Selective Activation of G Protein Signaling through the β2ar via Novel and Redefined Small Molecule Ligands

Michael Ippolito, Thomas Jefferson University


Emerging paradigms in G protein coupled receptor (GPCR) pharmacology like biased signaling or functional selectivity show promise for addressing areas of critical unmet medical need. Currently, GPCR targeted drugs make up a substantial portion of therapeutics used clinically for a variety of indications, however traditional drug discovery efforts focus on a narrow scope of pharmacological profiles and mechanisms of action. This necessarily limits potential outcomes and leaves known adverse effects of these compound classes unaddressed. The β2 – adrenergic receptor is a Gs coupled GPCR that promotes the reversal of airway constriction when activated, and β-agonists have been cornerstone therapies for asthma. Long-term use of these drugs leads to desensitization of therapeutic response, which is associated with severe adverse effects and an increased risk of sudden death during an asthma attack. This work takes a biased drug discovery approach to identifying and characterizing novel small-molecule β-ligands that promote a G protein biased signaling profile at the β2AR. We describe the characterization and classification of a set of G protein biased agonists of the β2AR. These molecules promote the selective activation of G proteins through β2AR, and demonstrate diminished agonist-promoted receptor phosphorylation and internalization relative to balanced agonists. We also describe the identification and characterization of an arrestin biased negative allosteric modulator (NAM) of β2AR, DFPQ. DFPQ was demonstrated to selectively inhibit arrestin recruitment to β2AR in the presence of a balanced agonist, and is the first small-molecule targeting the extracellular domains of the β2AR. Finally, we investigated a potential moleculardriver of biased signaling via mutagenesis. Here we show that the mutant receptor β2AR-F193A is intrinsically biased toward the G protein pathway with a phenotype consistent with pharmacologically promoted bias. All of the molecules characterized in this work promote a cAMP response downstream of Gs interaction with the receptor while showing diminished arrestin recruitment relative to balanced agonists. Additionally, these compounds protect from desensitization of response to agonist treatment in physiological airway systems. This thesis provides a proof-of- concept for the use of molecules that promote G protein biased signaling at the β2AR as next generation asthma therapeutics and provides a framework for future drug discovery toward this end.

Subject Area


Recommended Citation

Ippolito, Michael, "Selective Activation of G Protein Signaling through the β2ar via Novel and Redefined Small Molecule Ligands" (2021). ProQuest ETD Collection - Thomas Jefferson University. AAI28644296.