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
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
Author description:
PDF(KB) Free
Yihui Song
Min Zhao
Yahong Wu
Bin Yu
Hong-Min Liu

1. Chan RJ, Feng GS. PTPN11 is the first identified proto-oncogene that encodes a tyrosine phosphatase. Blood 2007;109:862-7.
2. Ostman A, Hellberg C, Bohmer FD. Protein-tyrosine phosphatases and cancer. Nat Rev Canc 2006;6:307-20.
3. Yang W, Klaman LD, Chen B, Araki T, Harada H, Thomas SM, et al. An Shp2/SFK/Ras/Erk signaling pathway controls trophoblast stem cell survival. Dev Cell 2006;10:317-27.
4. Liu Q, Qu J, Zhao M, Xu Q, Sun Y. Targeting SHP2 as a promising strategy for cancer immunotherapy. Pharmacol Res 2020;152:104595.
5. Marasco M, Berteotti A, Weyershaeuser J, Thorausch N, Sikorska J, Krausze J, et al. Molecular mechanism of SHP2 activation by PD-1 stimulation. Sci Adv 2020;6:4458.
6. Hof P, Pluskey S, Dhe-Paganon S, Eck MJ, Shoelson SE. Crystal structure of the tyrosine phosphatase SHP-2. Cell 1998;92:441-50.
7. Pádua RAP, Sun Y, Marko I, Pitsawong W, Stiller JB, Otten R, et al. Mechanism of activating mutations and allosteric drug inhibition of the phosphatase SHP2. Nat Commun 2018;9:4507.
8. Grossmann KS, Rosário M, Birchmeier C, Birchmeier W. The tyrosine phosphatase Shp2 in development and cancer. Adv Canc Res 2010; 106:53-89.
9. Bentires-Alj M, Paez JG, David FS, Keilhack H, Halmos B, Naoki K, et al. Activating mutations of the noonan syndrome-associated SHP2/PTPN11 gene in human solid tumors and adult acute myelogenous leukemia. Canc Res 2004;64:8816-20.
10. Xie J, Si X, Gu S, Wang M, Shen J, Li H, et al. Allosteric inhibitors of SHP2 with therapeutic potential for cancer treatment. J Med Chem 2017;60:10205-19.
11. Fodor M, Price E, Wang P, Lu H, Argintaru A, Chen Z, et al. Dual allosteric inhibition of SHP2 phosphatase. ACS Chem Biol 2018;13: 647-56.
12. Tang K, Jia YN, Yu B, Liu HM. Medicinal chemistry strategies for the development of protein tyrosine phosphatase SHP2 inhibitors and PROTACs degraders. Eur J Med Chem 2020;204:112657.
13. Song ZD, Wang MJ, Ge Y, Chen XP, Xu ZY, Sun Y, et al. Tyrosine phosphatase SHP2 inhibitors in tumor-targeted therapies. Acta Pharm Sin B 2021;11:13-29.
14. Bagdanoff JT, Chen Z, Acker M, Chen YN, Chan H, Dore M, et al. Optimization of fused bicyclic allosteric SHP2 inhibitors. J Med Chem 2019;62:1781-92.
15. Wu X, Xu G, Li X, Xu W, Li Q, Liu W, et al. Small molecule inhibitor that stabilizes the autoinhibited conformation of the oncogenic tyrosine phosphatase SHP2. J Med Chem 2019;62:1125-37.
16. Stanford SM, Bottini N. Targeting tyrosine phosphatases: time to end the stigma. Trends Pharmacol Sci 2017;38:524-40.
17. Chen YF, Fu LW. Mechanisms of acquired resistance to tyrosine kinase inhibitors. Acta Pharm Sin B 2011;1:197-207.
18. Chen YN, LaMarche MJ, Chan HM, Fekkes P, Garcia-Fortanet J, Acker MG, et al. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases. Nature 2016;535: 148-52.
19. Ruess DA, Heynen GJ, Ciecielski KJ, Ai J, Berninger A, Kabacaoglu D, et al. Mutant KRAS-driven cancers depend on PTPN11/SHP2 phosphatase. Nat Med 2018;24:954-60.
20. Dardaei L, Wang HQ, Singh M, Fordjour P, Shaw KX, Yoda S, et al. SHP2 inhibition restores sensitivity in ALK-rearranged non-small-cell lung cancer resistant to ALK inhibitors. Nat Med 2018;24:512-7.
