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Biological Description

Description: SCH 772984 is a potent inhibitor of ERK1/ERK2 (IC50: 4/1 nM) and has only weak inhibitory for other 300 tested kinases.

Targets&IC50: ERK2:1 nM (cell free), ERK1:4 nM (cell free)

In vitro: SCH772984 potently inhibited ERK1 and ERK2 activity (IC50s: 4/1 nmol/L). SCH772984 is highly selective, with only seven kinases of 300 tested showing more than 50% inhibition at a concentration of 1 μmol/L. Treatment of the BRAFV600E-mutant human melanoma cell line LOXIMV1 (LOX) with SCH772984 resulted in a dose-dependent inhibition of phosphorylation of the ERK substrate p90 ribosomal S6 kinase. SCH772984 also inhibited phosphorylation of residues in the activation loop of ERK itself. In a panel of 121 human tumor cell lines, SCH772984 showed EC50 values less than 500 nmol/L in approximately 88% and 49% of BRAF-mutant or RAS-mutant tumor lines, respectively.

In vivo: Treatment of BRAF-mutant LOX melanoma xenografts with SCH772984 (50 mg/kg twice daily) led to 98% tumor regression. Dose-dependent antitumor activity was also observed in the KRAS-mutant pancreatic MiaPaCa model, with 36% regression at 50 mg/kg twice daily.

Kinase Assay: SCH772984 was tested in 8-point dilution curves in duplicate against purified ERK1 or ERK2. The enzyme was added to the reaction plate and incubated with the compound before adding a solution of substrate peptide and ATP. Fourteen microliters of diluted enzyme (0.3 ng active ERK2 per reaction) was added to each well of a 384-well plate. The plates were gently shaken to mix the reagents and incubated for 45 minutes at room temperature. The reaction was stopped with 60 μL of IMAP Binding Solution (1:2,200 dilutions of IMAP beads in 1× binding buffer). The plates were incubated at room temperature for an additional 0.5 hours to allow complete binding of phosphopeptides to the IMAP beads. Plates were read on the LJL Analyst.

Cell Research: For resistant cell line creation, cells were grown in Dulbecco's modified Eagle medium with 10% heat-inactivated FBS media and increasing concentrations of inhibitor (PLX4032, 0.1–10 μmol/L; GSK1120212, 0.01–1 μmol/L) over approximately 4 to 8 months until resistant cells acquired growth properties similar to na?ve parental cells (at their top drug concentrations). For combination resistance, cells were incubated as above but with alternative dose escalation until a top concentration was acquired (PLX4032 10 μmol/L and GSK1120212 1 μmol/L). Stocks and dilutions of PLX4032, GSK1120212, and SCH772984 were made in DMSO solvent. Cell proliferation experiments were carried out in a 96-well format (six replicates), and cells were plated at a density of 4,000 cells per well. At 24 hours after cell seeding, cells were treated with DMSO or a 9-point IC50 dilution (0.001–10 μmol/L) at a final concentration of 1% DMSO for all concentrations. Viability was assayed 5 days after dosing using the ViaLight luminescence kit following the manufacturer's recommendations (n = 6, mean ± SE). For the cell line panel viability assay, cells were treated with SCH772984 for 4 days and assayed by the CellTiterGlo luminescent cell viability assay. For IncuCyte analysis, cells were plated as above in 96-well plates, and image-based cell confluence data were collected every 2 hours during live growth. For engineered resistant lines, cells were infected with lentivirus produced from lentiORF constructs expressing either RFP, KRASG13D, BRAFV600E, truncated BRAFV600E lacking exons 2–8 (Δ2-8), MEK1P124L, MEK1F129L, or constitutively active MEK1DD (S218D+S222D). Cells were selected in blasticidin (20 μg/mL) and used for ViaLight assays as described above.

Animal Research: Nude mice were injected subcutaneously with specific cell lines, grown to approximately 100 mm^3, randomized to treatment groups (10 mice/group), and treated intraperitoneally with either SCH772984 or vehicle according to the dosing schedule indicated in the figure legends. Tumor length (L), width (W), and height (H) were measured during and after the treatment periods by a caliper twice weekly on each mouse and then used to calculate tumor volume using the formula (L × W × H)/2. Animal body weights were measured on the same days twice weekly. Data were expressed as mean ± SEM. Upon completion of the experiment, vehicle- and SCH772984-treated tumor biopsies were processed for Western blot analysis.

Chemical Properties

Molecular Weight: 587.67

Formula: C33H33N9O2

CAS No.: 942183-80-4
 

Storage & Solubility Information

Storage

Powder: -20°C for 3 years

In solvent: -80°C for 2 years

Solubility Information

H2O: <1 mg/mL

Ethanol: <1 mg/mL

DMSO: 51 mg/mL(86.8 mM)

( < 1 mg/ml refers to the product slightly soluble or insoluble )

Catalog #: T6066

Please email us at cs@medikonia.com for any enquiry. To place an order, please include the catalog number(s) of the product(s) in the email.

References and Literature

1. Morris EJ, et al. Discovery of a novel ERK inhibitor with activity in models of acquired resistance to BRAF and MEK inhibitors. