Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries
Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries
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DOI：
10.1073/pnas.140132697
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发表时间：
2000-07-05
影响因子：
11.1
通讯作者：
Craik, CS
中科院分区：
文献类型：
--
作者：
Harris, JL;Backes, BJ;Craik, CS
本发明提供了一种制备和使用荧光肽底物的方法，该方法允许构造通用底物文库以快速鉴定蛋白酶的主要和扩展特异性。底物含有荧光离去基团7-氨基-4-氨基甲酰基甲基香豆素（ACC）。结合ACC离去基团的底物显示出与传统使用的7-氨基-4-甲基香豆素（AMC）离去基团的底物相当的动力学曲线。ACC的双功能性质允许通过使用基于9-芴甲氧羰基（Fmoc）的固相合成技术有效地生产单一底物和底物库。ACC的量子产率比AMC增加约3倍，允许酶和底物浓度降低。因此，在单个测定中可以耐受更大数量的底物，从而能够增加文库的多样性空间。构建了分别在P4-P3-P2-P1和P4-P3-P2位置具有氨基酸多样性的137，180和6，859个成员的可溶性定位蛋白酶底物文库。采用这种筛选方法，我们分析了底物特异性的各种蛋白酶，包括丝氨酸蛋白酶凝血酶，纤溶酶，因子Xa，尿激酶型纤溶酶原激活剂，组织纤溶酶原激活剂，颗粒酶B，胰蛋白酶，胰凝乳蛋白酶，人中性粒细胞弹性蛋白酶，和半胱氨酸蛋白酶木瓜蛋白酶和cruzain。由此产生的配置文件创建一个pharmacophoric描绘的蛋白酶，以帮助设计的选择性底物和有效的抑制剂。
A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors.