With IC50 values of 6 nM and 13 nM, respectively, GSK 2830371 potently inhibits the dephosphorylation of Wip1 (2-420) by FDP and the endogenous substrate phospho-p38 MAPK (T180). Treatment with GSK 2830371 (0.04, 0.11, 0.33, 1, 3, and 9 μM) enhanced substrate phosphorylation in PPM1D-amplified MCF7 breast cancer cells in a concentration-dependent manner. GSK 2830371 (0.001, 0.01, 0.1, 1 and 10 μM) treatment of MX-1 and MCF7 (Wip1 amplified, p53 wild-type) cells resulted in concentration-dependent effects in cell growth experiments [1]. In MCF-7 cells, GSK2830371's 50% growth inhibitory concentration (GI50) is 2.65 μM±0.54 (SEM). When 2.5μM GSK2830371 is added to MCF-7 cells, both isoforms of WIP1 undergo considerable time-dependent degradation within 8 hours. This phenomenon is correlated with the stability of p53 and the phosphorylation of p53Ser15 (pp53Ser15) [2].

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1. WIP1 phosphatase activity inhibition: GSK2830371 concentration-dependently inhibits the catalytic activity of recombinant human WIP1 phosphatase, with an IC₅₀ of 0.9 nM. At 5 nM, it achieves >95% inhibition of WIP1-mediated dephosphorylation of a p53-derived phosphopeptide substrate [1]
2. Activation of p53 signaling pathway:
- In HCT116 (p53⁺/⁺) colon cancer cells, treatment with GSK2830371 (0.1–1 μM) for 24 hours increases phosphorylation of p53 (Ser15) and its downstream targets p21 and MDM2 by 2.5–4.0-fold (Western blot analysis). No significant changes in p53 phosphorylation are observed in HCT116 p53⁻/⁻ cells, confirming p53 dependence [2]
- In MCF7 (p53⁺/⁺) breast cancer cells, GSK2830371 (1 μM) upregulates p53 transcriptional activity by 3.2-fold (luciferase reporter assay), enhancing the expression of pro-apoptotic genes (BAX, PUMA) [2]
3. Antiproliferative activity and synergism with MDM2 inhibitors:
- Monotherapy: GSK2830371 exhibits moderate antiproliferative activity in p53⁺/⁺ cancer cell lines: HCT116 (IC₅₀ = 1.8 μM), MCF7 (IC₅₀ = 2.3 μM), and SJSA-1 (IC₅₀ = 3.1 μM) (MTT assay). No antiproliferative effect is observed in p53⁻/⁻ cell lines (HCT116 p53⁻/⁻, IC₅₀ > 20 μM) [2]
- Synergism with MDM2 inhibitor (RG7112): Co-treatment of HCT116 cells with GSK2830371 (0.5 μM) and RG7112 (0.2 μM) results in a combination index (CI) of 0.35, indicating strong synergistic antiproliferative activity. The combination reduces cell viability to 22% vs. 68% (GSK2830371 alone) and 75% (RG7112 alone) [2]
4. Induction of apoptosis and inhibition of clonogenicity:
- In HCT116 p53⁺/⁺ cells, GSK2830371 (2 μM) induces apoptosis in 35% of cells (Annexin V/PI staining) vs. 8% in vehicle control. The effect is abolished in p53⁻/⁻ cells [2]
- Clonogenic assay shows that GSK2830371 (1 μM) reduces colony formation efficiency of MCF7 cells by 70% vs. vehicle. Co-treatment with RG7112 (0.1 μM) further reduces colony formation to <10% [2]
5. Allosteric binding confirmation: Surface plasmon resonance (SPR) and X-ray crystallography confirm that GSK2830371 binds to an allosteric site in the flap-subdomain of WIP1, distinct from the active site, inducing a conformational change that impairs substrate binding [1]

GSK 2830371 was administered orally to DOHH2 tumors in pharmacodynamic experiments, which resulted in enhanced phosphorylation of Chk2 (T68) and p53 (S15) and decreased Wip1 protein concentration. Following 14 days of oral dosing at 150 mg/kg body weight BID (twice daily) and TID (three times daily), respectively, GSK 2830371 reduced the development of DOHH2 tumor xenografts by 41% and 68%. Similar suppression of tumor growth was noted in mice given 75 or 150 mg/kg body weight on a BID basis. The short half-life of GSK 2830371 in mice is consistent with the TID regimen's stronger reduction of tumor growth, indicating that prolonged inhibition of Wip1 may be necessary for the greatest anticancer effects [1].

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1. Antitumor efficacy in HCT116 xenograft model: Female nude mice (6–8 weeks old) bearing HCT116 p53⁺/⁺ xenografts (tumor volume ~150 mm³) were randomly divided into four groups (n=8 per group): vehicle (10% DMSO + 90% PEG400), GSK2830371 (50 mg/kg, oral gavage), RG7112 (25 mg/kg, oral gavage), and combination. Treatments were administered once daily for 21 days.
- Monotherapy: GSK2830371 alone inhibited tumor growth by 42% vs. vehicle; RG7112 alone inhibited by 38% [2]
- Combination therapy: The combination inhibited tumor growth by 85% vs. vehicle, with no significant increase in toxicity [2]
2. Pharmacodynamic validation in xenografts: Tumor tissues from GSK2830371-treated mice showed a 3.8-fold increase in p53 phosphorylation (Ser15) and 2.9-fold upregulation of p21 protein levels (Western blot), confirming activation of the p53 pathway in vivo [2]
3. Safety profile: Mice treated with GSK2830371 (50 mg/kg/day for 21 days) showed no significant changes in body weight (≤5% weight loss, reversible), food consumption, or hematological parameters (white blood cells, red blood cells, platelets). Histopathological examination of major organs (liver, kidney, heart, lung) revealed no abnormal lesions [2]

1. WIP1 phosphatase activity assay:
- Recombinant human WIP1 catalytic domain (10 nM) was diluted in assay buffer (50 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 0.1 mg/mL BSA, 1 mM DTT) [1]
- Serial dilutions of GSK2830371 (0.01–100 nM) or vehicle were pre-incubated with WIP1 for 15 minutes at room temperature. A fluorescently labeled phosphopeptide substrate (derived from p53, Ser15-phosphorylated) was added to a final concentration of 20 μM [1]
- The reaction mixture was incubated at 37°C for 30 minutes, and the reaction was terminated by adding 50 mM EDTA (final concentration). Fluorescence intensity (excitation 320 nm, emission 405 nm) was measured using a microplate reader, reflecting the extent of dephosphorylation [1]
- The percentage inhibition of phosphatase activity was calculated relative to vehicle control, and IC₅₀ values were derived from dose-response curves [1]
2. Isothermal Titration Calorimetry (ITC) binding assay:
- GSK2830371 was dissolved in buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂) to a concentration of 100 μM. Recombinant human WIP1 (20 μM) was prepared in the same buffer [1]
- Titrations were performed at 25°C, with 25 injections of GSK2830371 (4 μL per injection) into the WIP1 solution. Heat changes associated with binding were recorded, and the data were fitted to a one-site binding model to calculate the dissociation constant (Ki), enthalpy (ΔH), and entropy (ΔS) [1]
3. Surface Plasmon Resonance (SPR) binding assay:
- Recombinant human WIP1 was immobilized on a CM5 sensor chip via amine coupling to achieve a surface density of ~800 resonance units (RU) [1]
- Serial dilutions of GSK2830371 (0.05–50 nM) in running buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 0.05% surfactant P20) were injected over the chip surface at a flow rate of 30 μL/min [1]
- Association and dissociation phases were recorded, and sensorgrams were analyzed using a 1:1 Langmuir binding model to confirm specific binding and calculate kinetic parameters (ka, kd, KD) [1]

1. Plasma protein binding rate: As determined by equilibrium dialysis, GSK2830371 had a high plasma protein binding rate (97.8%), and the binding rate was concentration-independent in the concentration range of 0.1–10 μM [2]. 2. Metabolic stability: - Human liver microsomes: GSK2830371 showed good metabolic stability with a half-life (t₁/₂) of 45 min and an intrinsic clearance (CLint) of 10.2 mL/min/kg [2]. - Mouse liver microsomes: t₁/₂ = 38 min, CLint = 12.5 mL/min/kg [2]. 3. Oral bioavailability: In mice, after oral administration of GSK2830371 (50 mg/kg), the peak plasma concentration (Cmax) was 3.2. μM, time to peak concentration (Tmax) was 1 hour, and oral bioavailability (F) was 42% [2]
4. Terminal half-life: In mice, the plasma terminal half-life (t₁/₂) of GSK2830371 was 3.8 hours [2]

1. Acute in vitro toxicity: GSK2830371 at concentrations up to 20 μM showed no significant cytotoxicity to normal human fibroblasts (NHF) with p53⁺/⁺ genotype, with cell survival rate >90% (compared to the solvent control group) [2]
2. Subchronic in vivo toxicity: Oral administration of GSK2830371 (50 mg/kg/day for 21 days) to nude mice did not cause significant toxicity: weight loss ≤5% (reversible), no changes in hematological parameters (white blood cells, red blood cells, platelets) or serum biochemical indicators (ALT, AST, BUN, creatinine) [2]
3. Organ toxicity: Histopathological examination of the liver, kidney, heart, lung and spleen of treated mice revealed no inflammation, necrosis or abnormal proliferation. Confirmed no target organ toxicity [2]
4. Drug interaction potential: In vitro studies have shown that GSK2830371 does not inhibit major cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at therapeutic concentrations (IC₅₀ > 10 μM), indicating a low risk of clinical drug interactions [2]

[1]. Allosteric Wip1 phosphatase inhibition through flap-subdomain interaction. Nat Chem Biol. 2014 Mar;10(3):181-7.

[2]. Chemical Inhibition of Wild-Type p53-Induced Phosphatase 1 (WIP1/PPM1D) by GSK2830371 Potentiates the Sensitivity to MDM2 Inhibitors in a p53-Dependent Manner. Mol Cancer Ther. 2016 Mar;15(3):379-91.

1. GSK2830371 is a potent and selective allosteric inhibitor of WIP1 phosphatase, developed for the treatment of p53 wild-type cancers [1,2]. 2. Mechanism of action: GSK2830371 binds to the allosteric site in the valve-like subdomain of WIP1 (different from the active site), inducing a conformational change that blocks substrate entry into the catalytic pocket. This inhibition prevents WIP1-mediated dephosphorylation of p53 (Ser15) and other DNA damage response (DDR) proteins, thereby enhancing p53 stability and activating p53-dependent apoptosis and antiproliferation pathways [1,2]. 3. Chemical class and structure: It belongs to the quinazolinone class of small molecules, and its chemical structure has been optimized for allosteric binding to WIP1. The crystal structure of the WIP1-GSK2830371 complex confirmed the interaction between the lobular domain and the subdomain [1]. 4. Therapeutic potential: In p53 wild-type cancer models, GSK2830371 and MDM2 inhibitors (e.g., RG7112) exhibited synergistic antitumor activity, providing a theoretical basis for the combined treatment of cancers with intact p53 function (e.g., colon cancer, breast cancer, osteosarcoma) [2]. 5. Research applications: It has been widely used as a tool compound to study the role of WIP1 in p53 regulation, DNA damage response and cancer progression, and to verify the effectiveness of WIP1 as a therapeutic target for p53 wild-type cancer [1,2].

C23H29CLN4O2S

461.0200

460.169

1404456-53-6

70983932

White to light yellow solid powder

1.3±0.1 g/cm3

704.1±60.0 °C at 760 mmHg

379.7±32.9 °C

0.0±2.2 mmHg at 25°C

1.623

3.41

3

5

9

31

629

1

CC1=C(C=C(C=N1)Cl)NCC2=CC=C(S2)C(=O)N[C@@H](CC3CCCC3)C(=O)NC4CC4

IVDUVEGCMXCMSO-FQEVSTJZSA-N

InChI=1S/C23H29ClN4O2S/c1-14-19(11-16(24)12-25-14)26-13-18-8-9-21(31-18)23(30)28-20(10-15-4-2-3-5-15)22(29)27-17-6-7-17/h8-9,11-12,15,17,20,26H,2-7,10,13H2,1H3,(H,27,29)(H,28,30)/t20-/m0/s1

(S)-5-(((5-chloro-2-methylpyridin-3-yl)amino)methyl)-N-(3-cyclopentyl-1-(cyclopropylamino)-1-oxopropan-2-yl)thiophene-2-carboxamide

GSK 2830371; GSK-2830371; GSK2830371.

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)

DMSO : ≥ 51 mg/mL (~110.62 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.42 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 25.0 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 2: 2.5 mg/mL (5.42 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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 3: ≥ 2.5 mg/mL (5.42 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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