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.


CONCLUSIONS
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.
Smith
G N
GN
Rheumatology Division, Indiana University School of Medicine, Indianapolis 46202-5103, USA.
Mickler
E A
EA
Hasty
K A
KA
Brandt
K D
KD
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D003094:Collagen / Q000187:drug effects; D004318:Doxycycline / Q000494:pharmacology*; D006801:Humans; D020781:Matrix Metalloproteinase 1; D053509:Matrix Metalloproteinase 13; D020784:Matrix Metalloproteinase 8; D061965:Matrix Metalloproteinase Inhibitors; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D061965:Matrix Metalloproteinase Inhibitors; D011994:Recombinant Proteins; D003094:Collagen; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; D020784:Matrix Metalloproteinase 8; D020781:Matrix Metalloproteinase 1; D004318:Doxycycline