Suppression of Pathogenicity ofPorphyromonas gingivalisby Newly Developed Gingipain Inhibitors

Abstract

Arg-gingipain (Rgp) and Lys-gingipain (Kgp) are cysteine proteinases produced by Porphyromonas gingivalis, a major etiological bacterium of periodontal diseases. Here we show a series of small peptide analogs able to inhibit either Rgp or Kgp, which are synthesized on the basis of the cleavage site specificity of human salivary histatins by each enzyme. Among this series of compounds, carbobenzoxy-Lys-Arg-CO-Lys-N-(CH 3 ) 2 (KYT-1) and carbobenzoxy-Glu(NHN(CH 3 )Ph)-Lys-CO-NHCH 2 Ph (KYT-36) were found to be the most potent inhibitors of Rgp and Kgp, respectively, with K i values of 10 Ϫ11 to 10 Ϫ10 M order. Both inhibitors exhibited slight or no inhibition on mammalian proteinases such as trypsin and cathepsins B, L, and H. All of the virulence induced by the culture supernatant of P. gingivalis tested, including the degradation of various host proteins such as human type I collagen, immunoglobulins, fibronectin, and fibrinogen, disruption of the bactericidal activity of polymorphonuclear leukocytes, and enhancement of the vascular permeability, were strongly inhibited by a combined action of both inhibitors. The functions essential for the bacterium to grow and survive in the periodontal pocket, such as coaggregation and acquisition of amino acids, were also strongly inhibited by the combined action of both inhibitors. The disruption of the adhesion and viability of human fibroblasts and hemagglutination by the organism were strongly suppressed by a single use of KYT-1. These results thus indicate that the newly developed KYT-1 and KYT-36 both should provide a broader application in studies of this important class of enzymes and facilitate the development of new approaches to periodontal diseases.

Cleavage of protease-activated receptors on an immortalized oral epithelial cell line by Porphyromonas gingivalis gingipains

“…Rgp can be found as three variants: a 50 kDa RgpB, a 50 kDa RgpA cat and a 95 kDa HRgpA (Potempa et al, 2000). In a mouse model, Kgp and RgpB, but not RgpA, contribute to periodontal disease (Pathirana et al, 2007a), findings that were confirmed by the use of novel gingipain inhibitors Kadowaki et al, 2004).…”

A Functional Virulence Complex Composed of Gingipains, Adhesins, and Lipopolysaccharide Shows High Affinity to Host Cells and Matrix Proteins and Escapes Recognition by Host Immune Systems

“…A monoclonal antibody that recognizes lipid A of LPS was from HyCult Biotechnology B.V. (Uden, The Netherlands). Rgp-and Kgp-specific inhibitors, designated KYT-1 and KYT-36, respectively, were synthesized by procedures described previously elsewhere (22). All other chemicals were reagent grade or better.…”

“…In addition, gingipains are also shown to be important for the bacterium to proliferate and survive in the periodontal pockets (26,34,39). The pathophysiological importance of gingipains has been further substantiated by newly developed gingipain inhibitors (22).…”

Deubiquitylation of histone H2A activates transcriptional initiation via trans-histone cross-talk with H3K4 di- and trimethylation

“…For these experiments, we found that a chemical compound termed KYT-36, which was developed as a cysteine protease inhibitor (Kadowaki et al 2004), inhibits deubiquitylation but does not affect transcription. KYT-36 can inhibit both recombinant USP21 and the intrinsic ubiquitin-specific protease activity present in Drosophila nuclear extract preparations, used as sources of GTFs (Supplemental Fig.…”