De novo Protein Design of mini-binders
modulating Wnt receptors and HCV infection
Hyun-Soo Cho
a Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea
Email: hscho8@gmail.com
G protein-coupled receptors (GPCRs) are key mediators of extracellular signaling and represent highly validated drug targets in diverse pathological contexts. And despite the curative success of direct-acting antivirals (DAAs), the lack of prophylaxis against hepatitis C virus (HCV) reinfection—particularly to prevent systemic viral reseeding in high-risk populations—remains a critical gap in global elimination efforts. In this study, we employed a structure-guided de novo design strategy to develop mini protein binders that selectively engage the extracellular domain of a target GPCR (Frizzled receptor) and targeting the large extracellular loop (LEL) of a HCV co-receptor. Using a combination of RFdiffusion for backbone generation, ProteinMPNN for sequence design, and AlphaFold2 for structural validation, we engineered small, stable binders with high predicted affinity. These mini protein binders act as functional antagonists by competitively blocking ligand access to the receptor, thereby suppressing downstream signaling activity. Computational evaluations and preliminary biophysical characterizations support their binding specificity and inhibitory potential. Our approach establishes a versatile design framework for creating GPCR-targeted biologics and blocking the viral infection with therapeutic relevance.
