New chromogenic substrates of human neutrophil cathepsin G containing non-natural aromatic amino acid residues in position P1 selected by combinatorial chemistry methods

Specificity of human cathepsin G was explored using combinatorial chemistry methods. Deconvolution of a tetrapeptide library, where 5-amino-2-nitrobenzoic acid served as a chromophore attached at the C-terminus, yielded the active sequence Phe-Val-Thr-Tyr-Anb(5,2)-NH2. This sequence was used for a second-generation library with the general formula Ac-Phe-Val-Thr-X-Anb(5,2)-NH2, where position X was replaced with several amino acids: L-pyridylalanine (Pal), 4-nitro-L-phenylalanine (Nif), 4-amino-L-phenylalanine (Amf), 4-carboxy-L-phenylalanine (Cbf),4-guanidine-L-phenylalanine (Gnf), 4-methyloxycarbonyl-L-phenylalanine (Mcf), 4-cyano-L-phenylalanine (Cyf), Phe, Tyr, Arg and Lys. Specificity ligand parameters, k (cat) and K (M), with human cathepsin G were determined for all chromogenic substrates synthesized. The highest value of the specificity constant (k (cat)/K (M)) was obtained for a substrate with the Gnf residue in position P-1. This peptide was 10 times more active than the second most active substrate which contained the Amf residue. The following order of potency was established: Gnf > > Amf > Tyr = Phe > Arg= Lys > Cyf. Substrate specificity for cathepsin G is greatly enhanced when an aromatic side chain and a strong positive charge are incorporated in residue P-1.