10779/crick.11550900.v1 Laura E de Vries Laura E de Vries Mateo I Sanchez Mateo I Sanchez Katarzyna Groborz Katarzyna Groborz Laurie Kuppens Laurie Kuppens Marcin Poreba Marcin Poreba Christine Lehmann Christine Lehmann Neysa Nevins Neysa Nevins Chrislaine Withers-Martinez Chrislaine Withers-Martinez David J Hirst David J Hirst Fang Yuan Fang Yuan Shirin Arastu-Kapur Shirin Arastu-Kapur Martin Horn Martin Horn Michael Mares Michael Mares Matthew Bogyo Matthew Bogyo Marcin Drag Marcin Drag Edgar Deu Edgar Deu Characterization of P. falciparum dipeptidyl aminopeptidase 3 specificity identifies differences in amino acid preferences between peptide-based substrates and covalent inhibitors. The Francis Crick Institute 2020 dipeptidyl aminopeptidase malaria positional scanning proteases specificity Deu Blackman FC001043 GSK 0601 Biochemistry and Cell Biology 1101 Medical Biochemistry and Metabolomics 0304 Medicinal and Biomolecular Chemistry Biochemistry & Molecular Biology 2020-01-08 17:02:37 Journal contribution https://crick.figshare.com/articles/journal_contribution/Characterization_of_P_falciparum_dipeptidyl_aminopeptidase_3_specificity_identifies_differences_in_amino_acid_preferences_between_peptide-based_substrates_and_covalent_inhibitors_/11550900 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. Finally, our homology modelling and docking studies provide potential structural explanations of the differences in specificity between DPAP1, DPAP3, and CatC, and between substrates and inhibitors in the case of DPAP3. 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. This article is protected by copyright. All rights reserved.