A cellular target engagement assay for the characterization of SHP2 (PTPN11) phosphatase inhibitors

J Biol Chem. 2020 Feb 28;295(9):2601-2613. doi: 10.1074/jbc.RA119.010838. Epub 2020 Jan 17.


The nonreceptor protein-tyrosine phosphatase (PTP) SHP2 is encoded by the proto-oncogene PTPN11 and is a ubiquitously expressed key regulator of cell signaling, acting on a number of cellular processes and components, including the Ras/Raf/Erk, PI3K/Akt, and JAK/STAT pathways and immune checkpoint receptors. Aberrant SHP2 activity has been implicated in all phases of tumor initiation, progression, and metastasis. Gain-of-function PTPN11 mutations drive oncogenesis in several leukemias and cause developmental disorders with increased risk of malignancy such as Noonan syndrome. Until recently, small molecule-based targeting of SHP2 was hampered by the failure of orthosteric active-site inhibitors to achieve selectivity and potency within a useful therapeutic window. However, new SHP2 allosteric inhibitors with excellent potency and selectivity have sparked renewed interest in the selective targeting of SHP2 and other PTP family members. Crucially, drug discovery campaigns focusing on SHP2 would greatly benefit from the ability to validate the cellular target engagement of candidate inhibitors. Here, we report a cellular thermal shift assay that reliably detects target engagement of SHP2 inhibitors. Using this assay, based on the DiscoverX InCell Pulse enzyme complementation technology, we characterized the binding of several SHP2 allosteric inhibitors in intact cells. Moreover, we demonstrate the robustness and reliability of a 384-well miniaturized version of the assay for the screening of SHP2 inhibitors targeting either WT SHP2 or its oncogenic E76K variant. Finally, we provide an example of the assay's ability to identify and characterize novel compounds with specific cellular potency for either WT or mutant SHP2.

Keywords: PTPN11; SHP2; allosteric inhibitors; anticancer drug; cellular target engagement; cellular thermal shift assay; drug discovery; protein drug interaction; small molecule; tyrosine-protein phosphatase (tyrosine phosphatase).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carcinogenesis / genetics
  • Cell Line
  • Drug Discovery / methods*
  • Enzyme Inhibitors / metabolism*
  • Gain of Function Mutation
  • Humans
  • Protein Binding
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / antagonists & inhibitors*
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / genetics
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / metabolism
  • Proto-Oncogene Mas


  • Enzyme Inhibitors
  • MAS1 protein, human
  • Proto-Oncogene Mas
  • PTPN11 protein, human
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11

Associated data

  • PDB/5EHR