Summary of Work G protein-coupled receptors (GPCRs) are one of the largest group of therapeutic targets in cardiovascular disease. Ligands for GPCRs are characterized as agonists: those that stabilize a receptor conformation that promote activation of heterotrimeric G-proteins to generate second-messenger signaling; or antagonists such as β-blockers that block such activation. While β-blockers are standard of therapy in heart failure, there is evidence for a positive relationship between higher β-blocker dose and improved clinical outcomes. However, achieving a maximal dose is often difficult due to the development of intolerable adverse effects. Thus developing novel ligands that have improved efficacy and reduced untoward effects is a huge unmet need. Studies over the past 2 decades, in a large part from investigators on this proposal, have shown that orthosteric ligands, i.e. the binding site of the endogenous ligand, stabilize distinct active receptor conformations to selectively engage either G-protein or β-arrestin, to induce distinct signaling pathways. This concept of orthosteric ligand-dependent transducer selectivity is known as biased signaling and has been shown to activate cardioprotective pathways. While the majority of GPCR ligands target orthosteric binding sites, an emerging area of receptor biology and drug development is the identification of ligands that bind to allosteric, or topographically distinct, sites on receptors. Allosteric ligands that potentiate activity of orthosteric agonists are termed positive allosteric modulators (PAMs), while those that noncompetitively decrease the activity of orthosteric agonists are termed negative allosteric modulators (NAMs). It is now apparent that allosteric ligands may offer great benefit as therapeutics because of enhanced receptor specificity and efficacy. The objective of this R01 renewal is to identify, characterize, and translate newly identified allosteric modulators on the β1 adrenergic receptor (β1AR) from our recently performed DNA- encoded library (DEL) screen of 1 billion unique chemical entities with the ultimate goal of identifying β-arrestin biased PAMs. To accomplish this ambitious goal, I have assembled a team of experts in GPCR biology and organic chemistry that are known for innovative, paradigm-shifting research. I propose the following specific aims.