A novel, potent dual inhibitor of Arg-gingipains and Lys-gingipain as a promising agent for periodontal disease therapy.

The periodontal pathogen Porphyromonas gingivalis produces a unique class of cysteine proteinases termed gingipains that comprises Arg-gingipain (Rgp) and Lys-gingipain (Kgp). Growing evidence indicates that these 2 types of gingipains synergistically contribute to the entire virulence of the organism and increase the risk of periodontal disease (PD) by disrupting the host immune system and degrading the host tissue and plasma proteins. Therefore, a dual inhibitor of both gingipains would have attractive clinical potential for PD therapy. In this study, a novel, potent, dual inhibitor of Rgp and Kgp was developed through structure-based drug design, and its biological potency was evaluated in vitro and in vivo. This inhibitor had low nanomolar inhibitory potency (Ki=40 nM for Rgp, Ki=0.27 nM for Kgp) and good selectivity for host proteases and exhibited potent antibacterial activity against P. gingivalis by abrogating its manifold pathophysiological functions. The therapeutic potential of this inhibitor in vivo was also verified by suppressing the vascular permeability that was enhanced in guinea pigs by the organism and the gingival inflammation in beagle dog PD models. These findings suggest that a dual inhibitor of Rgp and Kgp would exhibit noteworthy anti-inflammatory activity in the treatment of PD.

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.

Arg-gingipain (Rgp) and Lys-gingipain (Kgp) are Porphyromonas gingivalis cysteine proteinases implicated as major virulence factors in pathologies of periodontitis. We purified a 660-kDa cell-associated gingipain complex existing as a homodimer of two catalytically active monomers which comprises their catalytic and adhesin domains. Electron microscopy revealed that the complex was composed of a globular particle with a 10-nm external diameter possessing one or two electron-dense hole-like structures. Two-dimensional gel electrophoresis and immunoblot analyses revealed the association of lipopolysaccharide (LPS) with the catalytic domains and a hemagglutinin domain, Hgp44, of Rgp and Kgp in the complex. The complex significantly degraded human type I collagen and elastin and strongly disrupted viability of human gingival fibroblasts and umbilical vein endotherial cells with an efficiency which was higher than that of the monomeric gingipains. The native complex produced only a small amount of nitrogen dioxide, tumor necrosis factor alpha, and interleukin-6 by macrophages, whereas the heat-denatured complex resulted in increased production. Inhibition of the proteolytic activities of the gingipain complex did not up-regulate the cytokine production, indicating that the functional domains in LPS are structurally masked by the complex proteins. These results indicate the importance of the complex in evasion of host defense mechanisms as well as in host tissue breakdown.

[Gingipains as the determinants of periodontopathogenicity].

The arginine-specific cysteine proteinase (Arg-gingipain, Rgp) and lysine-specific cysteine proteinase (Lys-gingipain, Kgp) are produced by Porphyromonas gingivalis, an etiological bacterium of periodontal disease. Rgp and Kgp have been implicated as the major virulent factors because of their degrading activity to a broad range of host proteins and of the essential roles in bacterial cell viability. Recent studies have demonstrated the association of P. gingivalis with systemic diseases such as cardiovascular diseases, preterm birth, and low birth weight. The majority of gingipains exist as the membrane-associated complexes composed of the proteinase domains of both Rgp and Kgp, the C-terminal adhesin domains of RgpA and Kgp, phospholipids, and LPS. The complex induced potent viability loss of human endothelial cells and fibroblasts. As the suppression of Rgp and Kgp seems to be the most important to overcome the P. gingivalis-induced systemic disorders as well as the periodontal disease, we have thus designed and synthesized novel proteinase inhibitors specific to Rgp and Kgp on the basis of cleavage sites. Some of them suppressed the characteristic features of P. gingivalis associated with its pathogenicity such as degradation of host proteins, hemagglutination, enhancement of vascular permeability, disruption of leukocytes function, and induction of host cell death.

Roles for Arg- and Lys-gingipains in the disruption of cytokine responses and loss of viability of human endothelial cells by Porphyromonas gingivalis infection.

