shp2 (IC50 = 0.318 μM); shp1 (IC50 = 0.355 μM)

NSC-87877 is a novel, potent, and cell-permeable small molecule inhibitor of SHP-1 and SHP-2 PTP (protein tyrosine phosphatase) that is soluble in cells. Its IC50 values are 55 and 318 nM, respectively. Protein phosphorylation is essential to many regulatory processes that govern cellular activity and, consequently, to a number of disorders. The functions of protein kinases and phosphatases control the equilibrium of phosphorylation within cells. These regulatory proteins have therefore become attractive targets for pharmaceutical development. NSC-87877 reduced the phosphotase activity of dual-specificity protein phosphatase 26 (DUSP26) in a dose-dependent way. NSC-87877 and DUSP26 kinetic studies showed a competitive inhibition. DUSP26-mediated dephosphorylation of p38, a member of the mitogen-activated protein kinase (MAPK) family, was efficiently inhibited by NSC-87877. NSC-87877 may be a therapeutic reagent for the treatment of anaplastic thyroid cancer (ATC) because DUSP26 is involved in the survival of ATC cells.

NSC-87877 treatment inhibits tumor growth and increases p53 and p38 activity in an intrarenal mouse model of NB. NSC-87877's DUSP26 inhibition works well to activate the p53 and p38 mitogen-activated protein kinase (MAPK) tumor-suppressor pathways, which in turn causes NB cell cytotoxicity both in vitro and in vivo.

Prior to being further incubated with active phosphorylated p38 (10 ng) for 15 min at 37 °C, the six-His-tagged DUSP26 (1 μg) was pre-mixed with various NSC-87877 concentrations (0, 10, or 50 μM) in PTP assay buffer. Beginning with the pre-incubated samples, kinase reaction buffer (20 mM Tris–HCl (pH 7.5), 20 mM MgCl2, 0.1 mM sodium orthovanadate, and 1 mM DTT) was added, along with 20 μM ATP/10 μCi [γ-32P]ATP and 1 μg of GST-activating transcription factor 2 (ATF2) as a substrate, to start the kinase assay reactions. The kinase reaction products were separated by SDS-PAGE for autoradiography and the reactions were stopped after 30 minutes at 30 °C by adding SDS-PAGE sample buffer. In order to verify that DUSP26 does not dephosphorylate ATF2, ATF2 underwent 32P labeling through p38 incubation. Samples were incubated for an additional 30 minutes at 30 °C with or without DUSP26, and SDS-PAGE was used to resolve the results. After drying, the gels were exposed to X-ray film.
Immune complex kinase tests. Co-transfection of HA-p38 and FLAG-DUSP26 expression plasmids into HEK 293 cells was done for the immune complex kinase assay. Cells were stimulated with H2O2 (1 mM, 30 min) after 48 h of transfection, and NSC-87877 (0–100 μM, 3 h) was pretreated. Using anti-HA agarose beads, cell extracts were immunoprecipitated after being centrifuged to remove excess liquid. The beads were treated with the PTP lysis buffer once, 150 mM NaCl, 5 mM EDTA, 2 mM DTT, and 1 mM PMSF twice, and once more with a mixture of 20 mM Tris–HCl (pH 7.5), 20 mM MgCl2, and 5 mM EDTA. Subsequently, the beads were again suspended in kinase reaction buffer (20 mM Tris–HCl (pH 7.5), 20 mM MgCl2, and 1 mM DTT) containing 20 μM ATP, 0.3 μCi of [γ-32P]ATP, and 1 μg of GST-ATF2 for 1 hour at 30 degrees Celsius. The SDS–PAGE technique was used to separate the kinase reaction products. Film was exposed to the gels after they had dried.
Assays for phosphatase in vitro and kinetic evaluation. Using a 96-well microtiter plate assay based on previously published techniques, the activity of phosphatases was assessed using the substrate 3-O-methylfluorescein Phosphate (OMFP; Sigma, St. Louis, MO) at concentrations varying with the Km of each enzyme. H2O and DMSO were used to solubilize NSC-87877 (Calbiochem, San Diego, CA) and OMFP, respectively. Every reaction was carried out with 1% DMSO as the final concentration. The final incubation mixture (150 μl) contained 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 PTPs. This mixture was optimized for enzyme activity. OMFP was added to start the reaction, and it was incubated for 30 minutes at 37 °C. Using a multi-well plate reader (GENios Pro; excitation filter, 485 nm; emission filter, 535 nm), the product's fluorescence emission was measured. Throughout the course of the experiment, the reaction was linear and directly correlated with the concentrations of the enzyme and substrate. An inhibitor's half-maximal inhibition constant (IC50) was defined as the concentration at which PTP activity decreased by 50%. The best curve fit and half-maximal inhibition constants for Lineweaver–Burk plots were found using the curve fitting application Prism 3.0 (GraphPad Software, San Diego, CA). Every experiment was run through at least three repetitions in triplicate.
Assays for dephosphorylation using phosphorylated MAPKs that are active. In the PTP assay buffer (30 mM Tris–HCl (pH 7), 75 mM NaCl, 1 mM EDTA, 0.1 mM DTT, and 0.33% BSA), the six-His-tagged DUSP26 (1 μg) was mixed with active phosphorylated p38 (10 ng), ERK (10 ng), or JNK (50 ng). The mixture was then incubated for 30 minutes at 37 °C. In a 30-μl reaction volume, 1 μg of DUSP26 was combined with 10 ng of active phosphorylated p38 and different concentrations of NSC-87877 (0, 10, or 100 μM) to see if NSC-87877 down-regulates the DUSP26 effect on p38 in vitro. The mixture was then incubated for 30 minutes at 37 °C. Using phospho-MAPK antibodies, the samples were subjected to Western blotting analysis to determine the phosphorylation state of MAPKs.

