Figure 8.

Experimental validation of the hit compounds acting on GCGR and VIPR

A. Validation of the nine putative modulators identified in Figure 4C using cAMP accumulation assay. CD3400-G008 displayed a stable and remarkable antagonist effect on GCGR. B. Dose-dependent effect of CD3400-G008 on GCGR with respect to the inhibition of cAMP accumulation. GCGR-expressing cells were incubated with 0.01 nM glucagon and treated with different concentrations of CD3400-G008 to generate the dose–response curve (IC₅₀ = 22.6 µM). C. Glucagon dose–response curves, in which its concentration was gradually increased from 0.01 pM to 10 nM, whereas that of CD3400-G008 was set to 20 µM; DMSO was used as negative control. D. Effect of CD3400-G008 on forskolin-induced cAMP accumulation. E. Dose–response curves of CD3349-F005 in CHO-K1 cells overexpressing VIPR and in the parental CHO-K1 cells. CD3349-F005 concentration was gradually decreased from 100 µM to 0.78 µM. F. PDE inhibitor exclusion assay was conducted using CHO-K1 cells to detect the agonist effects of CD3349-F005 on forskolin-induced cAMP accumulation; a potent PDE inhibitor IBMX was used as positive control. All measurements were performed with at least three independent experiments in quadruplicate. Data are shown as means ± SEM and fitted to a four-parameter logistic regression model. Max, maximum response; PDE, phosphodiesterase; IBMX, 3-isobutyl-1-methylxanthine.