De Novo-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 (Mpro) 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 Mpro 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 Mpro with a dissociation constant (KD) of 0.567 μM and the lowest IC50 reported to date for the dimer interface. Functional assays further demonstrated inhibition of Mpro catalytic activity, with 51.1%. These findings highlight HB3-Core25 as a stable inhibitor of Mpro activity by interfering with its dimerization, offering a complementary strategy to classical active-site inhibition in antiviral drug development.