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膜型1基质金属蛋白酶(Membrane type-1 matrix metalloproteinase,MT1-MMP,MMP14)在肿瘤的发生发展及转移中起着重要作用,是肿瘤潜在理想的药物靶标。为了获得MT1-MMP结合肽,我们首先采用生物信息学方法分析MMPs序列,获得MT1-MMP差异大且特异的序列。以此为正义肽靶标,设计反义肽库,然后通过分子对接、分子动力学模拟以及体外细胞实验等多种方法,进行靶向MT1-MMP反义肽的筛选与活性研究。多序列比对确定了位于MT1-MMP环区的特异序列AYIREGHE(简称MT1-loop),并构建1 536条反义肽。经两轮虚拟筛选,选取打分位于前五的反义肽用于后续研究。该五条反义肽与MT1-MMP存在较强的相互作用且能很好地对接于正义肽区域。进一步分析其与MMPs其他家族成员(MMP1-3,MMP7-13,MMP14HPX,MMP16)的亲和力,发现反义肽FVTFPYIR对MT1-MMP具有更强的特异性。分子动力学模拟表明,反义肽FVTFPYIR可能是通过影响受体MT1-MMP的构象稳定性,进而影响其功能活性。体外细胞实验初步确定反义肽FVTFPYIR可选择性地抑制表达MT1-MMP的人成骨肉瘤细胞MG63和乳腺癌MDA-MB-231细胞的增殖。本研究为抗肿瘤反义肽先导药物的研发提供了一种新的思路与途径。
Membrane type 1 matrix metalloproteinase (MT1-MMP, MMP14) plays an important role in tumor development and metastasis, and is a potential drug target for tumors. In order to obtain MT1-MMP binding peptides, we first analyzed the sequences of MMPs using bioinformatics methods to obtain large and specific MT1-MMP sequences. As the target of the sense peptide, the antisense peptide library was designed. Then, the screening and the activity study of targeting MT1-MMP antisense peptide were carried out by a variety of methods such as molecular docking, molecular dynamics simulation and in vitro cell experiment. Multiple sequence alignments identified a specific sequence AYIREGHE (MT1-loop) located in the MT1-MMP loop region and constructed 1 536 antisense peptides. After two rounds of virtual screening, the top five antisense peptides were selected for further study. The five antisense peptides have strong interaction with MT1-MMP and can dock well with the region of the positive peptide. Further analysis of its affinity with other members of the MMPs (MMP1-3, MMP7-13, MMP14HPX, MMP16), antisense peptide FVTFPYIR found more specific MT1-MMP. Molecular dynamics simulations showed that the antisense peptide FVTFPYIR may affect its functional activity by affecting the conformational stability of MT1-MMP. In vitro experiments confirmed that antisense FVTFPYIR could selectively inhibit the proliferation of human osteosarcoma cells MG63 and breast cancer MDA-MB-231 cells expressing MT1-MMP. This study provides a new way of thinking for the development of anti-tumor antisense peptide leader.