-Arrestin-Biased Allosteric Modulator Potentiates Carvedilol-Stimulated Adrenergic Receptor Cardioprotection

beta(1) adrenergic receptors (beta(1)ARs) are central regulators of cardiac function and a drug target for cardiac disease. As a member of the G protein-coupled receptor family, beta(1)ARs activate cellular signaling by primarily coupling to Gs proteins to activate adenylyl cyclase, cAMP-dependent pathways, and the multifunctional adaptor-transducer protein beta-arrestin. Carvedilol, a traditional beta-blocker widely used in treating high blood pressure and heart failure by blocking beta adrenergic receptor-mediated G protein activation, can selectively stimulate Gs-independent beta-arrestin signaling of beta adrenergic receptors, a process known as beta-arrestin-biased agonism. Recently, a DNA-encoded small-molecule library screen against agonist-occupied beta(2) adrenergic receptors (beta(2)ARs) identified Compound-6 (Cmpd-6) to be a positive allosteric modulator for agonists on beta(2)ARs. Intriguingly, it was further discovered that Cmpd-6 is positively cooperative with the beta-arrestin-biased ligand carvedilol at beta(2)ARs. Here we describe the surprising finding that at beta(1)ARs unlike beta(2)ARs, Cmpd-6 is cooperative only with carvedilol and not agonists. Cmpd-6 increases the binding affinity of carvedilol for beta(1)ARs and potentiates carvedilolstimulated, beta-arrestin-dependent beta(1)AR signaling, such as epidermal growth factor receptor transactivation and extracellular signal-regulated kinase activation, whereas it does not have an effect on Gs-mediated cAMP generation. In vivo, Cmpd-6 enhances the antiapoptotic, cardioprotective effect of carvedilol in response to myocardial ischemia/reperfusion injury. This antiapoptotic role of carvedilol is dependent on beta-arrestins since it is lost in mice with myocyte-specific deletion of beta-arrestins. Our findings demonstrate that Cmpd-6 is a selective beta-arrestin-biased allosteric modulator of beta(1)ARs and highlight its potential clinical utility in enhancing carvedilol-mediated cardioprotection against ischemic injury. SIGNIFICANCE STATEMENT This study demonstrates the positive cooperativity of Cmpd-6 on beta(1)ARs as a beta-arrestin-biased positive allosteric modulator. Cmpd-6 selectively enhances the affinity and cellular signaling of carvedilol, a known beta-arrestin-biased beta-blocker for beta(1)ARs, whereas it has minimal effect on other ligands tested. Importantly, Cmpd-6 enhances the beta-arrestin-dependent in vivo cardioprotective effect of carvedilol during ischemia/reperfusion injury-induced apoptosis. The data support the potential therapeutic application of Cmpd-6 to enhance the clinical benefits of carvedilol in the treatment of cardiac disease.