PT - JOURNAL ARTICLE AU - Laura E. de Vries AU - Mateo I. Sanchez AU - Katarzyna Groborz AU - Laurie Kuppens AU - Marcin Poreba AU - Christine Lehmann AU - Fang Yuan AU - Shirin Arastu-Kapur AU - Martin Horn AU - Michael Mares AU - Matthew Bogyo AU - Marcin Drag AU - Edgar Deu TI - Characterization of <em>P. falciparum</em> dipeptidyl aminopeptidase 3 specificity reveals structural factors responsible for differences in amino acid preferences between peptide-based substrates and covalent inhibitors AID - 10.1101/246124 DP - 2018 Jan 01 TA - bioRxiv PG - 246124 4099 - http://biorxiv.org/content/early/2018/08/10/246124.short 4100 - http://biorxiv.org/content/early/2018/08/10/246124.full AB - Malarial dipeptidyl aminopeptidases (DPAPs) are cysteine proteases important for parasite development thus making them attractive drug targets. In order to develop inhibitors specific to the parasite enzymes it is necessary to map the determinants of substrate specificity of the parasite enzymes and its mammalian homologue cathepsin C (CatC). Here, we screened peptide-based libraries of substrates and covalent inhibitors to characterize the differences in specificity between parasite DPAPs and CatC, and used this information to develop highly selective DPAP1 and DPAP3 inhibitors. Interestingly, while the primary amino acid specificity of a protease is often used to develop potent inhibitors, we show that equally potent and highly specific inhibitors can be developed based on the sequences of non-optimal peptide substrates. Importantly, analysis of previously published data about the specificity of other proteases also unveiled significant discrepancies in the amino acid preference between substrates and inhibitors. In this article, we also discuss important structural and theoretical reasons that might account for these discrepancies. Overall, this study illustrates that focusing the development of protease inhibitors solely on substrate specificity might overlook important structural features that can be exploited to develop highly potent and selective compounds.