RT Journal Article SR Electronic T1 Elucidation of the Contribution of Active Site and Exosite Interactions to Affinity and Specificity of Peptidylic Serine Protease Inhibitors Using Non-Natural Arginine Analogs JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 585 OP 597 DO 10.1124/mol.111.072280 VO 80 IS 4 A1 Masood Hosseini A1 Longguang Jiang A1 Hans Peter Sørensen A1 Jan K. Jensen A1 Anni Christensen A1 Sarah Fogh A1 Cai Yuan A1 Lisbeth M. Andersen A1 Mingdong Huang A1 Peter A. Andreasen A1 Knud J. Jensen YR 2011 UL http://molpharm.aspetjournals.org/content/80/4/585.abstract AB There is increasing interest in developing peptides for pharmacological intervention with pathophysiological functions of serine proteases. From phage-displayed peptide libraries, we previously isolated peptidylic inhibitors of urokinase-type plasminogen activator, a potential target for intervention with cancer invasion. The two peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), are competitive inhibitors of human and murine urokinase-type plasminogen activator, respectively. Both have an Arg as the P1 residue, inserting into the S1 pocket in the active site of the enzymes, but their specificity depends to a large extent on interactions outside the enzymes' active sites, so-called exosite interactions. Here we describe upain-2 (CSWRGLENHAAC) and the synthesis of a number of upain-2 and mupain-1 variants in which the P1 Arg was substituted with novel non-natural Arg analogs and achieved considerable improvement in the affinity of the peptides to their targets. Using chimeras of human and murine urokinase-type plasminogen activator as well as X-ray crystallography, we delineated the relative contribution of the P1 residue and exosite interactions to the affinity and specificity of the inhibitors for their target enzyme. The effect of inserting a particular non-natural amino acid into the P1 position is determined by the fact that changes in interactions of the P1 residue in the S1 pocket lead to changed exosite interactions and vice versa. These findings are of general interest when the affinities and specificities of serine protease inhibitors to be used for pharmacological intervention are considered and could pave the way for potential drug candidates for the treatment of cancer.