Table 3.
Pharmacological data for the inhibition by FR900359 on Gs mutants designed with select high-impact mutations, based on the results of the restored mutants (see Table 2)
The Gs mutants were expressed in ΔGs-HEK293 cells to enable assessment of their potential inhibition by FR900359. The cells were stimulated with isoproterenol (5 nm) to activate the endogenous Gs-coupled β2-adrenergic receptor after treatment with FR900359 (associated concentration-inhibition curves in Fig. S6 (A–C)). The extent of the high-potency inhibition component for partial and biphasic inhibition curves is given in parentheses as a percentage of full inhibition. The Lys-77–restored mutant has been included for comparison.
| Name/Mutation(s) | FR900359 pIC50 ± S.E. | n | FR900359 IC50 (nm) (95% confidence interval) |
|---|---|---|---|
| Gs WT | 3 | >100,000 | |
| T76G, F194I | 3 | >100,000 | |
| T76G, D80L, F194I | 3 | >100,000 | |
| T76G, D80L, F194I, T196Y | 3 | >100,000 | |
| T76G, D80L, F194I, K197P | 3 | >100,000 | |
| T76G, K77F, D80L, F194I | 3 | >100,000 | |
| K77F, D80L, C186V | 7.31 ± 0.14 | 6 | 49.0 (21.2–111) (42%) |
| K77F, D80L, C186V, F194I | 7.01 ± 0.16 | 5 | 97.5 (34.0–279) (47%) |
| K77F, D80L, F194I, K197P | 7.41 ± 0.06 | 3 | 38.9 (22.2–68.2) (49%) |
| K77F, F194I, T196Y | 7.12 ± 0.08 | 8 | 76.0 (50.0–116) (56%) |
| K77F, D80L, F194I, T196Y | 7.17 ± 0.11 | 8 | 67.0 (37.6–119) (48%) |
| 4.77 ± 0.08 | 16,900 (11,100–25,600)a,b | ||
| K77F, D80L, F194I, T196Y, K197P | 7.18 ± 0.09 | 8 | 65.8 (40.6–106) (56%) |
| 5.04 ± 0.01 | 9,162 (8,490–9,890)a,b | ||
| K77F, D80L, C186V, F194I, T196Y | 7.07 ± 0.09 | 7 | 84.5 (50.8–141) (50%) |
| 4.86 ± 0.10 | 11,041 (5,350–19,200)a,b | ||
| Lys-77–restored/V57I, F68Y, E73D, T76G, D80L, L189P, S191T, F194I, T196Y, K197P | 7.87 ± 0.20 | 1 | 13.5 (9.29–36.3) (52%) |
| 4.84 ± 0.08 | 9 | 14,600 (9,840–21,500)a,b |
a Biphasic inhibition observed.
b Bottom-plateau of concentration-inhibition curves constrained to buffer level to allow curve fitting of low potent mutant.