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Targeted enzyme-prodrug systems deliver an exogenous enzyme to a disease site to generate active drug locally, thus increasing therapeutic efficacy and decreasing systemic toxicity. The majority of such systems have used bacterial enzymes, which are subject to immune recognition and inactivation or clearance in vivo. To address this problem, we report herein the development of a new enzyme-prodrug system that uses a human enzyme, peptide deformylase (PDF), which can be supplied exogenously while the endogenous enzyme is restricted to the mitochondria. The prodrugs feature an optimized masking group comprised of an N-formyldipeptide and a self-immolative m-fluoro-m'-pyridinyl amide (FPA) linker that enables a variety of functional groups to be masked. The mask is designed such that enzymatic deformylation triggers spontaneous dipeptide cyclorelease and linker self-immolation to release the active drug. We demonstrate the effectiveness of this human enzyme-prodrug system in vitro with two cytotoxic drugs, 5'-O-sulfamoyladenosine (AMS) and doxorubicin, masked at distinct functional groups.