21. Zhao M, Guo W, Wu Y, Yang C, Zhong L, Deng G, et al. SHP2 inhibition triggers anti-tumor immunity and synergizes with PD-1 blockade. Acta Pharm Sin B 2019;9:304-15.
22. Wang M, Lu J, Wang M, Yang CY, Wang S. Discovery of SHP2-D26 as a first, potent, and effective PROTAC degrader of SHP2 protein. J Med Chem 2020;63:7510-28.
23. Nichols RJ, Haderk F, Stahlhut C, Schulze CJ, Hemmati G, Wildes D, et al. RAS nucleotide cycling underlies the SHP2 phosphatase dependence of mutant BRAF-, NF1- and RAS-driven cancers. Nat Cell Biol 2018;20:1064-73.
24. LaRochelle JR, Fodor M, Xu X, Durzynska I, Fan L, Stams T, et al. Structural and functional consequences of three cancer-associated mutations of the oncogenic phosphatase SHP2. Biochemistry 2016; 55:2269-77.
25. Walters WP, Namchuk M. Designing screens: how to make your hits a hit. Nat Rev Drug Discov 2003;2:259-66.
26. Copeland RA. Evaluation of enzyme inhibitors in drug discovery: a guide for medicinal chemists and pharmacologists. 2nd ed. New Jersey: John Wiley & Sons, Inc.; 2013.
27. Renaud JP, Chung CW, Danielson UH, Egner U, Hennig M, Hubbard RE, et al. Biophysics in drug discovery: impact, challenges and opportunities. Nat Rev Drug Discov 2016;15:679-98.
28. Lo MC, Aulabaugh A, Jin G, Cowling R, Bard J, Malamas M, et al. Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery. Anal Biochem 2004;332:153-9.
29. Lucet IS, Hildebrand JM, Czabotar PE, Zhang JG, Nicola NA, Silke J, et al. Determination of pseudokinase-ligand interaction by a fluorescence-based thermal shift assay. Bio-protocol 2014;4:1135.
30. Niesen FH, Berglund H, Vedadi M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2007;2:2212-21.
31. Irving E, Stoker AW. Vanadium compounds as PTP inhibitors. Molecules 2017;22:2269.
32. LaRochelle JR, Fodor M, Vemulapalli V, Mohseni M, Wang P, Stams T, et al. Structural reorganization of SHP2 by oncogenic mutations and implications for oncoprotein resistance to allosteric inhibition. Nat Commun 2018;9:4508.
33. Yuan S, Yu B, Liu HM. Palladium-catalyzed ‘on-water’ tandem cyclization reactions for the synthesis of biologically important 4-arylquinazolines. Chem Eur J 2019;25:13109-13.
34. Yuan S, Chang J, Yu B. Construction of biologically important biaryl scaffolds through direct C—H bond activation: advances and prospects. Top Curr Chem 2020;378:23.
35. Yuan S, Wang S, Zhao M, Zhang D, Chen J, Li JX, et al. Brønsted acid-promoted ‘on-water’ C(sp3)-H functionalization for the synthesis of isoindolinone/[1,2,4]triazolo[1,5-a]pyrimidine derivatives targeting the SKP2eCKS1 interaction. Chin Chem Lett 2020;31:349-52.
36. Yuan S, Yu B, Liu HM. Brønsted acid-catalyzed direct C(sp2)-H heteroarylation enabling the synthesis of structurally diverse biaryl derivatives. Adv Synth Catal 2019;361:59-66.
37. Welte S, Baringhaus KH, Schmider W, Muller G, Petry S, Tennagels N. 6,8-Difluoro-4-methylumbiliferyl phosphate: a fluorogenic substrate for protein tyrosine phosphatases. Anal Biochem 2005; 338:32-8.
38. Vazhappilly CG, Saleh E, Ramadan W, Menon V, Al-Azawi AM, Tarazi H, et al. Inhibition of SHP2 by new compounds induces differential effects on RAS/RAF/ERK and PI3K/AKT pathways in different cancer cell types. Invest New Drugs 2019;37:252-61.
39. Chen C, Xue T, Fan P, Meng L, Wei J, Luo D. Cytotoxic activity of Shp2 inhibitor fumosorinone in human cancer cells. Oncol Lett 2018; 15:10055-62.
Similar articles: