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 variabl e 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), an d MMP-13 (collagenase 3), truncated forms of MMP-8 and MMP-13 lacking the h emopexin-like domain, and a mutant form of truncated MMP-13 were used in th ese studies. The activity of the full-length MMP in the presence of doxycyc line was tested against type II collagen, a natural substrate for the enzym es. A small peptolide substrate was used to determine which structural feat ures of the MMPs were related to sensitivity to doxycycline inhibition. Results. The activity of MMP-13 and MMP-8 against type II collagen was inhi bited by 50-60% by 30 mu M doxycycline, while that of MMP-1 was inhibited o nly 18% by 50 mu M doxycycline. In contrast, in experiments with the peptol ide substrate, neither full-length nor truncated MMP-13 was inhibited until the concentration of the drug exceeded 90 mu M. MMP-8 and truncated MMP-8 were sensitive to inhibition by 30 mu M doxycycline, while MMP-1 was slight ly inhibited (14%) by 90 mu M doxycycline, For MMP-8, inhibition was revers ible upon dilution and was independent of the order in which the reagents w ere added. Kinetic analysis of the inhibition constant (K-i) of MMP-8 (K-i = 36 mu M) and truncated MMP-8 (K-i = 77 mu M) indicated that inhibition wa s noncompetitive, Conclusion. Significant inhibition of MMP-13 and MMP-8 activity against col lagen 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 hemope xin-like domain of MMP-13 and the catalytic domain of MMP-8.