Original articles
Yihui Song, Min Zhao, Yahong Wu, Bin Yu, Hong-Min Liu. A multifunctional cross-validation high-throughput screening protocol enabling the discovery of new SHP2 inhibitors[J]. Acta Pharmaceutica Sinica B, 2021, 11(3): 750-762

A multifunctional cross-validation high-throughput screening protocol enabling the discovery of new SHP2 inhibitors
Yihui Songa, Min Zhaoa, Yahong Wub, Bin Yua, Hong-Min Liua
a School of Pharmaceutical Sciences & Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education, Zhengzhou University, Zhengzhou 450001, China;
b School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China
Abstract:
The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2 (SHP2) is implicated in various cancers, and targeting SHP2 has become a promising therapeutic approach. We herein described a robust cross-validation high-throughput screening protocol that combined the fluorescence-based enzyme assay and the conformation-dependent thermal shift assay for the discovery of SHP2 inhibitors. The established method can effectively exclude the false positive SHP2 inhibitors with fluorescence interference and was also successfully employed to identify new protein tyrosine phosphatase domain of SHP2 (SHP2-PTP) and allosteric inhibitors. Of note, this protocol showed potential for identifying SHP2 inhibitors against cancer-associated SHP2 mutation SHP2-E76A. After initial screening of our in-house compound library (~2300 compounds), we identified 4 new SHP2-PTP inhibitors (0.17% hit rate) and 28 novel allosteric SHP2 inhibitors (1.22% hit rate), of which SYK-85 and WS-635 effectively inhibited SHP2-PTP (SYK-85: IC50 = 0.32 μmol/L; WS-635: IC50 = 4.13 μmol/L) and thus represent novel scaffolds for designing new SHP2-PTP inhibitors. TK-147, an allosteric inhibitor, inhibited SHP2 potently (IC50 = 0.25 μmol/L). In structure, TK-147 could be regarded as a bioisostere of the well characterized SHP2 inhibitor SHP-099, highlighting the essential structural elements for allosteric inhibition of SHP2. The principle underlying the cross-validation protocol is potentially feasible to identify allosteric inhibitors or those inactivating mutants of other proteins.
Key words:    SHP2    High-throughput screening    Enzyme assay    Thermal shift assay    Allosteric inhibitors   
Received: 2020-05-26     Revised: 2020-08-01
DOI: 10.1016/j.apsb.2020.10.021
Funds: We sincerely acknowledge the financial support from the National Natural Science Foundation of China (Nos. 31900875, 81773562, 81973177, and 81703326), Program for Science & Technology Innovation Talents in Universities of Henan Province (No. 21HASTIT045, China), China Postdoctoral Science Foundation (Nos. 2019M662518, 2018M630840, and 2019T120641, China), and Postdoctoral Starting Foundation of Henan Province (No. 201903007, China). We thank Prof. Stephen C. Blacklow at Department of Biological Chemistry & Molecular Pharmacology of Harvard Medical School (Boston, MA, USA) for providing useful suggestions in designing the enzyme assay, and Kai Tang, Yunkai Shi, and Shuai Wang (Zhengzhou University, China) for providing the compound library for screening.
Corresponding author: Bin Yu, Hong-Min Liu     Email:yubin@zzu.edu.cn;liuhm@zzu.edu.cn
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Yihui Song
Min Zhao
Yahong Wu
Bin Yu
Hong-Min Liu

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