Abstract

Inhibition of Trypsin-Like Cysteine Proteinases (Gingipains) from Porphyromonas gingivalis by Tetracycline and Its Analogues Takahisa Imamura 1 , * , Kenji Matsushita 2 , James Travis 3 , and Jan Potempa 4 Division of Molecular Pathology, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, Kumamoto 860-0811, 1 and Department of Operative Dentistry and Endodontology, Kagoshima University Dental School, Kagoshima 890-8544, 2 Japan; Department of Biochemistry, University of Georgia, Athens, Georgia 30602 3 ; and Department of Microbiology and Immunology, Institute of Molecular Biology, Jagiellonian University, 31-120 Kraków, Poland 4 ABSTRACT Extracellular cysteine proteinases, referred to as gingipains, are considered important virulence factors for Porphyromonas gingivalis , a bacterium recognized as a major etiologic agent of chronic periodontitis. We investigated the effect of tetracycline and its analogues, doxycycline and minocycline, on the enzymatic activities of gingipains. Tetracyclines at 100 μM totally inhibited the amidolytic activity of arginine-specific gingipains (HRgpA and RgpB). In contrast, inhibition of Kgp was less efficient and required a somewhat higher concentration of the antibiotic to achieve the same effect. Among tetracycline derivatives, the most potent gingipain inhibitor was doxycycline, followed by tetracycline and minocycline. RgpB was inhibited by doxycycline in an uncompetitive and reversible manner with a 50% inhibitory concentration of 3 μM. Significantly, inhibition was unaffected by calcium, excluding the chelating activity of tetracyclines as the mechanism of gingipain inactivation. In contrast, the inhibitory activities of the tetracyclines were reduced by cysteine, a reducing agent, suggesting an interference of the drug at the oxidative region with the catalytic system of the enzyme. Doxycycline, at 10 μM, significantly inhibited the RgpB-mediated production of vascular permeability-enhancing activity from human plasma, thus proving an effective inhibition of gingipain in vivo. These results indicate a new activity of tetracyclines as cysteine proteinase inhibitors and may explain the therapeutic efficiency of these antibiotics in the treatment of periodontitis.