Following cell harvesting, lysing was done by sonication in 50 mM Tris–HCl (pH 8), 300 mM NaCl, 1% NP-40, and 1 mM PMSF (phenylmethylsulphonyl fluoride). For 30 minutes at 4 °C, the lysates were clarified at 4000 rpm. A column of Ni-NTA resin (PEPTRON, Daejon, Korea) received the supernatant by gravity flow. Following an elution step using 20 mM Tris–HCl (pH 8), 500 mM NaCl, and 200–300 mM imidazole, the resin was rinsed with 20 mM Tris–HCl (pH 8), 50 mM imidazole, and 500 mM NaCl. After being dialyzed overnight against 20 mM Tris–HCl, 100 mM NaCl, 30% glycerol, and 0.5 mM PMSF, the eluted proteins were stored at −80 °C.

Intrarenal neuroblastoma (NB) tumor mouse model in female nude mice.
30 mg/kg.
IP once daily for 15 days.

[1]. Discovery of a novel shp2 protein tyrosine phosphatase inhibitor. Mol Pharmacol. 2006 Aug;70(2):562-70.

[2]. NSC-87877, inhibitor of SHP-1/2 PTPs, inhibits dual-specificity phosphatase 26 (DUSP26). Biochem Biophys Res Commun. 2009 Apr 17;381(4):491-5.

[3]. NSC-87877 inhibits DUSP26 function in neuroblastoma resulting in p53-mediated apoptosis. Cell Death Dis. 2015 Aug 6;6(8):e1841.

C19H13N3O7S2

459.45

459.02

C, 49.67; H, 2.85; N, 9.15; O, 24.38; S, 13.96

56990-57-9

NSC-87877 disodium;56932-43-5

Solid powder

C1=CC2=C(C=C(C(=C2N=C1)O)N=NC3=CC4=C(C=C3)C=C(C=C4)S(=O)(=O)O)S(=O)(=O)O

XGMFVZOKHBRUTL-UHFFFAOYSA-N

InChI=1S/C19H13N3O7S2/c23-19-16(10-17(31(27,28)29)15-2-1-7-20-18(15)19)22-21-13-5-3-12-9-14(30(24,25)26)6-4-11(12)8-13/h1-10,23H,(H,24,25,26)(H,27,28,29)

8-hydroxy-7-[(6-sulfonaphthalen-2-yl)diazenyl]quinoline-5-sulfonic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.53 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 1.67 mg/mL (3.63 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

View More

Solubility in Formulation 3: 3.33 mg/mL (7.25 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

---

 (Please use freshly prepared in vivo formulations for optimal results.)