NSC 663284 Inhibits SHP-2

Experimental Section

Cell Culture. Human embryonic kidney (HEK) 293 cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific) and penicillin/streptomycin (Life Technologies Corporation, Carlsbad, CA) in the presence of 5% CO2.

Transfection and Plasmid Constructs. HEK 293 cells were transfected by polyethylenimine (Polysciences, Inc., Warrington, PA) with 1.5 μg of FLAG-tagged SHP-2 or empty vector (Mock). FLAG-tagged SHP-2 (residues 205- 593) was constructed in pcDNA3.1 (Invitrogen, Carlsbad, CA). Bacterial expression His-tagged SHP-2 was constructed in pET28a (Novagen, Darmstadt, Germany).

Recombinant PTP Proteins. PTP expression plasmids constructed in pET-28a were transformed into BL21 (DE3)- RIL E. coli. Expression of recombinant protein was induced with 1 mM isopropyl-β-D-thiogalactopyranoside at 37 ℃ for 3 h or 30 ℃ for 6 h or 22 ℃ for overnight. Cells were harvested and then lysed by sonication in E. coli lysis buffer (50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1% Tergitol-type NP-40, 1 mM phenylmethylsulphonyl fluoride (PMSF)). After centrifuging at 10,000 rpm for 30 min at 4 ℃, the supernatant was applied by gravity flow to a column of Ni- NTA resin (PEPTRON). The resin was washed twice with washing buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 50 mM imidazole) and eluted with elution buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 200-300 mM imidazole). The recombinant proteins were dialyzed with dialysis buffer overnight (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 5 mM β-mercaptoethanol, 50% glycerol) before storage at −80 ℃.

In vitro Phosphatase Assays and Kinetic Analysis. The activity of phosphatases was measured using the substrate 3- Omethylfluorescein phosphate (OMFP; Sigma-Aldrich) in a 96-well microtiter plate assay based on methods described periviously.15 NSC 663284 and OMFP were solubilized in DMSO and H2O. All reactions were performed at a final concentration of 1% DMSO. The final incubation mixture (100 μL) were optimized for enzyme activity and composed of 30 mM Tris-HCl (pH 7), 75 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.33% bovine serum albumin (BSA), 100 nM of PTP. Reactions were initiated by addition of OMFP and incubated for 30 min at 37 ℃. Fluorescence emission from product was measured with a multi-well plate reader (GENios Pro; excitation filter, 485 nm; emission filter, 535 nm). Half-maximal inhibition constant (IC50) was defined as the concentration of an inhibitor that caused a 50% decrease in the PTP activity. Half-maximal inhibition constants and best curve fit for Lineweaver-Burk plots were determined by using the curve fitting program Prism 3.0 (GraphPad Software). The inhibition constant (Ki) to SHP-2 for NSC 663284 was calculated using the equations from the Lineweaver-Burk plots by measuring the initial rates at several OMFP concentrations for each fixed concentration of the inhibitor. The slopes showed the noncompetitive inhibition pattern and the Ki value was obtained from the below equations of noncompetitive inhibition. All experiments were performed in triplicate and were repeated at least three times.

1/V = Km(1 + [I]/Ki)/Vmax [S] + 1/Vmax (1 + [I]/Ki)

Effects of NSC 663284 on SHP-2 Purified from HEK 293 Cells. HEK 293 cells were transfected with 1.5 μg of FLAG-SHP-2 phosphatase expression plasmid. After 48 h of transfection, cells were washed twice with phosphate buffered saline (PBS) buffer and lysed in PTP lysis buffer containing 0.5% NP-40, 0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% glycerol, 1 mM PMSF for 30 min at 4 ℃. Cleared cell lysates were mixed with FLAG M2-agarose (Sigma-Aldrich, St.Louis, MO) and incubated for 1h at 4 ℃ using rotation device. FLAG M2-agarose was washed three times with PTP lysis buffer. Then, the phosphatase activities of the immunoprecipitated SHP-2 with various concentrations of NSC 663284 were measured as described above.

In vivo Effect of NSC 663284 on SHP-2 Phosphataseregulated Phospho-ERK. After HEK 293 cells were transiently transfected with or without FLAG tagged SHP-2 (residues 205-593) for 48 h, cells were treated with NSC 663284 (0, 10, 50 μM ) for 3 h. Cells were washed twice with phosphate buffered saline (PBS) buffer and lysed on ice in lysis buffer containing 150 mM NaCl, 20 mM Tris (pH 8.0) 0.5% NP-40, 0.5% Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 10 mM NaF. Samples were boiled at 100 ℃ for 5 min and run in SDS-10% polyacrylamide gels and transferred onto nitrocellulose membrane. Then the membranes were blocked with 5% nonfat skim milk for 1 h and incubated with an appropriate antibody with 5% BSA, followed by incubation with a HRP-conjugated secondary antibody with 5% skim milk. The immunoreactive bands were visualized by the ECL detection system (Pierce, Rockford, IL).