Lauer-Fields Janelle L, Whitehead John K, Li Shunzi, Hammer Robert P, Brew Keith, Fields Gregg B
Department of Chemistry and Biochemistry and College of Biomedical Sciences, Florida Atlantic University, Boca Raton, FL 33431, USA.
J Biol Chem. 2008 Jul 18;283(29):20087-95. doi: 10.1074/jbc.M801438200. Epub 2008 May 22.
Unregulated activities of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix components, such as collagen and laminin. However, clinical trials with small molecule MMP inhibitors have been largely unsuccessful, with a lack of selectivity considered particularly problematic. Enhanced selectivity could be achieved by taking advantage of differences in substrate secondary binding sites (exosites) within the MMP family. In this study, triple-helical substrates and triple-helical transition state analog inhibitors have been utilized to dissect the roles of potential exosites in MMP-9 collagenolytic behavior. Substrate and inhibitor sequences were based on either the alpha1(V)436-450 collagen region, which is hydrolyzed at the Gly (downward arrow) Val bond selectively by MMP-2 and MMP-9, or the Gly (downward arrow) Leu cleavage site within the consensus interstitial collagen sequence alpha1(I-III)769-783, which is hydrolyzed by MMP-1, MMP-2, MMP-8, MMP-9, MMP-13, and MT1-MMP. Exosites within the MMP-9 fibronectin II inserts were found to be critical for interactions with type V collagen model substrates and inhibitors and to participate in interactions with an interstitial (types I-III) collagen model inhibitor. A triple-helical peptide incorporating a fibronectin II insert-binding sequence was constructed and found to selectively inhibit MMP-9 type V collagen-based activities compared with interstitial collagen-based activities. This represents the first example of differential inhibition of collagenolytic activities and was achieved via an exosite-binding triple-helical peptide.
基质金属蛋白酶（MMP）家族的无节制活动与原发性和转移性肿瘤生长、血管生成以及细胞外基质成分（如胶原蛋白和层粘连蛋白）的病理性降解有关。然而，小分子MMP抑制剂的临床试验大多未成功，缺乏选择性被认为是特别棘手的问题。利用MMP家族内底物二级结合位点（外位点）的差异可以实现更高的选择性。在本研究中，三螺旋底物和三螺旋过渡态类似物抑制剂已被用于剖析潜在外位点在MMP-9胶原溶解行为中的作用。底物和抑制剂序列基于α1（V）436-450胶原区域，该区域在Gly（向下箭头）Val键处被MMP-2和MMP-9选择性水解，或者基于共有间隙胶原序列α1（I-III）769-783内的Gly（向下箭头）Leu切割位点，该位点被MMP-1、MMP-2、MMP-8、MMP-9、MMP-13和MT1-MMP水解。发现MMP-9纤连蛋白II插入片段内的外位点对于与V型胶原模型底物和抑制剂的相互作用至关重要，并参与与间隙（I-III型）胶原模型抑制剂的相互作用。构建了一种包含纤连蛋白II插入片段结合序列的三螺旋肽，发现与基于间隙胶原的活性相比，它能选择性抑制基于V型胶原的MMP-9活性。这代表了胶原溶解活性差异抑制的首个实例，并且是通过外位点结合三螺旋肽实现的。
