Specificity of inhibition of matrix metalloproteinase activity by doxycycline: Relationship to structure of the enzyme

Gerald N. Smith, Elizabeth A. Mickler, Karen Hasty, Kenneth D. Brandt

Research output: Contribution to journalArticle

116 Citations (Scopus)

Abstract

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 (K(i)) of MMP-8 (K(i) = 36 μM) and truncated MMP-8 (K(i) = 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.

Original languageEnglish (US)
Pages (from-to)1140-1146
Number of pages7
JournalArthritis and rheumatism
Volume42
Issue number6
DOIs
StatePublished - Jun 1 1999

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Doxycycline
Matrix Metalloproteinases
Enzymes
Hemopexin
Matrix Metalloproteinase 1
Collagen Type II
Matrix Metalloproteinase 8

All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Rheumatology
  • Immunology
  • Pharmacology (medical)

Cite this

Specificity of inhibition of matrix metalloproteinase activity by doxycycline : Relationship to structure of the enzyme. / Smith, Gerald N.; Mickler, Elizabeth A.; Hasty, Karen; Brandt, Kenneth D.

In: Arthritis and rheumatism, Vol. 42, No. 6, 01.06.1999, p. 1140-1146.

Research output: Contribution to journalArticle

Smith, Gerald N. ; Mickler, Elizabeth A. ; Hasty, Karen ; Brandt, Kenneth D. / Specificity of inhibition of matrix metalloproteinase activity by doxycycline : Relationship to structure of the enzyme. In: Arthritis and rheumatism. 1999 ; Vol. 42, No. 6. pp. 1140-1146.
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abstract = "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 (K(i)) of MMP-8 (K(i) = 36 μM) and truncated MMP-8 (K(i) = 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.",
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AU - Mickler, Elizabeth A.

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AU - Brandt, Kenneth D.

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N2 - 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 (K(i)) of MMP-8 (K(i) = 36 μM) and truncated MMP-8 (K(i) = 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.

AB - 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 (K(i)) of MMP-8 (K(i) = 36 μM) and truncated MMP-8 (K(i) = 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.

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