Mentor: Biswaranjan Pani, PhD; Department of Medicine, Duke University Medical Center
PI: Robert Lefkowitz, MD; Department of Medicine, Duke University Medical Center; Howard Hughes Medical Institute, Duke University Medical Center
GPCRs constitute the largest class of receptors in humans, ultimately serving as important targets for drug discovery. Ligands bind to GPCRs to drive signaling pathways via two signal transducers, G proteins and β-arrestins; biased ligands can preferentially activate one transducer pathway over the other. Carvedilol, a drug displaying bias toward the β-arrestin pathway, targets the β-2 adrenergic receptor (β2AR), but the mechanisms behind this biased signaling remains unknown due to the dynamism of GPCRs. Nanobodies have been commonly employed to stabilize certain confirmations of GPCRs. With this in mind, we hope to isolate nanobodies and test their abilities to stabilize the β2AR while bound to Carvedilol. We used yeast display surface technology to create an enriched yeast library containing nanobodies of interest. Then, we identified a single nanobody, B7, that displayed weak affinity to the Carvedilol bound β2AR. Using B7 as the template, we employed site-directed mutagenesis to purify new nanobodies, and we will use ELISA and competition binding and pull-down assays to test their abilities to stabilize the confirmation. The results of these have not been completed, but we hope that one nanobody will serve as a hit for stabilizing the β2AR in its biased state.
