Figure 7. A cell-permeable GD20 analog, cpGD20, is a dual-effect G protein modulator.

(A) Illustration of Gαs/Gβγ PPI inhibitors acting as dual-effect G protein modulators in cells.

(B) CAPA assay results of ct-GD20 and ct-GD20-F10L. Mean ± SD, n = 3.

(C) 25 μM cpGD20, but not 25 μM cpGD20-F5A, inhibited Gαs/Gβγ reassociation in HEK293 cells transfected with β2AR and Gαs/Gβ1γ2. Gαs/Gβγ dissociation was measured by BRET signal reduction after 10 nM ISO application. BRET signal was normalized to cells that were not treated with ISO. Mean ± SD, n = 3. Two-tailed unpaired t tests, **p < 0.01, ns p > 0.05.

(D) cpGD20 did not inhibit Gαi/Gβγ reassociation in HEK293 cells transfected with M2R and Gαi/Gβ1γ2. Gαi1/Gβγ dissociation was measured by BRET signal reduction after 100 nM ACh application. BRET signal was normalized to cells that were not treated with Ach. Mean ± SD, n = 3. Two-tailed unpaired t tests, ns p > 0.05.

(E) Representative voltage-clamp recordings of HEK293 cells transiently transfected with β2AR, GIRK4, Gβγ-Venus, and Gαs. Membrane potential was held at −80 mV. 1 μM ISO was applied as indicated. 25 μM of cpGD20, cpGD20-F5A, or DMSO were added to the pipette solution prior to recordings.

(F) The amounts of residual ISO-activated currents after 60 s of washout normalized to the maximum ISO-activated currents. Mean ± SD, n = 6. Two-tailed unpaired t tests with Welch’s correction, *p < 0.05, ***p < 0.001, ns p > 0.05.

(G) Maximum ISO-activated currents normalized to the capacitance of the cells. Mean ± SD, n = 6. Two-tailed unpaired t tests with Welch’s correction, *p < 0.05, ns p > 0.05.

See also Figure S7.