Discovery of D‐peptides that allosterically enhance SARS ‐ CoV ‐2 3CLpro activity by stabilizing its monomeric state

Abstract The SARS‐CoV‐2 main protease (3CLpro) is an essential enzyme for viral replication and a major target for antiviral drug development. While its catalytic activity is known to require dimer formation, the mechanism by which it cleaves itself from the monomeric viral polyprotein remains to be further studied. In this study, we engineered several D‐peptides based on a de novo designed D‐peptide inhibitor. These peptides bind to the 3CLpro monomer and unexpectedly activate its catalytic function. By enhancing the turnover number, the peptides significantly boost the catalytic efficiency of 3CLpro. Among them, LY11 stands out with high binding affinity ( K D  = 117 nM) and strong potency of enzymatic activation (EC 200  < 1 μM). Through biophysical and computational approaches, we show that LY11 binds in 1:1 stoichiometry, stabilizing the monomeric state while allosterically remodeling the inter‐domain linker of 3CLpro to expand the substrate binding pocket and switch on the protease. These findings suggest new insights into the maturation and catalytic mechanism of 3CLpro. The LY11‐regulated 3CL protease may serve as a versatile molecular tool for synthetic biology applications.