shp2 (IC50 = 0.318 μM); shp1 (IC50 = 0.355 μM)
SHP2 (protein tyrosine phosphatase, PTP) ( binds to the catalytic cleft) [1]
- SHP1 (protein tyrosine phosphatase, PTP) (cross-inhibited in vitro) [1]
- Dual-specificity phosphatase 26 (DUSP26) (competitive inhibition mode) [2][3]

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.

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NSC-87877 (chemical name: 8-hydroxy-7-(6-sulfonaphthalen-2-yl)diazenyl-quinoline-5-sulfonic acid) is a potent and selective inhibitor of SHP2 PTP. It cross-inhibits SHP1 in vitro but shows selectivity over other PTPs including PTP1B, HePTP, DEP1, CD45, and LAR [1]
- It inhibits epidermal growth factor (EGF)-induced activation of SHP2 PTP, Ras, and Erk1/2 in cell cultures without blocking EGF-induced Gab1 tyrosine phosphorylation or Gab1-Shp2 association. It also inhibits Erk1/2 activation by a Gab1-Shp2 chimera but does not affect Shp2-independent Erk1/2 activation by phorbol 12-myristate 13-acetate [1]
- NSC-87877 inhibits DUSP26 phosphatase activity in a dose-dependent manner and exhibits competitive inhibition kinetics with DUSP26. It effectively blocks DUSP26-mediated dephosphorylation of p38 MAP kinase [2]
- In neuroblastoma (NB) cell lines (IMR32, NB-19, SH-SY5Y, SK-N-SH, SK-N-AS, SHEP), NSC-87877 shows dose- and time-dependent growth inhibition and induces apoptosis. The IC₅₀ values vary among different NB cell lines, with measurable cytotoxicity observed at concentrations starting from 0.25 μM [3]
- Treatment with NSC-87877 increases p53 phosphorylation at Ser37 and Ser46, activates p53, and enhances the activation of downstream p38 effector proteins (HSP27 and MAPKAPK2) in NB cells. It also induces cleavage of PARP and caspase-3, markers of apoptosis [3]
- The cytotoxicity of NSC-87877 in NB cells is partially reversed by knocking down p53 expression with shRNA or inhibiting p38 activity with SB203580. It also suppresses anchorage-independent growth of NB cells in soft agar assays [3]

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.

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NSC-87877 affects NB tumor growth in vivo[3]
In order to test the effect of small molecule inhibition of DUSP26 in vivo, we used a well-established intrarenal NB tumor mouse model.30 SH-SY5Y cells with luciferase expression levels were injected into the left kidney of female nude mice. After 10 days of tumor growth, mice were treated with i.p. injection of placebo control (control) or 30 mg/kg of NSC-87877 once daily for 15 days. Mice were monitored weekly with i.p. injection of luciferin and bioluminescence imaging. Figure 7a demonstrates equivalent tumor burden with similar bioluminescent signal at day 1 of therapy and a difference in signal at 15 days of therapy. On day 15, necropsy was performed and the tumors were weighed. The control group tumors were significantly larger than the NSC-87877-treated group (P<0.01; Figure 7b).

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In an intrarenal xenograft mouse model of NB (female NCr nude mice implanted with luciferase-tagged SH-SY5Y cells), intraperitoneal injection of NSC-87877 at 30 mg/kg for 15 days results in significant tumor growth inhibition, as evidenced by reduced tumor weight and bioluminescent signal [3]
- Tumor tissues from NSC-87877-treated mice show increased activation of p53 (phospho-p53 Ser46) and p38 pathway (phospho-MAPKAPK2, phospho-HSP27) compared to vehicle control. Cleavage of PARP and caspase-7 is also detected in tumor tissues from treated mice [3]

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.
\n\nImmune 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.
\n\nAssays 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 at concentrations varying with the Km of each enzyme. H2O and DMSO were used to solubilize NSC-87877 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 . Every experiment was run through at least three repetitions in triplicate.
\n\nAssays 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.

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PTP activity inhibition assay: Prepare reaction mixtures containing purified SHP2, SHP1, or other PTPs (PTP1B, HePTP, DEP1, CD45, LAR) with a phosphopeptide substrate. Add different concentrations of NSC-87877 to the reaction mixtures and incubate for a specified time. Measure the release of phosphate to determine the inhibitory effect on PTP activity and assess selectivity [1]
- DUSP26 phosphatase activity assay: Prepare reaction mixtures with purified DUSP26 and a p38 MAP kinase substrate. Incubate with varying concentrations of NSC-87877 and measure the phosphatase activity by detecting the dephosphorylation of the substrate. Perform kinetic analysis to determine the inhibition mode (competitive inhibition) [2]

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 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.

