Conditional and Reversible Activation of Class A and B G Protein-Coupled Receptors Using Tethered Pharmacology

Abstract

Understanding the activation and internalization of G protein-coupled receptors (GPCRs) using conditional approaches is paramount to developing new therapeutic strategies. Here, we describe the design, synthesis, and testing of <b>ExONatide</b>, a benzylguanine-linked peptide agonist of the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR required for maintenance of glucose levels in humans. <b>ExONatide</b> covalently binds to SNAP-tagged GLP-1R-expressing cells, leading to prolonged cAMP generation, Ca²⁺ rises, and intracellular retention of the receptor. These effects were readily switched OFF following cleavage of the introduced disulfide bridge using the cell-permeable reducing agent <i>beta</i>-mercaptoethanol (BME). A similar approach could be extended to a class A GPCR using <b>GhrelON</b>, a benzylguanine-linked peptide agonist of the growth hormone secretagogue receptor 1a (GHS-R1a), which is involved in food intake and growth. Thus, <b>ExONatide</b> and <b>GhrelON</b> allow SNAP-tag-directed activation of class A and B GPCRs involved in gut hormone signaling in a reversible manner. This tactic, termed reductively cleavable agONist (RECON), may be useful for understanding GLP-1R and GHS-R1a function both <i>in vitro</i> and <i>in vivo</i>, with applicability across GPCRs.