Specific inhibitors of the protein tyrosine phosphatase Shp2 identified by high-throughput docking

Abstract

The protein tyrosine phosphatase Shp2 is a positive regulator of growth factor signaling. Gain-of-function mutations in several types of leukemia define Shp2 as a bona fide oncogene. We performed a high-throughput in silico screen for small-molecular-weight compounds that bind the catalytic site of Shp2. We have identified the phenylhydrazonopyrazolone sulfonate PHPS1 as a potent and cell-permeable inhibitor, which is specific for Shp2 over the closely related tyrosine phosphatases Shp1 and PTP1B. PHPS1 inhibits Shp2-dependent cellular events such as hepatocyte growth factor/scatter factor (HGF/SF)-induced epithelial cell scattering and branching morphogenesis. PHPS1 also blocks Shp2-dependent downstream signaling, namely HGF/SF-induced sustained phosphorylation of the Erk1/2 MAP kinases and dephosphorylation of paxillin. Furthermore, PHPS1 efficiently inhibits activation of Erk1/2 by the leukemia-associated Shp2 mutant, Shp2-E76K, and blocks the anchorage-independent growth of a variety of human tumor cell lines. The PHPS compound class is therefore suitable for further development of therapeutics for the treatment of Shp2-dependent diseases.

Fig. 1.

PHPS1 is as an active site-directed small molecule inhibitor of Shp2. (a) Homology model of the active site of Shp2 as generated by WHAT_IF and visualized by Accelrys Viewer Lite v5.0. Selectivity-determining amino acid residues in the periphery of the active center are indicated by three-letter code; negatively charged areas are colored in red, and positively charged are in blue. Red arrow, active center. (b) Chemical structure and drug-like parameters (31) of PHPS1. (c) Fitting score values for PHPS1 in the active sites of Shp2, PTP1B, and Shp1, as generated by GOLD v2.1. (d) Ligand-interaction diagram of PHPS1 in the active site of Shp2, as generated by MOE v2006.3, modified. Blue arrows, backbone donor H bonds (Ser-460, Ala-461, Ile-463, Gly-464, Arg-465); green arrows, sidechain donor H bonds (Asn-281, Arg-362, Arg-465); purple circles, polar residues; purple/red circles, acidic residues; purple/blue circles, basic residues; light green circles, hydrophobic residues; blue spheres, ligand exposure; cyan spheres, receptor exposure.

Fig. 2.

PHPS1 inhibits HGF/SF-induced scattering and branching morphogenesis of MDCK epithelial cells. (a–c) MDCK cells were cultivated without stimulation (a) or stimulated with 1 unit/ml HGF/SF (b) or with 1 unit/ml HGF/SF and treated with 5 μM PHPS1 for 20 h (c). (d–f) MDCK cells were cultivated in a collagen gel without stimulation (d) or stimulated with 1 unit/ml HGF/SF (e) or with 1 unit/ml HGF/SF and treated with 10 μM PHPS1 (f). (g) Total cell lysates of wild-type or daSHP2 MDCK cells (stably expressing the dominant-active myr-SHP2ΔN-flag fusion protein) were immunoblotted with antibodies specific for flag epitope or Shp2, respectively. (h and i) daSHP2 MDCK cells were cultivated in a collagen gel and either untreated (h) or treated with 20 μM PHPS1 (i).

Fig. 3.

PHPS1 inhibits Shp2-dependent signaling pathways. (a) PHPS1 inhibits Erk1/2 but not Akt and Stat3 phosphorylation in a dose-dependent manner. MDCK cells were pretreated with PHPS1 and stimulated with 1 unit/ml of HGF/SF. Total cell lysates were immunoblotted with antibodies specific for phospho-Erk1/2 (P-Erk1/2), Erk1/2, phospho-Akt (P-Akt), or phospho-Stat3 (P-Stat3), respectively. (b) PHPS1 inhibits HGF/SF-induced paxillin dephosphorylation. MDCK cells were either untreated or treated with 20 μM PHPS1 and stimulated with 1 unit/ml of HGF/SF. Paxillin was immunoprecipitated from total cell lysates and immunoblotted with antibodies specific for paxillin and for phosphotyrosine. (c) PHPS1 does not inhibit Ras-V12-induced MAP kinase activation. NIH 3T3 cells were transfected with an expression vector for RasV12 and either untreated or treated with 10 μM PHPS1 or PD98059 for 2 days, respectively. Total cell lysates were immunoblotted with antibodies specific for phospho-Erk1/2 (P-Erk1/2) and Erk1/2. (d) PHPS1 does not inhibit PMA-induced MAP kinase activation. HEK293 cells were pretreated with serial dilutions of PHPS1 and stimulated with 1 unit/ml of HGF/SF or PMA, as indicated. Total cell lysates were immunoblotted with antibodies specific for phospho-Erk1/2 (P-Erk1/2) or Erk1/2. (e) PHPS1 inhibits Shp2-dependent signaling. HEK293 cells were transfected with expression vectors for HA-GAB2, HA-SHP2-E76K, or ERBB2 and either untreated or treated with 20 μM PHPS1. Total cell lysates were immunoblotted with antibodies specific for hemagglutinin epitope (HA), phospho-Erk1/2 (P-Erk1/2), and Erk1/2.

Fig. 4.

PHPS1 inhibits soft agar colony formation of human tumor cells. The indicated human cancer cell lines were grown in soft agar for 4 weeks and treated with solvent control or PHPS1 every 7 days. Representative pictures are shown (see Fig. S4b for quantification).