Smith G N, Mickler E A, Hasty K A, Brandt K D
Rheumatology Division, Indiana University School of Medicine, Indianapolis 46202-5103, USA.
Arthritis Rheum. 1999 Jun;42(6):1140-6. doi: 10.1002/1529-0131(199906)42:6<1140::AID-ANR10>3.0.CO;2-7.
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.
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.
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.
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.
研究强力霉素对基质金属蛋白酶1（MMP-1）、MMP-8和MMP-13的抑制作用，并确定每种MMP可变的血色素结合蛋白样结构域是否是这些胶原酶对强力霉素抑制敏感性差异的原因。
本研究使用了重组人MMP-1（胶原酶1）、MMP-8（胶原酶2）和MMP-13（胶原酶3）、缺乏血色素结合蛋白样结构域的MMP-8和MMP-13截短形式，以及截短MMP-13的突变形式。在强力霉素存在的情况下，测试全长MMP对II型胶原（这些酶的天然底物）的活性。使用一种小肽底物来确定MMP的哪些结构特征与对强力霉素抑制的敏感性相关。
30μM强力霉素可使MMP-13和MMP-8对II型胶原的活性抑制50%-60%，而50μM强力霉素仅使MMP-1的活性抑制18%。相反，在使用肽底物的实验中，直到药物浓度超过90μM，全长和截短的MMP-13均未受到抑制。MMP-8和截短的MMP-8对30μM强力霉素的抑制敏感，而MMP-1在90μM强力霉素作用下仅受到轻微抑制（14%）。对于MMP-8，稀释后抑制作用可逆，且与试剂添加顺序无关。MMP-8（抑制常数（K(i)）=36μM）和截短MMP-8（K(i)=77μM）的抑制常数动力学分析表明抑制作用为非竞争性。
在体外，接近口服给药后血清中达到的浓度时，MMP-13和MMP-8对胶原的活性受到显著抑制。对截短酶和两种底物的研究表明，强力霉素会破坏MMP-13血色素结合蛋白样结构域的构象以及MMP-8的催化结构域。
