Transcending stereochemical boundaries: ambidextrous cleavage of d- and l-peptide enantiomers by natural eukaryotic proteases

Proteins have predominantly evolved in the l-form, favoring homochiral interactions with other l-proteins while avoiding their d-enantiomer counterparts. Reflecting this principle, natural proteases are known to hydrolyze l-peptides but not their mirror-image d-enantiomers. In this study, we designed and screened a combinatorial d-peptide library tailored for proteases and unexpectedly revealed that several eukaryotic proteases — including papain, human cathepsin B, and mouse carboxylesterase 1c — efficiently cleave d-peptide substrates through sequence-specific recognition. The corresponding enantiomeric l-peptide substrates were also cleaved, indicating ambidextrous substrate proteolysis. Structural analyses show that cathepsin B achieves activity through enantiomer-specific substrate-binding modes, underscoring its ability to accommodate alternative stereochemistries for substrate recognition and processing. Leveraging this activity, we developed a d-peptide substrate of cathepsin B as an efficient cleavable linker for antibody–drug conjugates, demonstrating potent antitumor efficacy in vitro and in vivo. These results reveal an unanticipated capacity for substrate recognition in natural eukaryotic proteases, broadening the framework of protease specificity and enabling new possibilities for therapeutic design.