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.