Improving the Selectivity of Engineered Protease Inhibitors: Optimizing the P2 Prime Residue Using a Versatile Cyclic Peptide Library
Improving the Selectivity of Engineered Protease Inhibitors: Optimizing the P2 Prime Residue Using a Versatile Cyclic Peptide Library
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DOI：
10.1021/acs.jmedchem.5b01148
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发表时间：
2015-10-22
影响因子：
7.3
通讯作者：
Swedberg, Joakim E.
中科院分区：
文献类型：
--
作者：
de Veer, Simon J.;Wang, Conan K.;Swedberg, Joakim E.
标准机制抑制剂是工程可逆丝氨酸蛋白酶抑制剂的有吸引力的设计模板。当优化抑制剂和靶蛋白酶之间的相互作用时，许多研究集中在抑制剂结合环的非引发片段（包括接触β链）。然而，目前很少有用于筛选引物片段上的残基的方法。在这里，我们设计了一个合成的抑制剂库（基于向日葵胰蛋白酶I），用于表征各种丝氨酸蛋白酶的P2'特异性。针对13种不同蛋白酶筛选文库揭示了对胰蛋白酶、糜蛋白酶、间质蛋白酶、纤溶酶、凝血酶、四种激肽释放酶相关肽酶和几种凝血因子的独特P2'偏好。使用这些信息来修饰现有的工程化抑制剂产生了新的变体，这些变体显示出显著改善的选择性，达到了某些脱靶蛋白酶的7000倍选择性。我们的研究证明了P2'残基在标准机制抑制中的重要性，并揭示了一种筛选P2'取代的新方法，这将有利于未来的抑制剂工程研究。
Standard mechanism inhibitors are attractive design templates for engineering reversible serine protease inhibitors. When optimizing interactions between the inhibitor and target protease, many studies focus on the nonprimed segment of the inhibitor's binding loop (encompassing the contact beta-strand). However, there are currently few methods for screening residues on the primed segment. Here, we designed a synthetic inhibitor library (based on sunflower trypsin inhibitorI) for characterizing the P2' specificity of various serine proteases. Screening the library against 13 different proteases revealed unique P2' preferences for trypsin, ch-ymotrypsin, matriptase, plasmin, thrombin, four kallikrein-related peptidases, and several clotting factors. Using this information to modify existing engineered inhibitors yielded new variants that showed considerably improved selectivity, reaching up to 7000-fold selectivity over certain off-target proteases. Our study demonstrates the importance of the P2' residue in standard mechanism inhibition and unveils a new approach for screening P2' substitutions that will benefit future inhibitor engineering studies.