Project Summary/Abstract Here we develop a platform for protease-activated peptide prodrugs. We initially focus the effort on antimicrobial peptides (AMP), which have a broad spectrum of cell-penetrating, antimicrobial and cytotoxic activities. However, they have multiple limitations, including off-target cytotoxicity and proteolytic instability. To circumvent these unwanted effects, linear protease-activated prodrugs have been employed. Although effective at enhancing the activity of the active payload, it does not adequately address the side effects stated above. To mitigate the side effects of the traditional protease-activated prodrug approach, the proposed research strategy herein seeks to design a branched protease-activated prodrug by coupling a cleavable linker to the sidechains of amino acid residue that significantly impacts antimicrobial peptide activity. As a proof-of-concept, bombolitin (an AMP that is cytotoxic to mammalian cells) will be used as the payload, masked with caspase-3 and fibroblast activation protein (cancer biomarkers) substrate-specific linkers. By modifying the sidechains of Lys, Ser, Thr, and Tyr with these linkers, we hypothesize that the physicochemical properties of bombolitin will be significantly altered, thus reducing peptide-membrane interaction until activation. In this research, we propose to 1) design and synthesize single and double-branched protease-activated prodrugs cleaved in the presence of either caspase-3 and/or fibroblast activation protein to yield the active peptide, 2) confirm caspase-3 and fibroblast activation protein cleavage of linker from prodrugs by performing in vitro enzymatic assays and biophysical analysis, and 3) perform cell-based assays to validate prodrug cleavage, target specificity, activity enhancement, and reduction of off-target effects.