Accumulating evidence indicates that periodontal disease is associated with human cardiovascular diseases. The periodontal pathogen Porphyromonas gingivalis was shown to be present in atherosclerotic plaques in addition to periodontal pockets. This bacterium is known to produce two individual cysteine proteinases, Arg-gingipain (Rgp) and Lys-gingipain (Kgp). Here we show that these two enzymes are responsible for either the disruption of cytokine responses in human umbilical vein endothelial cells (HUVEC) to the bacterium infection or the loss of cell viability. The expression of interleukin-8 and monocyte chemoattractant protein-1 mRNA in HUVEC was greatly induced when infected with the wild-type strain, nevertheless, their protein levels in the culture medium were markedly decreased. This decrease was completely abolished in the cells infected with the Rgp/Kgp-null mutant, but not in either the Rgp- or Kgp-null mutants. Loss of the adhesion activity and viability of HUVEC were greatly induced by the culture supernatant of the wild-type strain and strongly inhibited by either a combination of the Rgp- and the Kgp-specific inhibitors or the deficiency of the Rgp- and Kgp-encoding genes. These findings indicate that P. gingivalis modulates the cytokine response in the cells and disrupts the adhesion activity and the viability through the cooperative action of Rgp and Kgp and thereby may contribute to pathogenesis of cardiovascular diseases as well as periodontal disease.

Isolation and characterization of a novel and potent inhibitor of Arg-gingipain from Streptomyces sp. strain FA-70.

Arg-gingipain (Rgp) is a major cysteine proteinase produced by the oral bacterium Porphyromonas gingivalis, which is a major pathogen of advanced periodontal diseases. This enzyme is important for the bacterium both to exhibit its virulence and to survive in periodontal pockets. The development of Rgp inhibitors thus provides new therapeutic approaches to periodontal diseases. In this study, we first isolated and purified a novel and potent inhibitor of Rgp from the culture supernatant of Streptomyces species strain FA-70, now designated as FA-70C1. This compound was found to be an antipain analog composed of phenylalanyl-ureido-citrullinyl-valinyl-cycloarginal (C27H43N9O7). The Ki value was calculated to be 4.5x10(-9) M when benzyloxycarbonyl-phenylalanyl-arginine-4-methly-coumaryl-7-amide was used as a substrate. This compound also inhibited cathepsins B, L, and H, though their Ki values were much higher than that of Rgp. FA-70C1 had little or no inhibitory activity on Lys-gingipain, another cysteine proteinase of P. gingivalis. The Rgp-induced degradation of various human proteins was completely blocked by this inhibitor. Disruption of both the bactericidal activity of polymorphonuclear leukocytes and the viability of human fibroblasts and umbilical vein endothelial cells induced by the culture supernatant of P. gingivalis was suppressed by the inhibitor in a dose-dependent manner. The enhancement of vascular permeability induced by in vivo administration of the culture supernatant of P. gingivalis was strongly inhibited by the inhibitor. Furthermore, the growth of P. gingivalis was suppressed by FA-70C1 in a dose-dependent manner. These results strongly suggest that FA-70C1 is a useful tool to prevent the virulence of P. gingivalis.

Roles of Arg- and Lys-gingipains in coaggregation of Porphyromonas gingivalis: identification of its responsible molecules in translation products of rgpA, kgp, and hagA genes.

Arg- (Rgp) and Lys-gingipains (Kgp) are two individual cysteine proteinases produced by Porphyromonas gingivalis , an oral anaerobic bacterium, and are implicated as major virulence factors in a wide range of pathologies of adult periodontitis. Coaggregation of this bacterium with other oral bacteria is an initial and critical step in infectious processes, yet the factors and mechanisms responsible for this process remain elusive. Here we show that the initial translation products of the rgpA , kgp and hemagglutinin hagA genes are responsible for coaggregation of P. gingivalis and that the proteolytic activity of Rgp and Kgp is indispensable in this process. The rgpA rgpB kgp- and rgpA kgp hagA -deficient triple mutants exhibited no coaggregation activity with Actinomyces viscosus , whereas the kgp -null and rgpA rgpB -deficient double mutants significantly retained this activity. Consistently, the combined action of Rgp- and Kgp-specific inhibitors strongly inhibited the coaggregation activity of the bacterium, although single use of Rgp- or Kgp-specific inhibitor significantly retained this activity. We also demonstrate that the 47- and 43-kDa proteins produced from the translation products of the rgpA , kgp , and hagA genes by proteolytic activity of both Rgp and Kgp are responsible for the coaggregation of P. gingivalis.

Suppression of pathogenicity of Porphyromonas gingivalis by newly developed gingipain inhibitors.

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-(CH2)2 (KYT-1) and carbobenzoxy-Glu(NHN(CH3)Ph)-Lys-CO-NHCH2Ph (KYT-36) were found to be the most potent inhibitors of Rgp and Kgp, respectively, with Ki values of 10(-11) to 10(-10) 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.