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 μM doxycycline, while that of MMP‐1 was inhibited only 18% by 50 μM 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 μM. MMP‐8 and truncated MMP‐8 were sensitive to inhibition by 30 μM doxycycline, while MMP‐1 was slightly inhibited (14%) by 90 μM 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 (Ki) of MMP‐8 (Ki = 36 μM) and truncated MMP‐8 (Ki= 77 μM) 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.