Chk2 (IC50 = 15 nM)
Checkpoint Kinase 2 (CHK2) [1]
Checkpoint Kinase 2 (CHK2) (2-arylbenzimidazole analog 2h: IC50 = 15 nM, ATP-competitive inhibitor;) [2]

No significant weight loss or gross abnormalities were observed in SUDHL6 DLBCL xenograft mice treated intraperitoneally with either vehicle, ERK inhibitor (5 mg kg−1), CHK2 inhibitor II (1 mg kg−1), or both for 20 days. While both 1 mg/kg CHK2 inhibitor II and 5 mg/kg ERK inhibitor modestly inhibit tumor growth, combined treatment with both inhibitors produces a statistically significant suppression of tumor growth[2].

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1. Combined treatment with Chk2 Inhibitor II and ERK inhibitor significantly reduced subcutaneous tumour growth of SUDHL6 cells in xenograft mice (28 mice, 4 groups: vehicle, ERK inhibitor, Chk2 Inhibitor II, combination; n=7 per group); average tumour volume was lower in the combination group (P < 0.05/P < 0.01 vs single treatments); tumour tissue analysis showed reduced Ki67 expression (proliferation marker) and increased TUNEL-positive cells (apoptosis) in the combination group (immunohistochemistry, X400 for Ki67, X600 for TUNEL); western blot of tumour extracts confirmed altered pCHK2, pERK1/2, γH2AX, and cleaved PARP levels. [1]

In order to ascertain the activity of chk2 inhibitors, the following conditions must be met: 5 nM recombinant human Chk2, 50 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl2, 25 μM synthetic peptide substrate (biotin-SGLYRSPSMPENLNRPR), 1 μM ATP, 50 μCi/mL [γ-³³P] ATP, and a combination of protease inhibitors. The peptide substrate is bound to streptavidin-conjugated agarose beads by the reaction mixtures after three hours of incubation at 37°C. A 0.1% Tween-20 solution in phosphate-buffered saline (pH 7.4) is used to repeatedly wash the agarose beads. The amount of radioactive phosphate bound to the substrate peptide at various concentrations of BML-277 (6.25, 12.5, 25, 50, 100, and 200 nM) is measured by scintillation counting, which is used to determine the enzyme activity. Kinetic experiments involve varying the concentration of ATP while maintaining a constant ratio between unlabeled and [γ-³³P] labeled ATP. Samples are kept on ice for additional processing and reactions are halted at various times by adding 50 mM cold ATP[1].

Purified T-cells are incubated at 100,000 cells per well in BML-277 (10^2.5 nM, 1 μM, 10^0.5 μM, 10 μM, and 10^1.5 μM) or vehicle (DMSO) at different concentrations in 96-well stripwells for one hour in order to assess the radioprotective effect of Chk2 inhibitors. The cells are then placed back into the incubator for an additional 24 hours after being exposed to a dose of 0 or 10 Gy gamma irradiation from a ¹³⁷Cs source at a dose rate of 3.65 Gy/min. Propidium iodide and Annexin V-FITC are used to stain cells in accordance with the manufacturer's instructions. Using a FACSCalibur FACS instrument, apoptotic and surviving cells are quantified. The number of survivors from treatment groups less the number of cells surviving in the irradiated control group divided by the number of surviving cells in the untreated control groups is the percentage of recovery used to report data[1].

