Peptidic　inhibitors　of　proteases　are　attracting　increasing　interest　not　only　as　drug　candidates　but　also　for　studying　the　function　and　regulation　mechanisms　of　these　enzymes.　Previously,　we　screened　out　a　cyclic　peptide　inhibitor　of　human　uPA　(Figure　presented.)　and　found　that　Ala　substitution　of　P2　residue　turns　upain-1　to　a　substrate.　To　further　investigate　the　effect　of　P2　residue　on　the　peptide　behavior　transformation,　we　constructed　upain-1-W3F,　which　has　Phe　replacement　in　the　P2　position.　We　determined　KD　and　Ki　of　upain-1-W3F　and　found　that　upain-1-W3F　might　still　exist　as　an　inhibitor.　Furthermore,　the　high-resolution　crystal　structure　of　upain-1-W3F·uPA　reveals　that　upain-1-W3F　indeed　stays　as　an　intact　inhibitor　bind　to　uPA.　We　thus　propose　that　the　P2　residue　plays　a　nonnegligible　role　in　the　conversion　of　upain-1　to　a　substrate.　These　results　also　proposed　a　strategy　to　optimize　the　pharmacological　properties　of　peptide-based　drug　candidates　by　hydrophobicity　and　steric　hindrance.　Abbreviations　:　uPA:　urokinase-type　plasminogen　activator;　SPD:　serine　protease　domain;　S1　pocket:　specific　substrate-binding　pocket.　©　2020,　©　2020　Japan　Society　for　Bioscience,　Biotechnology,　and　Agrochemistry.