Elucidation of the Contribution of Active Site and Exosite Interactions to Affinity and Specificity of Peptidylic Serine Protease Inhibitors Using Non-Natural Arginine Analogs

Abstract

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.

Oxidizing Electrode Potentials Decrease Current Production and Coulombic Efficiency through Cytochrome c Inactivation in Shewanella oneidensis MR‐1

“…It should be noted that in addition to accessibilities of cleavage sites, the amino acid sequences adjacent to the dibasic residue cleavage sites are also important for determining the preferences of proteases for cleavage sites 29–32. Therefore, while the cleavage sites #1, 4, 5, 11, and 12 show similar H‐D exchange compared with the most readily cleaved sites #6 and #7, the different amino acid sequences at such sites may participate in the lower extent of their proteolytic processing 15, 16, 27…”

The Impact of Nanoparticle Protein Corona on Cytotoxicity, Immunotoxicity and Target Drug Delivery

“…On the other hand, the physicochemical characterization of bare NPs before exposure to body fluids remains crucial to reveal the correlations between the properties of NPs and PC composition (Figure 1). During the recent years, a large number of studies have been devoted to the characterization of NP-PC complexes [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23]. Furthermore, the scientific community is now moving from the The impact of nanoparticle protein corona on cytotoxicity, immunotoxicity and target drug delivery The bare NP (synthetic identity) that has a specific shape, size and charge is injected into the body.…”

Discovery of Novel Inhibitors of a Disintegrin and Metalloprotease 17 (ADAM17) Using Glycosylated and Non-glycosylated Substrates

“…Secondary binding site (exosite) inhibitors provide a viable alternative to active site inhibitors (11)(12)(13)(14)(15)(16). Selective exosite inhibitors of MMP-13 that were discovered by several groups (17)(18)(19)(20)(21), including ours, are currently being investigated for arthritis therapy.…”