<i> De <i>N</i> ovo </i> -Designed Miniprotein Inhibits the Enzymatic Activity of the SARS-CoV-2 Main Protease

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. <i>In vitro</i> 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 (<i>K</i>_D) of 0.567 μM 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.