Activating mutations in (encoding SHP2), a protein tyrosine phosphatase (PTP) that

Activating mutations in (encoding SHP2), a protein tyrosine phosphatase (PTP) that plays an overall positive role in growth factor and cytokine signaling, are directly associated with the pathogenesis of Noonan syndrome and childhood leukemias. 50% of patients with Noonan syndrome, as well as in 35% of juvenile myelomonocytic leukemia (JMML), 10% of myelodysplastic syndrome, 7% of B cell acute lymphoblastic leukemia/lymphoma, and 4% of acute myeloid leukemia cases (9C13). Additionally, activating mutations of have been identified in sporadic solid tumors (14). Such mutations disrupt the inhibitory intramolecular interaction of SHP2, leading to gain-of-function by allowing constitutive access to the phosphatase catalytic site on the enzyme (12, 15). mutations and other JMML-associated gain-of-function buy Amyloid b-Peptide (1-43) (human) buy Amyloid b-Peptide (1-43) (human) mutations are sufficient to induce Noonan syndrome, JMML-like myeloproliferative disease, and acute leukemias in mice (18C23), suggesting that the mutations play a causal role in the development of these diseases. The direct connection between hyperactivation of SHP2 and human diseases points to SHP2 as a potential target for mechanism-based therapeutics. Selective and potent SHP2 inhibitors are needed. Discovery of SHP2-specific inhibitors would not only facilitate research on SHP2 signaling in model systems but could also buy Amyloid b-Peptide (1-43) (human) lead to the development of new drugs for mutated mouse and human leukemia cells, providing evidence buy Amyloid b-Peptide (1-43) (human) that SHP2 is a druggable target for the treatment of Phosphatase Activity Assay Glutathione S-transferase (GST) fusion proteins of SHP2 purified in-house were used as the enzyme and a phosphopeptide corresponding to the surrounding sequence of pTyr1146 in the insulin receptor (Thr-Arg-Asp-Ile-Tyr[PO3H2]-Glu-Thr-Asp-Tyr-Tyr) was used as the substrate. The assay determines free phosphate generated by dephosphorylation of the substrate using the Malachite Green reagent (Sigma, St. Louis, MO). Briefly, 0.5 g of GST-SHP2 PTP was incubated in 40 L assay buffer (25 mM Tris-HCl, pH 7.4, 50 mM NaCl, 5 mM DTT, and 2.5 mM EDTA) with test compounds at various concentrations at room temperature for 30 min. The substrate was then added to a final concentration of 0.2 mM. The system was incubated at buy Amyloid b-Peptide (1-43) (human) 30C for 30 min. Finally, 50 L of Malachite Green solution was added and OD620 was measured after 10 min. The protocols for the phosphatase assays for SHP1, CD45, LAR, MEG2, and TC-PTP were similar, with the exception that GST-SHP1, GST-CD45 cytoplasmic domain, GST-LAR, GST-MEG2, and GST-TC-PTP enzymes purchased from Biomol International, L.P. (Plymouth Meeting, PA), were used in the respective assays. For IC50 determinations, 5 concentrations of #220C324 were tested. Each experiment was performed in triplicate. Fluorescence Titrations For all experiments, purified SHP2 PTP domain GST-fusion protein was diluted into 20 mM Tris-HCl, pH 7.5. Fluorescence spectra were recorded with a Luminescence Spectrometer LS50 (Perkin-Elmer, Boston, MA). Titrations were performed by increasing the test compound concentrations while maintaining the SHP2 protein concentration at 3 M. Contributions from background fluorescence of the inhibitor were accounted for by subtracting the fluorescence of the inhibitor alone Mouse monoclonal to BLK from the protein-inhibitor solution. The excitation wavelength was 295 nm and fluorescence was monitored from 360 to 500 nm. All reported fluorescence intensities were relative values and were not corrected for wavelength variations in detector response. Cell Proliferation Assay Ba/F3 cells and mouse embryonic fibroblasts (MEFs) were seeded into 96-well plates at a density of 1104 cells/well (Ba/F3) and 5103 cells/well (MEFs) in RPMI-1640 containing 10% FBS plus recombinant mouse IL-3 (1.0 ng/mL) and DMEM containing 10% FBS, respectively. Cells were grown overnight and then treated with either test compounds or the same concentrations of DMSO. Three days later, the number of viable cells was determined using a CellTiter 96? AQueous One Solution Cell Proliferation Assay kit. Western Blotting Ba/F3 cells were starved in serum and cytokine-free RPMI1640 overnight. Cells were then treated with #220C324 for 3 hrs prior to IL-3 stimulation. Stimulated cells were harvested and lysed on ice with RIPA buffer containing 50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.25% Na-deoxycholate;.