In vitro activity: NSC-87877 is a novel, potent, and cell-permeable small molecule inhibitor of SHP-1 and SHP-2 PTP (protein tyrosine phosphatase) with IC50 of 55 and 318 nM, respectively. Protein phosphorylation plays critical roles in many regulatory mechanisms controlling cell activities and thus involved in various diseases. The cellular equilibrium of phosphorylation is regulated through the actions of protein kinases and phosphatases. Therefore, these regulatory proteins have emerged as promising targets for drug development. Phosphatase activity of dual-specificity protein phosphatase 26 (DUSP26) was decreased by NSC-87877 in a dose-dependent manner. Kinetic studies with NSC-87877 and DUSP26 revealed a competitive inhibition. NSC-87877 effectively inhibited DUSP26-mediated dephosphorylation of p38, a member of mitogen-activated protein kinase (MAPK) family. Since DUSP26 is involved in survival of anaplastic thyroid cancer (ATC) cells, NSC-87877 could be a therapeutic reagent for treating ATC.

Kinase Assay: The six-His-tagged DUSP26 (1 μg) was pre-mixed with various NSC-87877 concentrations (0, 10, or 50 μM) in PTP assay buffer for 15 min at 37 °C and then further incubated in the presence of active phosphorylated p38 (10 ng) for 15 min at 37 °C. Kinase assay reactions were initiated by mixing the pre-incubated samples with kinase reaction buffer (20 mM Tris–HCl (pH 7.5), 20 mM MgCl2, 0.1 mM sodium orthovanadate, and 1 mM DTT) supplemented with 20 μM ATP/10 μCi [γ-32P]ATP and 1 μg of GST-activating transcription factor 2 (ATF2) as a substrate. After 30 min at 30 °C, reactions were terminated by addition of SDS–PAGE sample buffer and the products of kinase reactions were separated by SDS–PAGE for autoradiography. To confirm that DUSP26 does not dephosphorylate ATF2, ATF2 was 32P-labeled by incubation with p38. After incubation, samples were further incubated with or without DUSP26 for 30 min at 30 °C and then resolved by SDS–PAGE. The gels were dried and exposed to X-ray film.

Immune complex kinase assays. For the immune complex kinase assay, HEK 293 cells were co-transfected with HA-p38 and FLAG-DUSP26 expression plasmids. After 48 h of transfection, cells were pretreated with NSC-87877 (0–100 μM, 3 h) and then stimulated with H2O2 (1 mM, 30 min). Cell extracts were clarified by centrifugation, and the supernatants were immunoprecipitated with anti-HA agarose beads. The beads were washed once with the PTP lysis buffer, twice with a solution containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 2 mM DTT, and 1 mM PMSF, and then once with a solution containing 20 mM Tris–HCl (pH 7.5) and 20 mM MgCl2. The beads were then resuspended in kinase reaction buffer (20 mM Tris–HCl (pH 7.5), 20 mM MgCl2, and 1 mM DTT) containing 20 μM ATP and 0.3 μCi of [γ-32P]ATP with 1 μg of GST-ATF2 for 30 min at 30 °C. The products of kinase reactions were separated by SDS–PAGE. The gels were dried and exposed to film.

In vitro Phosphatase assays and Kinetic analysis. The activity of phosphatases was measured using the substrate 3-O-methylfluorescein Phosphate (OMFP; Sigma, St. Louis, MO) at concentrations varying with the Km of each enzyme in a 96-well microtiter plate assay based on methods described previously. The NSC-87877 (Calbiochem, San Diego, CA) and OMFP were solubilized in H2O and DMSO, respectively. All reactions were performed at a final concentration of 1% DMSO. The final incubation mixture (150 μl) was optimized for enzyme activity and comprised of 30 mM Tris–HCl (pH 7), 75 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM dithiothreitol (DTT), 0.33% bovine serum albumin (BSA) and 100 nM of PTPs. Reactions were initiated by addition of OMFP and the incubation time was 30 min at 37 °C. Fluorescence emission from product was measured with a multi-well plate reader (GENios Pro; excitation filter, 485 nm; emission filter, 535 nm). The reaction was linear over the time period of the experiment and was directly proportional to both enzyme and substrate concentration. 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, San Diego, CA). All experiments were performed in triplicate and were repeated at least three times.

Dephosphorylation assays with active phosphorylated MAPKs. The six-His-tagged DUSP26 (1 μg) was combined with active phosphorylated p38 (10 ng), ERK (10 ng), or JNK (50 ng) in PTP assay buffer (30 mM Tris–HCl (pH 7), 75 mM NaCl, 1 mM EDTA, 0.1 mM DTT, and 0.33% BSA), and incubated for 30 min at 37 °C. To determine whether NSC-87877 down-regulates DUSP26 effect on p38 in vitro, 1 μg of DUSP26 was mixed with 10 ng active phosphorylated p38 and various NSC-87877 concentrations (0, 10, or 100 μM) in a 30-μl reaction volume and incubated for 30 min at 37 °C. The samples were subjected to Western blotting analysis to examine the phosphorylation state of MAPKs using the phospho-MAPK antibodies.

Cell Assay: Cells were harvested and then lysed by sonication in 50 mM Tris–HCl (pH 8), 300 mM NaCl, 1% NP-40, and 1 mM phenylmethylsulphonyl fluoride (PMSF). The lysates were clarified at 4000 rpm for 30 min at 4 °C. The supernatant was applied by gravity flow to a column of Ni–NTA resin (PEPTRON, Daejon, Korea). The resin was washed with 20 mM Tris–HCl (pH 8), 500 mM NaCl, 50 mM imidazole and eluted with 20 mM Tris–HCl (pH 8), 500 mM NaCl, 200–300 mM imidazole. The eluted proteins were dialyzed overnight against 20 mM Tris–HCl, 100 mM NaCl, 30% glycerol, 0.5 mM PMSF before storage at –80 °C.