Identification of Porphyromonas gingivalis cysteine protease inhibitors from casein peptides

Methods: &Kappa-casein(109-137) was identified with the most protease inhibitory activity from a chymosin digest of milk caseins. This peptide was characterised by synthesising and testing it in proteolytic assays with chromogenic substrates or fluorescently-labelled bovine serum albumin. The assays were carried out against P. gingivalis whole-cell proteases, purified outer-membrane protease complexes, or purified RgpB.

Results: &Kappa-casein(109-137) exhibited ~90% inhibition of Arg- and Lys-gingipain activity with 200 µM of peptide and inhibited the gingipains in an uncompetitive manner with a K_i' value of 40.2 µM. The active region was determined by synthesising several shorter peptides and analysing for protease inhibitory activity. The peptides analysed were &Kappa-casein(117-123), &Kappa-casein(127-137) and &Kappa-casein(117-137). &Kappa-casein(117-137) inhibited both Arg- and Lys-gingipain activity with similar potency to the original peptide, demonstrating that the Lys-rich residues in the N-terminal region are nonessential for gingipain inhibitory activity. Molecular modeling of the peptide supported the uncompetitive mechanism of inhibition of the peptide.

Conclusion: The casein-derived &Kappa-casein(109-137) peptide demonstrates significant protease inhibitory activity against the P. gingivalis gingipains. This could lead to the development of a novel therapeutic strategy for the treatment or prevention of chronic periodontitis.