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. 2008 May 20;105(20):7275-80.
doi: 10.1073/pnas.0710468105. Epub 2008 May 14.

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

Klaus Hellmuth  1 , Stefanie Grosskopf, Ching Tung Lum, Martin Würtele, Nadine Röder, Jens Peter von Kries, Marta Rosario, Jörg Rademann, Walter Birchmeier
Affiliations

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

Klaus Hellmuth et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
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.
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.
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.
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).
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