5 ± 0.2 (4) ns p = 0.9969 |
1 ↓ |
6.9 ± 0.0 (4) ** p = 0.0021 |
6 ↓ |
7.4 ± 0.1 (4) ns p = 0.9351 |
2 ↓ |
7.4 ± 0.1 (3) ** p = 0.0038 |
13 ↓ |
| Y3196.51A |
6.6 ± 0.0 (3) **** p < 0.0001 |
13 ↓ |
6.5 ± 0.2 (4) *** p = 0.0004 |
80 ↓ |
7.1 ± 0.0 (3) *** p = 0.0001 |
8 ↓ |
ND (4) |
6.7 ± 0.0 (4) ns p = 0.1629 |
2 ↑ |
6.4 ± 0.1 (3) ns p = 0.9125 |
2 ↓ |
7.6 ± 0.1 (4) ns p = 0.9845 |
1 ↓ |
7.2 ± 0.2 (4) ns p = 0.6146 |
3 ↓ |
7.0 ± 0.0 (3) *** p = 0.0002 |
32 ↓ |
|
| F3447.39A |
4.6 ± 0.0 (3) **** p < 0.0001 |
1259↓ |
5.3 ± 0.2 (4) **** p < 0.0001 |
1259↓ |
5.9 ± 0.1 (3) **** p < 0.0001 |
126 ↓ |
6.0 ± 0.3 (4) *** p = 0.0005 |
126 ↓ |
5.8 ± 0.0 (4) **** p < 0.0001 |
5 ↓ |
6.1 ± 0.1 (4) ns p = 0.2316 |
3 ↓ |
6.7 ± 0.1 (4) *** p = 0.0003 |
10 ↓ |
6.7 ± 0.1 (4) * p = 0.0121 |
8 ↓ |
6.7 ± 0.2 (3) **** p < 0.0001 |
63 ↓ |
| Q3477.42A |
6.3 ± 0.2 (3) **** p < 0.0001 |
25 ↓ |
6.9 ± 0.1 (4) ** p = 0.0048 |
32 ↓ |
7.8 ± 0.2 (3) ns p = 0.4132 |
2 ↓ |
9.2 ± 0.2 (4) ns p = 0.0872 |
13 ↑ |
7.7 ± 0.1 (4) **** p < 0.0001 |
16 ↑ |
8.4 ± 0.1 (4) **** p < 0.0001 |
63 ↑ |
7.3 ± 0.2 (4) ns p = 0.2886 |
3 ↓ |
8.1 ± 0.2 (4) ns p = 0.1077 |
3 ↑ |
6.6 ± 0.2 (3) **** p < 0.0001 |
80 ↓ |
| W3487.43A | ND (3) | ND (4) | ND (3) | ND (4) | ND (3) | ND (3) | ND (3) | ND (4) | ND (3) | |||||||||
Data were shown as mean ± sem of at least three independent experiments which were performed in duplicate. Binding affinity (pKi) was determined with [3H] JNJ7777120 displacement assay. Potency (pEC50) was determined with a BRET-based Gi activation biosensor. Fold decrease (↓) or increase (↑) in binding affinity or potency compared to WT H4R is indicated. Statistical difference (p < 0.05) in pKi or pEC50 for the mutants in comparison to WT H4R was analyzed for each ligand using One-way AVONA followed by Dunnett’s multiple comparison test. Statistical differences are indicated with asterisk and corresponding p-values are shown in roman. ns not significant. ND not detectable.