Among β-blockers that are clinically prescribed for heart failure, carvedilol is a first-choice agent with unique pharmacological properties. Carvedilol is distinct from other β-blockers in its ability to elicit β-arrestin-biased agonism, which has been suggested to underlie its cardioprotective effects. Augmenting the pharmacologic properties of carvedilol thus holds the promise of developing more efficacious and/or biased β-blockers. We recently identified compound-6 (cmpd-6), the first small molecule positive allosteric modulator of the β2-adrenergic receptor (β2AR). Cmpd-6 is positively cooperative with orthosteric agonists at the β2AR and enhances agonist-mediated transducer (G-protein and β-arrestin) signaling in an unbiased manner. Here, we report that cmpd-6, quite unexpectedly, displays strong positive cooperativity only with carvedilol among a panel of structurally diverse β-blockers. Cmpd-6 enhances the binding affinity of carvedilol for the β2AR and augments its ability to competitively antagonize agonist-induced cAMP generation. Cmpd-6 potentiates β-arrestin1- but not Gs-protein-mediated high-affinity binding of carvedilol at the β2AR and β-arrestin-mediated cellular functions in response to carvedilol including extracellular signal-regulated kinase phosphorylation, receptor endocytosis, and trafficking into lysosomes. Importantly, an analog of cmpd-6 that selectively retains positive cooperativity with carvedilol acts as a negative modulator of agonist-stimulated β2AR signaling. These unprecedented cooperative properties of carvedilol and cmpd-6 have implications for fundamental understanding of G-protein-coupled receptor (GPCR) allosteric modulation, as well as for the development of more effective biased beta blockers and other GPCR therapeutics. SIGNIFICANCE STATEMENT: This study reports on the small molecule-mediated allosteric modulation of the β-arrestin-biased β-blocker, carvedilol. The small molecule, compound-6 (cmpd-6), displays an exclusive positive cooperativity with carvedilol among other β-blockers and enhances the binding affinity of carvedilol for the β2-adrenergic receptor. Cooperative effects of cmpd-6 augment the β-blockade property of carvedilol while potentiating its β-arrestin-mediated signaling functions. These findings have potential implications in advancing G-protein-coupled receptor allostery, developing biased therapeutics and remedying cardiovascular ailments.
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