Table 2.
Pharmacological data for FR900359 inhibition of the Gs-11 (V57I, F68Y, E73D, T76G, K77F, D80L, L189P, S191T, F194I, T196Y, and K197P) and derived restored mutants
The G proteins 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 shown in Fig. 3 (A and B)).
| Name/Mutation(s) | FR900359 pIC50 ± S.E. | n | FR900359 − IC50 (nm) (95% confidence interval) |
|---|---|---|---|
| Gs-11/V57I, F68Y, E73D, T76G, K77F, D80L, L189P, S191T, F194I, T196Y, K197P | 6.64 ± 0.07 | 5 | 231 (146–363) |
| Val-57–restored/F68Y, E73D, T76G, K77F, D80L, L189P, S191T, F194I, T196Y, K197P | 6.55 ± 0.08 | 3 | 282 (132–604) |
| Phe-68–restored/V57I, E73D, T76G, K77F, D80L, L189P, S191T, F194I, T196Y, K197P | 6.26 ± 0.14 | 3 | 546 (135–2200) |
| Glu-73–restored/V57I, F68Y, T76G, K77F, D80L, L189P, S191T, F194I, T196Y, K197P | 6.85 ± 0.15 | 3 | 143 (33.9–601) |
| Thr-76–restored/V57I, F68Y, E73D, K77F, D80L, L189P, S191T, F194I, T196Y, K197P | 5.94 ± 0.17 | 3 | 1,140 (213–6,080) |
| Lys-77–restored/V57I, F68Y, E73D, T76G, D80L, L189P, S191T, F194I, T196Y, K197P | 7.87 ± 0.20 | 19 | 13.5 (9.29–36.3) |
| 4.84 ± 0.08 | 14,600 (9,840–21,500)a,b | ||
| Asp-80–restored/V57I, F68Y, E73D, T76G, K77F, L189P, S191T, F194I, T196Y, K197P | 5.55 ± 0.15 | 3 | 2,850 (656–12,400) |
| Leu-189–restored/V57I, F68Y, E73D, T76G, K77F, D80L, S191T, F194I, T196Y, K197P | 6.59 ± 0.10 | 3 | 255 (93.3–698) |
| Ser-191–restored/V57I, F68Y, E73D, T76G, K77F, D80L, L189P, F194I, T196Y, K197P | 6.52 ± 0.11 | 7 | 302 (160–570) |
| Phe-194–restored/V57I, F68Y, E73D, T76G, K77F, D80L, L189P, S191T, T196Y, K197P | 4.36 ± 0.07 | 3 | 43,400 (20,800–90,200)b |
| Thr-196–restored/V57I, F68Y, E73D, T76G, K77F, D80L, L189P, S191T, F194I, K197P | 5.05 ± 0.09 | 4 | 8,970 (4,670–17,300)b |
| Lys-197–restored/V57I, F68Y, E73D, T76G, K77F, D80L, L189P, S191T, F194I, T196Y | 5.75 ± 0.07 | 3 | 1,780 (935–3,400) |
a Biphasic inhibition observed.
b Bottom-plateau of concentration-inhibition curve constrained to buffer level to allow curve fitting of low-potency mutant.