Targeting viral proteases is a well-established antiviral strategy and a promising approach that has been actively explored against SARS-CoV-2. The SARS-CoV-2 main protease (M pro ) is essential for viral replication and functions as a homodimer, making its dimerization interface an attractive therapeutic target. In this study, we report the rational design of HB3-Core25, a miniprotein computationally engineered to disrupt M pro dimerization and inhibit its catalytic activity. In vitro production followed by biophysical characterization showed that HB3-Core25 folds into a compact trimeric helical bundle, exhibiting high solubility and thermal stability. Biophysical assays confirmed binding to M pro with a disso... More
Targeting viral proteases is a well-established antiviral strategy and a promising approach that has been actively explored against SARS-CoV-2. The SARS-CoV-2 main protease (M pro ) is essential for viral replication and functions as a homodimer, making its dimerization interface an attractive therapeutic target. In this study, we report the rational design of HB3-Core25, a miniprotein computationally engineered to disrupt M pro dimerization and inhibit its catalytic activity. In vitro production followed by biophysical characterization showed that HB3-Core25 folds into a compact trimeric helical bundle, exhibiting high solubility and thermal stability. Biophysical assays confirmed binding to M pro with a dissociation constant ( K D ) of 0.567 M and the lowest IC50 reported to date for the dimer interface. Functional assays further demonstrated inhibition of M pro catalytic activity, with 51.1%. These findings highlight HB3-Core25 as a stable inhibitor of M pro activity by interfering with its dimerization, offering a complementary strategy to classical active-site inhibition in antiviral drug development.
