The β-Blocker Carvedilol and Related Aryloxypropanolamines Promote ERK1/2 Phosphorylation in HEK293 Cells with Values Distinct From Their Equilibrium Dissociation Constants as -Adrenoceptor Antagonists: Evidence for Functional Affinity.

The determination of affinity by using functional assays is important in drug discovery because it provides a more relevant estimate of the strength of interaction of a ligand to its cognate receptor than radioligand binding. However, empirical evidence for so-called, "functional affinity" is limited. Herein, we determined whether the affinity of carvedilol, a -adrenoceptor antagonist used to treat heart failure that also promotes extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation, differed between these two pharmacological activities. Four structurally related -adrenoceptor antagonists (alprenolol, carazolol, pindolol, propranolol) that also activated ERK1/2 were included as comparators to enhance our understanding of how these drugs work in the clinical setting. In HEK293 cells stably expressing the human -adrenoceptor carvedilol and related aryloxypropanolamines were of ERK1/2 phosphorylation with potencies ([A]s) that were than their equilibrium dissociation constants ( s) as -adrenoceptor antagonists. As the [A] of a partial agonist is a good approximation of its , then these data indicated that the affinities of carvedilol and related ligands for these two activities were distinct. Moreover, there was a significant negative rank order correlation between the [A] of each ligand to activate ERK1/2 and their intrinsic activities (i.e., as intrinsic activity for ERK1/2 phosphorylation increased, so did affinity). Genome editing revealed that the transducer that coupled the -adrenoceptor to ERK1/2 phosphorylation in response to carvedilol and other -adrenoceptor antagonists was Gs. Collectively, these data support the concept of "functional affinity" and indicate that the ability of the -adrenoceptor to recruit Gs may influence the affinity of the activating ligand. SIGNIFICANCE STATEMENT: In HEK293 cells overexpressing the human β-adrenoceptor carvedilol and four related aryloxypropanolamines behaved as β-adrenoceptor antagonists partial agonists of ERK1/2 phosphorylation with rank orders of affinity that were distinct. These data imply that carvedilol and other β-blockers can stabilize the β-adrenoceptor in different affinity conformations that are revealed when functionally distinct responses are measured. This is the basis for the pharmacological concept of "functional affinity."