Section 1: Abstract Opioid-induced respiratory depression (OIRD) is responsible for thousands of overdose deaths annually. The μ-opioid receptor (MoR) mediates OIRD through heterotrimeric G-protein signaling, specifically via activation of Gβγ subunits, which subsequently modulate inwardly rectifying potassium channels (GIRK/Kir3) and other downstream effectors in the pre-Bötzinger complex, the brain’s primary respiratory control center. Current therapeutic approaches, such as naloxone, prevent OIRD by outcompeting an opioid to bind to the MoR ligand binding site, but because opioids are more fat soluble than naloxone, the risk of the opioid re-binding to the receptor and inducing delayed OIRD, known as renarcotization, is a serious risk without an existing therapeutic target. This has led to interest in developing a small molecule or protein capable of binding to the Gβγ subunits of the MoR rather than the ligand binding site to prevent OIRD without disrupting the analgesic effects of an opioid while also reducing the risk of renarcotization. This project aims to engineer a de novo peptide antagonist that selectively binds and inhibits Gβγ-mediated signaling, providing a molecular foundation for future therapeutic intervention in OIRD. Using rational computational design based on amphipathic α-helix architecture from known Gβγ-binding proteins (e.g., GRK2), we will design three peptide variants—a lead antagonist (BGA-1) and two alanine-scan controls—and express them as N-terminal GFP fusions in E. coli. Binding validation will be performed via co-immunoprecipitation (co-IP) with purified recombinant Gβγ protein and fluorescence plate reader detection. Structural validation employs AlphaFold2 predictions to confirm peptide-Gβγ interface interactions.