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1. CHK2 phosphorylation inhibition assay: Farage/SUDHL6 cells were cultured in RPMI 1640 complete medium and treated with different doses of Chk2 Inhibitor II for 4 h, or 5 μM Chk2 Inhibitor II for different time courses; total cell lysates were prepared, and immunoblotting was performed to detect pCHK1, CHK1, pCHK2, CHK2 levels (actin as loading control). [1]
2. Apoptosis assay (cell lines): SUDHL4/SUDHL6/Farage cells were exposed to 5 μM Chk2 Inhibitor II, 20 μM ERK inhibitor, or their combination for 72 h; Annexin V analysis was used to quantify apoptotic cells (data as mean ± s.d., ≥3 independent experiments, paired Student’s t-test). [1]
3. Apoptosis assay (primary cells): Single-cell suspensions from DLBCL patient lymph node biopsies were treated with DMSO, ERK inhibitor, Chk2 Inhibitor II, or combination for 48 h; Annexin V analysis (flow cytometry) and trypan blue dye uptake assay (hemocytometer) were used to determine apoptotic/non-viable cells (mean ± s.d., 3 independent experiments, Student’s t-test). [1]
4. Protein interaction assay: Farage/SUDHL4/SUDHL6 cells were lysed, and immunoprecipitation was performed with anti-ERK1/2 or anti-CHK2 antibody; immunoblotting was used to detect co-immunoprecipitated CHK2/ERK1/2; cytoplasmic/nuclear fractionation (tubulin for cytoplasm, HDAC1 for nucleus) followed by CHK2 immunoprecipitation and ERK1/2 immunoblotting confirmed subcellular interaction; GST-fused ERK2 pull-down assay with Farage/SUDHL6 lysates was used to verify in vitro ERK2-CHK2 binding (disrupted by Chk2 Inhibitor II). [1]
5. ERK phosphorylation assay: SUDHL6/Farage cells were treated with Chk2 Inhibitor II for 4 h; HEK293/HeLa cells were transfected with CHK2 plasmids (WT/T68D/KD); Farage cells were transfected with CHK2 siRNA/control siRNA; Farage/SUDHL6 cells were treated with 5 μM etoposide for 15/30 min; cell lysates were subjected to immunoblotting for pERK1/2, ERK1/2, pCHK2, CHK2 (actin as loading control); in vitro kinase assay with purified GST-CHK2 (WT/KD), ERK2, and MBP was performed to assess ERK2-mediated phosphorylation (autoradiography and Coomassie staining). [1]

Formulated in alcohol and diluted in PBS; 1 mg/kg; i.p.
mice bearing SUDHL6 DLBCL xenografts

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1. Xenograft tumour model: SUDHL6 cells were subcutaneously implanted into mice to establish tumours (28 mice total); mice were divided into 4 groups (vehicle, ERK inhibitor, Chk2 Inhibitor II, combination; n=7 per group); drug administration details (dose, frequency, route) for Chk2 Inhibitor II were not specified; tumour volume was measured over time (mean ± s.d., Student’s t-test); mice were euthanized, tumours excised, and protein extracts/preserved tissue sections were prepared for western blot, Ki67 immunohistochemistry, and TUNEL staining. [1]

[1]. J Med Chem . 2005 Mar 24;48(6):1873-85.

[1]. Nat Commun . 2011 Jul 19:2:402.

1. Chk2 inhibitors II target CHK2, a key mediator of double-strand breaks in DNA damage checkpoint reactions; ERK1/2 and CHK2 are co-localized/overexpressed in diffuse large B-cell lymphoma (DLBCL), and their physical interaction depends on the phosphorylation of CHK2 Thr68 (which Chk2 inhibitors II can disrupt). [1]
2. Chk2 inhibitors II increase the phosphorylation of ERK1/2 through a non-kinase-dependent mechanism (CHK2 directly inhibits ERK2-mediated MBP phosphorylation); the combined inhibition of ERK and CHK2 has a synergistic antitumor effect in DLBCL, supporting a dual-targeting therapy strategy. [1]
3. CHK2 inhibitors (2-arylbenzimidazoles) have radioprotective effects on T cells and have the potential to be used as adjuvants to radiotherapy (no data available for Chk2 inhibitors II). Homology models and molecular docking studies of CHK2 have guided the optimization of 2-arylbenzimidazole inhibitors (no information available for Chk2 inhibitors II). [2]

C20H14CLN3O2

363.80

363.077

C, 66.03; H, 3.88; Cl, 9.74; N, 11.55; O, 8.80

516480-79-8

516480-79-8; 516480-80-1;

9969021

White to off-white solid powder

1.4±0.1 g/cm3

637.1±63.0 °C at 760 mmHg

339.1±33.7 °C

0.0±1.9 mmHg at 25°C

1.703

4.61

2

3

4

26

489

0

ClC(C=C1)=CC=C1OC(C=C2)=CC=C2C3=NC4=CC(C(N)=O)=CC=C4N3

UXGJAOIJSROTTN-UHFFFAOYSA-N

InChI=1S/C20H14ClN3O2/c21-14-4-8-16(9-5-14)26-15-6-1-12(2-7-15)20-23-17-10-3-13(19(22)25)11-18(17)24-20/h1-11H,(H2,22,25)(H,23,24)

2-[4-(4-chlorophenoxy)phenyl]-3H-benzimidazole-5-carboxamide

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: ≥ 1.67 mg/mL (4.59 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 2: ≥ 1.67 mg/mL (4.59 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 16.7 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: ≥ 1.67 mg/mL (4.59 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 16.7 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.)