Objective: Inhibitors of benzamidine type have been proven to inhibit arginine specific cysteine proteinases, a well known virulence factor of the periodontopathogenic species Porphyromonas gingivalis. Of special interest is the inhibitor 2,6-bis-(4-amidinobenzyl)-cyclohexanone which has a Ki-value of 0.45 µM on RgpB. In-vitro-studies confirmed an enhanced phagocytosis of P. gingivalis and inhibited hemagglutination in association with the Ki-values and the inhibition of the arginine specific amidolytic activity. Contrary pentamidine with a Ki-value of 10.4 µM was most effective in growth inhibition as well as it totally eliminated intracellular P. gingivalis. The conclusion was that inhibitors of benzamidine type do not act only on Arg-gingipains. Methods: A purification of P. gingivalis suspension was made by means of a benzamidine column to find other ligands of P. gingivalis strains to benzamidine. This result was to specify. Furthermore the inhibitors were tested in a fertilized egg model. In all these experiments benzamidine, pentamidine and the inhibitor 2,6-bis-(4-amidinobenzyl)-cyclohexanone were studied against P. gingivalis ATCC 33277 (reference strain) and P. gingivalis M5-1-2 (clinical isolate). Results: Purification and subsequent amino acid sequencing showed that both HrgpA and RgpB as well as GroEL (HSP60) bind to benzamidine. The bacteria were stressed by heat (1 h at 45°C). The mRNA expression of HSP60 was not influenced, but the protein was found in higher quantities in stressed bacteria. Western blot analysis showed additional bands with a lower molecular weight after addition of 2,6-bis-(4-amidinobenzyl)-cyclohexanone and especially after addition of pentamidine indicating an interaction with HSP60. Using the fertilized egg model it was shown first that the clinical isolate was more virulent than the reference strain and second that the virulence of the bacteria was reduced by the inhibitors. Conclusion: Pentamidine and especially 2,6-bis-(4-amidinobenzyl)-cyclohexanone in local application might be promising compounds in supportive periodontitis treatment and should be included in clinical trials.

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