Inhibition of proteolytic, serpinolytic, and progelatinase-b activation activities of periodontopathogens by doxycycline and the non-antimicrobial chemically modified tetracycline derivatives.
BACKGROUND
Tetracyclines, particularly doxycycline (Doxy), and their non-antimicrobial chemically-modified derivatives (CMTs) inhibit the activities of human matrix metalloproteinases (MMPs), and reduce the severity and progression of periodontal disease in animal models and humans. In this study, the effects of Doxy and CMT-1, -3, and -5 on proteolytic, serpinolytic, and progelatinase-B activation activities of potent periodontopathogens were studied.


METHODS
The effect of Doxy and CMTs (0.5 to 50 microM) on proteolytic activities were investigated by incubating bacteria with chromogenic substrates or human serum albumin. A collagenolytic fraction of Porphyromonas gingivalis was used to evaluate the effect of these substances on collagenolytic (type I collagen) and serpinolytic (alpha1-proteinase inhibitor) activities. Lastly, the effect of Doxy on progelatinase-B (pro-MMP-9) activation by purified proteinases from P. gingivalis and Treponema denticola was investigated by SDS-PAGE/Western immunoblotting.


RESULTS
Doxy and CMTs, except CMT-5 which lacks the structural elements required for cation chelation, inhibited Arg- and Lys-gingipain activities as well as collagenolytic activity of P. gingivalis. Doxy and CMTs did not markedly affect the chymotrypsin-like activity of T. denticola but inhibited its trypsin-like activity. In addition, degradation of human serum albumin by cells of P. gingivalis and T. denticola was strongly inhibited by Doxy and CMT-1. Doxy and CMT-1 also inhibited the inactivation of alpha1-proteinase inhibitor (serpinolytic activity) by a collagenolytic fraction of P. gingivalis. Lastly, Doxy prevented the latent to active conversion of human neutrophil progelatinase-B (pro-MMP-9) by Arg-gingipains A/B of P. gingivalis but not by the chymotrypsin-like proteinase of T. denticola.


CONCLUSIONS
Data from this study suggest that Doxy and CMTs have the potential to inhibit the periodontopathogenic bacterial proteinases, which contribute to tissue destruction cascades during periodontitis directly and indirectly by triggering the host response.
Grenier
D
D
Groupe de Recherche en Ecologie Buccale, Faculté de Médecine Dentaire, Université Laval, Québec, Canada. Daniel.Grenier@greb.ulaval.ca
Plamondon
P
P
Sorsa
T
T
Lee
H M
HM
McNamara
T
T
Ramamurthy
N S
NS
Golub
L M
LM
Teronen
O
O
Mayrand
D
D
D018829:Adhesins, Bacterial / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002863:Chromogenic Compounds; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004318:Doxycycline / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors*; D018093:Gelatinases / Q000037:antagonists & inhibitors*; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins / Q000378:metabolism; D006801:Humans; D061965:Matrix Metalloproteinase Inhibitors; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D009504:Neutrophils / Q000201:enzymology; D016966:Porphyromonas gingivalis / Q000201:enzymology; D011480:Protease Inhibitors / Q000494:pharmacology*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D015843:Serpins / Q000494:pharmacology*; D012709:Serum Albumin; D013752:Tetracycline / Q000494:pharmacology; D013754:Tetracyclines / Q000494:pharmacology; D014208:Treponema / Q000201:enzymology; D014361:Trypsin Inhibitors / Q000494:pharmacology; D000515:alpha 1-Antitrypsin / Q000494:pharmacology
D018829:Adhesins, Bacterial; D000900:Anti-Bacterial Agents; D002863:Chromogenic Compounds; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D012709:Serum Albumin; D013754:Tetracyclines; D014361:Trypsin Inhibitors; D000515:alpha 1-Antitrypsin; C117155:tetracycline CMT-3; C053450:4-des-dimethylaminotetracycline; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; D018093:Gelatinases; D008666:Metalloendopeptidases; C069860:progelatinase; D013752:Tetracycline; D004318:Doxycycline