Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity
Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity
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DOI：
10.1042/bj20030582
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发表时间：
2004-02-01
影响因子：
4.1
通讯作者：
Matrisian, LM
中科院分区：
文献类型：
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作者：
McIntyre, JO;Fingleton, B;Matrisian, LM
本研究描述了使用一种新的基于聚合物的荧光底物PB-M7VIS对肿瘤相关的基质金属蛋白酶-7(基质金属蛋白酶-7或基质溶素)活性进行体内检测和成像，PB-M7vis作为这种金属蛋白酶的选择性‘蛋白分解灯塔’(PB)。PB-M7vis构建在14.2 kDa的PAMAM(聚酰胺氨基)树枝状大分子核心上，与F1(荧光素)标记的肽F1(AHX)RPLALWRS(AHX)C(其中AHX代表氨基己酸)和TMR(四甲基罗丹明)共价偶联。PB-M7vis可被基质金属蛋白酶-7选择性地切割，其k(CAT)/K-m值为1.9×10(5)M-1。S(-1)，以F1荧光增加速度(优化的PB-M7VIS裂解高达17倍)衡量，TMR荧光变化最小。PB-M7vis的K-m值约为。0.5微米，这大约是。与类似的可溶性多肽相比低了两个数量级，这表明基质金属蛋白酶-7与合成的聚合物底物有效地相互作用。对于基质金属蛋白酶-2或-3，PB-M7vis的k(Cat)/K-m值约为。与基质金属蛋白酶-7相比，分别低56倍和13倍。在PB-M7vis中，F1(AHX)RPLALWRS(AHX)C是一种选择性的基质金属蛋白酶-7活性光学传感器，TMR用于检测未裂解和裂解的试剂。其中每一个都可以在小鼠身上可视化为皮下荧光模体，并根据绿色/红色(F1/TMR)荧光的比率进行光学区分。在小鼠异种移植模型中检测PB-M7vis的体内特异性。静脉注射PB-M7vis可显著增强基质金属蛋白酶-7阳性肿瘤的F1荧光，但不能增强对照肿瘤的F1荧光(P<；0.0001)，这两种肿瘤最初都来自SW480人结肠癌细胞。预先用基质金属蛋白酶抑制剂BB-94{[4-(N-hydroxyamino)-2R-isobutyl-3S-(thienylthiomethyl)-succinyl]-L-phenylatanine-N-methylamide}对荷瘤小鼠进行系统治疗，可显著降低基质金属蛋白酶-7阳性转换率上的F1荧光约1倍。60%。因此，PB-M7vis起到PB的作用，用于体内检测基质金属蛋白酶-7的活性，该活性用于点燃这一光学信标，因此是一种选择性的体内光学分子成像造影剂。
The present study describes the in vivo detection and imaging of tumour-associated MMP-7 (matrix metalloproteinase-7 or matrilysin) activity using a novel polymer-based fluorogenic substrate PB-M7vis, which serves as a selective 'proteolytic beacon' (PB) for this metalloproteinase. PB-M7vis is built on a PAMAM (polyamido amino) dendrimer core of 14.2 kDa, covalently coupled with an F1 (fluorescein)-labelled peptide F1(AHX) RPLALWRS(AHX)C (where AHX stands for aminohexanoic acid) and with TMR (tetramethylrhodamine). PB-M7vis is efficiently and selectively cleaved by MMP-7 with a k(cat)/K-m value of 1.9 x 10(5) M-1 . s(-1) as measured by the rate of increase in F1 fluorescence (up to 17-fold for the cleavage of an optimized PB-M7VIS) with minimal change in the TMR fluorescence. The K-m value for PB-M7vis is approx. 0.5 muM, which is approx. two orders of magnitude lower when compared with that for an analogous soluble peptide, indicating efficient interaction of MMP-7 with the synthetic polymeric substrate. With MMP-2 or -3, the k(cat)/K-m value for PB-M7vis is approx. 56- or 13-fold lower respectively, when compared with MMP-7. In PB-M7vis, F1(AHX)RPLALWRS(AHX)C is a selective optical sensor of MMP-7 activity and TMR serves to detect both the uncleaved and cleaved reagents. Each of these can be visualized as subcutaneous fluorescent phantoms in a mouse and optically discriminated based on the ratio of green/red (F1/TMR) fluorescence. The in vivo specificity of PB-M7vis was tested in a mouse xenograft model. Intravenous administration of PB-M7vis gave significantly enhanced F1 fluorescence from MMP-7-positive tumours, but not from control tumours (P < 0.0001), both originally derived from SW480 human colon cancer cells. Prior systemic treatment of the tumour-bearing mice with an MMP inhibitor BB-94 {[4-(N-hydroxyamino)-2R-isobutyl-3S-(thienylthiomethyl)-succinyl]-L-phenylatanine-N-methylamide} markedly decreased the F1 fluorescence over the MMP-7-positive turnout by approx. 60%. Thus PB-M7vis functions as a PB for in vivo detection of MMP-7 activity that serves to light this optical beacon and is, therefore, a selective in vivo optical molecular imaging contrast reagent.