Inhibition of trypsin-like cysteine proteinases (gingipains) from Porphyromonas gingivalis by tetracycline and its analogues

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 microM 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 microM. 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 microM, 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.

FIG. 1

Inhibition of gingipains by tetracyclines. (A to C) Fifty microliters of a tetracycline analogue, dissolved in 0.1 M Tris-HCl (pH 7.6) containing 0.15 M NaCl, and an equal volume of a gingipain (2 nM) in the same buffer were mixed in a 96-well microplate and incubated for 5 min at 25°C. Then, 100 μl of 0.4 mM Z-Pyr-Gly-Arg-MCA for RgpB (A) and HRgpA (B) or Boc-Val-Leu-Lys-MCA for Kgp (C) in the same buffer was added to the mixture. ▵, tetracycline; □, minocycline; ○, doxycycline. The concentrations of tetracycline analogues in the initial mixture are shown. (D) Time course of RgpB inhibition by doxycycline. Fifty microliters of doxycycline (20 μM), dissolved in 0.1 M Tris-HCl (pH 7.6) containing 0.15 M NaCl, and an equal volume of RgpB (1 nM) in the same buffer were mixed and incubated at 25°C for various periods, followed by addition of 100 μl of Z-Pyr-Gly-Arg-MCA (0.4 mM). The amount of AMC released by the residual proteinase was measured fluorometrically.

FIG. 2

Mechanism of RgpB inhibition. Fifty microliters of doxycycline (●, 0 μM; ▵, 80 μM; □, 200 μM), dissolved in 0.1 M Tris-HCl (pH 7.6) containing 0.15 M NaCl, and 100 μl of Z-Pyr-Gly-Arg-MCA in the same buffer were mixed in a 96-well microplate, followed by addition of 50 μl of RgpB (2 nM) in the same buffer. The amount of AMC released by the residual proteinase was measured fluorometrically. (A) AMC release velocity (v) versus Z-Pyr-Gly-Arg-MCA concentration (s). (B) Plot of 1/v versus 1/[s]. The concentrations of Z-Pyr-Gly-Arg-MCA in the final mixture are shown.

FIG. 3

Effect of calcium and cysteine on RgpB inhibition by doxycycline. Fifty microliters of doxycycline, dissolved in 0.1 M Tris-HCl (pH 7.6) containing 0.15 M NaCl only (○) or further supplemented with 5 mM NaCl₂ (▴) or 10 mM cysteine (▪), and an equal volume of RgpB (2 nM) in the same buffer were mixed in a 96-well microplate and incubated at 25°C for 5 min. Then, 100 μl of Z-Pyr-Gly-Arg-MCA (0.4 mM) in the same buffer was added to the mixture. The amount of AMC released by the residual proteinase was measured fluorometrically.

FIG. 4

Inhibition by doxycycline of RgpB VPE activity production from human plasma. Normal human plasma (50 μl) supplemented with 1,10-phenanthroline (2 mM) was mixed with 25 μl of doxycycline, followed by addition of 25 μl of RgpB (40 nM) 15 s later and incubation at 25°C for 5 min. The reaction was stopped by adding 400 μl of TBS supplemented with 1,10-phenanthroline (1 mM), soybean trypsin inhibitor (20 μM), leupeptin (10 μM), or FPR-CK (10 μM). The VPE activity of each sample was measured. Activity was expressed in terms of mean micrograms of dye released in triplicate assays. Dye leakage at TBS-injected sites was used as a control and the value was subtracted from the value for each sample. Doxycycline concentrations during incubation are shown. ∗, P < 0.01 for VPE activity of RgpB-treated plasma in the absence of doxycycline. P, plasma alone; P + D, plasma plus doxycycline (1,000 μM); D, doxycycline alone (1,000 μM).