question: Furin plays an important role in various pathological states, especially in bacterial and viral infections A detailed understanding of the structural requirements for inhibitors targeting this enzyme is crucial to develop new therapeutic strategies in infectious diseases, including an urgent unmet need for SARS-CoV-2 infection Previously, we have identified a potent furin inhibitor, peptide Ac-RARRRKKRT-NH2 (CF1), based on the highly pathogenic avian influenza hemagglutinin The goal of this study was to determine how its N-terminal part (the P8-P5 positions) affects its activity profile To do so, the positional-scanning libraries of individual peptides modified at the selected positions with natural amino acids were generated Subsequently, the best substitutions were combined together and/or replaced by unnatural residues to expand our investigations The results reveal that the affinity of CF1 can be improved (2-2 5-fold) by substituting its P5 position with the small hydrophobic residues (Ile or Val) or a basic Lys © 2021American Chemical Society