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Cell viability and proliferation assay: Seed NB cell lines or other target cells in 96-well plates. Treat with NSC-87877 at concentrations ranging from 0.25 μM to 50 μM for 24–72 h. Assess cell viability using MTT assay (measuring absorbance at 540 nm) and calculate IC₅₀ values. For long-term proliferation analysis, monitor cell growth every 24 h for up to 5–9 days [3]
- Western blot analysis: Treat cells with NSC-87877 at specified concentrations and time points. Lyse cells, extract total proteins, separate by SDS-PAGE, and transfer to membranes. Probe with antibodies against target proteins (p-SHP2, Ras, Erk1/2, p-p53, p53, p-p38, HSP27, MAPKAPK2, PARP, caspase-3, caspase-7, β-actin) to detect protein expression, phosphorylation, or cleavage [1][3]
- Soft agar colony formation assay: Seed NB cells in six-well plates with agar, media, and different concentrations of NSC-87877. Incubate for 2–3 weeks, stain colonies with MTT, count colonies, and quantify anchorage-independent growth [3]
- shRNA knockdown rescue assay: Transduce NB cells with p53 shRNA or non-silencing control shRNA. Seed transduced cells in 96-well plates, treat with NSC-87877 for 48–72 h, and assess cell viability by MTT assay to verify the role of p53 in drug-induced cytotoxicity [3]
- p38 inhibition rescue assay: Seed NB cells in 96-well plates, pretreat with 5 μM SB203580 (p38 inhibitor) or vehicle, then add increasing concentrations of NSC-87877. Measure cell viability by MTT assay to evaluate the contribution of p38 pathway to drug-induced cytotoxicity [3]

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

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Effect of RNA interference and NSC-87877 on NB cell growth in an orthotopic mouse model[3]
Female Nu-nude mice (Taconic Biosciences, Hudson, NY, USA) were used for in vivo testing of NSC-87877 compared with control. SH-SY5Y transduced luciferase cells (SY5Y-Luc) and SH-SY5Y transduced with shDUSP26-1 were implanted into the left kidney as previously described.30 The mice were imaged 10 days after implantation and flux measured. A threshold of 5 × 107 total flux (p/s) was used to standardize the mice who would be treated. Two groups were treated, one with NSC-87877 using a dose of 30 mg/kg/day, and the other with a carrier control composed of an equivalent volume of 0.9% NaCl via i.p. injection. After 15 days, necropsy was performed and tumor weights measured. For phosphor-immunoblotting, SH-SY5Y-Luc were implanted into the kidneys of three mice, once the previously indicated threshold for flux was reached, two mice were treated with 30 mg/kg of NSC-87877 and one control mouse was treated with carrier control. The mice were killed, at 12 and 24 h after administration of NSC-87877, and necropsy was performed. Tumors were immediately flash frozen with liquid nitrogen for later protein extraction. Protein was extracted by grinding 10 mg of tumor tissue, which was mixed with protein lysis buffer, passed through a 22 G needle, and incubated on ice for 30 min.[3]

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NB intrarenal xenograft model: Implant luciferase-tagged SH-SY5Y cells into the left kidney of female NCr nude mice. After confirming tumor establishment via bioluminescent imaging, randomly divide mice into treatment and control groups (n ≥ 3 per group). Administer NSC-87877 at 30 mg/kg via intraperitoneal injection once daily for 15 days; the control group receives normal saline. Monitor tumor growth weekly using bioluminescent imaging. At the end of the study, euthanize mice, dissect tumors, weigh them, and extract proteins for western blot analysis [3]

[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.

8-O-7-[(6-sulfono-2-naphthyl)hydrazino]-5-quinoline sulfonic acid is a naphthalene sulfonic acid. Shp2 is a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. It is involved in the activation of growth factor-induced mitogen-activated protein (MAP) kinases Erk1 and Erk2 (Erk1/2) and is associated with the pathogenicity of the oncogenic bacterium Helicobacter pylori. Furthermore, gain-of-function mutations in Shp2 have been found in childhood leukemia and Noonan syndrome. Therefore, small-molecule Shp2 PTP inhibitors are essential for evaluating Shp2 as a therapeutic target and for conducting functional chemical biology research on Shp2. By screening the National Cancer Institute (NCI) diversity compound library, we identified 8-hydroxy-7-(6-sulfononaphthyl)diazoquinoline-5-sulfonic acid (NSC-87877) as a potent Shp2 PTP inhibitor. Molecular modeling and site-directed mutagenesis studies have shown that NSC-87877 binds to the catalytic cleft of Shp2 PTP. NSC-87877 exhibits cross-inhibition of Shp1 in vitro, but its selectivity for Shp2 is higher than that of other PTPs (PTP1B, HePTP, DEP1, CD45, and LAR). Notably, NSC-87877 inhibits epidermal growth factor (EGF)-induced activation of Shp2 PTP, Ras, and Erk1/2 in cell cultures, but does not block EGF-induced Gab1 tyrosine phosphorylation or Gab1-Shp2 binding. Furthermore, NSC-87877 inhibits Gab1-Shp2 chimera activation of Erk1/2, but has no effect on phorbol ester (PMA)-induced Shp2-independent Erk1/2 activation. These results indicate that NSC-87877 is the first PTP inhibitor capable of inhibiting Shp2 PTP in cell culture without significant off-target effects. Our study also provides the first pharmacological evidence for Shp2-mediated EGF-induced activation of Erk1/2 MAP kinases. [1] Protein phosphorylation plays a key role in many mechanisms regulating cellular activity and is therefore associated with a variety of diseases. Intracellular phosphorylation balance is regulated by protein kinases and phosphatases. Therefore, these regulatory proteins have become potential targets for drug development. In this study, protein tyrosine phosphatases (PTPs) were screened by in vitro phosphatase activity assays to identify PTPs that could be inhibited by 8-hydroxy-7-(6-sulfonnaphthalen-2-yl)diazoquinoline-5-sulfonic acid (NSC-87877). NSC-87877 is a potent inhibitor of SHP-1 and SHP-2 PTPs. The phosphatase activity of bispecific protein phosphatase 26 (DUSP26) can be reduced by this inhibitor in a dose-dependent manner. Kinetic studies of NSC-87877 and DUSP26 showed a competitive inhibitory relationship. NSC-87877 effectively inhibited DUSP26-mediated dephosphorylation of p38, a member of the mitogen-activated protein kinase (MAPK) family. Since DUSP26 is involved in the survival of anaplastic thyroid cancer (ATC) cells, NSC-87877 may be a potential drug for the treatment of ATC. [2]

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Bispecific protein phosphatase 26 (DUSP26) is overexpressed in high-risk neuroblastoma (NB) and leads to chemotherapy resistance by inhibiting p53 function. In vitro experiments showed that DUSP26 can also effectively inhibit p38 MAP kinase. We hypothesized that inhibition of DUSP26 would reduce the growth of NB cells in a p53 and/or p38-mediated manner. NSC-87877 (8-hydroxy-7-[(6-sulfo-2-naphthyl)azo]-5-quinoline sulfonic acid) is a novel small molecule inhibitor of DUSP26 that effectively inhibits the growth of NB cell lines and induces their apoptosis. NB cell lines treated with small hairpin RNA (shRNA) targeting DUSP26 exhibited proliferation defects both in vitro and in vivo. Treatment of NB cell lines with NSC-87877 led to increased p53 phosphorylation (Ser37 and Ser46) and activation, increased activation of downstream p38 effector proteins (heat shock protein 27 (HSP27) and MAP kinase-activated protein kinase 2 (MAPKAPK2)), and increased poly-ADP-ribose polymerase/caspase-3 cleavage. Knockdown of p53 expression with shRNA and inhibition of p38 activity with SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine partially reversed the cytotoxicity induced by DUSP26 inhibition. In an intrarenal mouse neuroblastoma (NB) model, NSC-87877 treatment reduced tumor growth and enhanced p53 and p38 activity. These results collectively suggest that inhibiting DUSP26 with NSC-87877 is an effective strategy to induce NB cytotoxicity in vitro and in vivo by activating the p53 and p38 mitogen-activated protein kinase (MAPK) tumor suppressor pathways. [3]

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NSC-87877 is a Shp2 PTP inhibitor identified by screening the National Cancer Institute (NCI) library of diverse compounds. Molecular modeling and site-directed mutagenesis studies have shown that it binds to the catalytic cleft of Shp2 [1]
- It is the first PTP inhibitor to inhibit Shp2 PTP in cell culture without significant off-target effects, providing pharmacological evidence for Shp2-mediated EGF-induced Erk1/2 MAP kinase activation [1]
- DUSP26 is overexpressed in high-risk neuroblastoma (NB) and leads to chemotherapy resistance by inhibiting p53 function. NSC-87877, which targets DUSP26, represents a potential strategy for treating neuroblastoma and undifferentiated thyroid carcinoma (ATC) [2][3]

C19H13N3O7S2

459.45

459.019

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

56990-57-9

NSC-87877 disodium;56932-43-5

92577

Light brown to black solid powder

1.7±0.1 g/cm3

1.758

0.12

3

10

4

31

883

0

N1C=CC=C2C(S(=O)(O)=O)=CC(/N=N/C3C=CC4C=C(S(O)(=O)=O)C=CC=4C=3)=C(O)C=12

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

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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.

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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.

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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).

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