Small MoleculeDrugs Triggered the Activation of MacrocycleMasked Proteins

On-demand activation of prodrugs represents an emerging and fast developing strategy to improve the therapeutic index of certain drugs. However, strategies to generate protein-based prodrugs with controllable activation are still limited. Here, we present a supramolecular masking strategy that enables on-demand activation of macrocycle-masked proteins with Food and Drug Administration (FDA)-approved oral drugs. Proteins of interest were engineered to incorporate two N-terminal peptide motifs, which were dimerized by cucurbit[8]­uril (CB[8]) to form a supramolecular mask that sterically blocks functional protein interfaces, inhibiting interactions with targets or substrates. The inhibitory effect was selectively reversed by amantadine or memantine to restore the protein activity. This masking strategy was validated across various protein classes, including antibodies, cytokines, and enzymes. Activation of CB[8]-masked proteins was further demonstrated in living mice via FDA-approved small molecule treatments. Our method provided a supramolecular strategy for the selective activation of protein-based prodrugs and the development of next-generation protein therapeutics.