Mallinckrodt Institute of Radiology, Washington University, School of Medicine, St. Louis, Missouri 63110, USA.
Bioconjug Chem. 2012 Mar 21;23(3):656-63. doi: 10.1021/bc300027y. Epub 2012 Feb 29.
We report a novel activatable NIR fluorescent probe for in vivo detection of cancer-related matrix metalloproteinase (MMP) activity. The probe is based on a triple-helical peptide substrate (THP) with high specificity for MMP-2 and MMP-9 relative to other members of the MMP family. MMP-2 and MMP-9 (also known as gelatinases) are specifically associated with cancer cell invasion and cancer-related angiogenesis. At the center of each 5 kDa peptide strand is a gelatinase sensitive sequence flanked by 2 Lys residues conjugated with NIR fluorescent dyes. Upon self-assembly of the triple-helical structure, the 3 peptide chains intertwine, bringing the fluorophores into close proximity and reducing fluorescence via quenching. Upon enzymatic cleavage of the triple-helical peptide, 6 labeled peptide chains are released, resulting in an amplified fluorescent signal. The fluorescence yield of the probe increases 3.8-fold upon activation. Kinetic analysis showed a rate of LS276-THP hydrolysis by MMP-2 (k(cat)/K(M) = 30,000 s(-1) M(-1)) similar to that of MMP-2 catalysis of an analogous fluorogenic THP. Administration of LS276-THP to mice bearing a human fibrosarcoma xenografted tumor resulted in a tumor fluorescence signal more than 5-fold greater than that of muscle. This signal enhancement was reduced by treatment with the MMP inhibitor Ilomostat, indicating that the observed tumor fluorescence was indeed enzyme mediated. These results are the first to demonstrate that triple-helical peptides are suitable for highly specific in vivo detection of tumor-related MMP-2 and MMP-9 activity.
我们报道了一种新型的可激活近红外荧光探针，用于体内检测癌症相关的基质金属蛋白酶（MMP）活性。该探针基于一种对 MMP-2 和 MMP-9 具有高度特异性的三螺旋肽底物（THP），相对于 MMP 家族的其他成员而言。MMP-2 和 MMP-9（也称为明胶酶）与癌细胞浸润和癌症相关的血管生成特异性相关。在每个 5 kDa 肽链的中心是一个明胶酶敏感序列，两侧是 2 个赖氨酸残基，与近红外荧光染料缀合。在三螺旋结构自组装后，3 个肽链相互交织，使荧光团紧密靠近并通过猝灭来降低荧光。在三螺旋肽的酶切后，释放出 6 个标记的肽链，从而产生放大的荧光信号。探针的荧光产率在激活时增加了 3.8 倍。动力学分析表明，MMP-2 对 LS276-THP 的水解速度（k(cat)/K(M) = 30,000 s(-1) M(-1)）与 MMP-2 催化类似的荧光 THP 的速度相似。将 LS276-THP 施用于携带人纤维肉瘤异种移植瘤的小鼠中，导致肿瘤荧光信号比肌肉高 5 倍以上。这种信号增强被 MMP 抑制剂 Ilomostat 的处理所降低，表明观察到的肿瘤荧光确实是酶介导的。这些结果是首次表明三螺旋肽适用于高度特异性的体内检测肿瘤相关的 MMP-2 和 MMP-9 活性。
