Zhang Huiya, Xing Yuyu, Yang Yixuan, Tang Bixi, Zong Zhaoyun, Li Jia, Zang Yi, Bogyo Matthew, Lu Xiaojie, Chen Shiyu
Biotech Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China.
University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing, 100049, China.
Chembiochem. 2024 Dec 2;25(23):e202400559. doi: 10.1002/cbic.202400559. Epub 2024 Nov 4.
Profiling the substrate sequence preferences of proteases is important for understanding both biological functions as well as for designing protease inhibitors. Several methods are available for profiling the sequence specificity of proteases. However, there is currently no rapid and high-throughput method to profile specificity of proteases for noncanonical substrates. In this study, we described a strategy to use a DNA-encoded noncanonical substrate library to identify the protease substrates composed of both canonical and noncanonical amino acids. This approach uses a DNA-encoded peptide library and introduces a biotin molecule at the N-terminus to immobilize the library on a solid support. Upon protease hydrolysis, the released DNA tag of the substrate peptides can be sequenced to identify the substrate structures. Using this approach, we profiled trypsin and fibroblast activation protein α and discovered noncanonical substrates that were more efficiently cleaved than the commonly used substrates. The identified substrates of FAP were further used to design corresponding covalent inhibitors containing non-canonical sequences with high potency for the target protease. Overall, our approach can aid in the development of new protease substrates and inhibitors.
剖析蛋白酶的底物序列偏好对于理解生物学功能以及设计蛋白酶抑制剂都很重要。有几种方法可用于剖析蛋白酶的序列特异性。然而，目前尚无快速且高通量的方法来剖析蛋白酶对非经典底物的特异性。在本研究中，我们描述了一种使用DNA编码的非经典底物文库来鉴定由经典和非经典氨基酸组成的蛋白酶底物的策略。这种方法使用DNA编码的肽文库，并在N端引入生物素分子以将文库固定在固体支持物上。蛋白酶水解后，可对底物肽释放的DNA标签进行测序以鉴定底物结构。使用这种方法，我们剖析了胰蛋白酶和成纤维细胞激活蛋白α，并发现了比常用底物更易被切割的非经典底物。所鉴定的FAP底物进一步用于设计相应的共价抑制剂，这些抑制剂含有对靶蛋白酶具有高效力的非经典序列。总体而言，我们的方法有助于开发新的蛋白酶底物和抑制剂。
