Background

SHP2 (Src homology 2 containing protein tyrosine phosphatase2) is a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. SHP2 plays an important role as a phosphatase in maintaining the homeostasis of protein tyrosine phosphorylation and is involved in the regulation of several signaling pathways (RAS/RAF/MEK/ERK, PI3K/AKT and JAK/STAT). Due to its involvement in the regulation of several important signaling pathways, the over-activation of SHP2 is associated with a variety of cancers. Therefore, it has become an important target for cancer drug development.

SHP2 contains two SH2 structural domains (N-SH2, C-SH2), a highly conserved PTP catalytic domain and a C-terminal tail region containing 2 tyrosine phosphorylation sites (Tyr542 and Tyr580). In the non-activated state, N-SH2 of SHP2 binds to the PTP structural domain to maintain the autoinhibited state of phosphatase activity; when SH2 binds to the phosphorylated tyrosine residues, the protein conformation is changed and the autoinhibited state is lifted to perform the function of phosphatase to dephosphorylate the target protein.

As a potential target for cancer therapy, the development of SHP2 inhibitors has become one of the popular areas for antitumor drug development. The traditional idea of SHP2 inhibitor design is to target its orthosteric site (PTP catalytic active center), however, due to PTP is high conserved, the small molecule inhibitors designed for the catalytic structural domain of PTP have low selectivity. Another idea is to develop allosteric inhibitors, and SHP099, developed by Novartis in 2016, is the first allosteric inhibitors for the SHP2 target. SHP099 binds to a tunnel-like allosteric pocket at the junction of the C-SH2, N-SH2 and PTP structural domains, which maintains SHP2 in an autoinhibited conformation and inhibits catalytic activity.

Results

1. Protein expression & purification

The expression system used in this study was the E. coli expression system (BL21), and then Ni-NTA column was used for protein purification followed by gel filtration chromatography.

Fig. 1 Results of gel filtration chromatography and SDS-PAGE

2. Protein crystallization and diffraction data collection

In this experiment, the protein crystals were crystallized by the sitting-drop method, and diffraction data information was collected by synchrotron radiation source.

Fig. 2 SHP099-SHP2 eutectic and diffraction image

3. Structure determination

Determination of the three-dimensional structure of the SHP099-SHP2 complex with a resolution of 1.9 Å.

Fig. 3 Three-dimensional structure of SHP099-SHP2 complex