Specificity of inhibition of matrix metalloproteinase activity by doxycycline: relationship to structure of the enzyme.
OBJECTIVE: To investigate the inhibition of matrix metalloproteinase 1 (MMP-1), MMP-8, and MMP-13 by doxycycline, and to determine whether the variable hemopexin-like domain of each MMP was responsible for the differences in susceptibility to doxycycline inhibition among these collagenases. METHODS: Recombinant human MMP-1 (collagenase 1), MMP-8 (collagenase 2), and MMP-13 (collagenase 3), truncated forms of MMP-8 and MMP-13 lacking the hemopexin-like domain, and a mutant form of truncated MMP-13 were used in these studies. The activity of the full-length MMP in the presence of doxycycline was tested against type II collagen, a natural substrate for the enzymes. A small peptolide substrate was used to determine which structural features of the MMPs were related to sensitivity to doxycycline inhibition. RESULTS: The activity of MMP-13 and MMP-8 against type II collagen was inhibited by 50-60% by 30 microM doxycycline, while that of MMP-1 was inhibited only 18% by 50 microM doxycycline. In contrast, in experiments with the peptolide substrate, neither full-length nor truncated MMP-13 was inhibited until the concentration of the drug exceeded 90 microM. MMP-8 and truncated MMP-8 were sensitive to inhibition by 30 microM doxycycline, while MMP-1 was slightly inhibited (14%) by 90 microM doxycycline. For MMP-8, inhibition was reversible upon dilution and was independent of the order in which the reagents were added. Kinetic analysis of the inhibition constant (K(i)) of MMP-8 (K(i) = 36 microM) and truncated MMP-8 (K(i) = 77 microM) indicated that inhibition was noncompetitive. CONCLUSION: Significant inhibition of MMP-13 and MMP-8 activity against collagen occurred in vitro at concentrations that were near the concentrations achieved in serum after oral dosing. Studies with truncated enzymes and 2 substrates suggest that doxycycline disrupts the conformation of the hemopexin-like domain of MMP-13 and the catalytic domain of MMP-8.[1]References
- Specificity of inhibition of matrix metalloproteinase activity by doxycycline: relationship to structure of the enzyme. Smith, G.N., Mickler, E.A., Hasty, K.A., Brandt, K.D. Arthritis Rheum. (1999) [Pubmed]
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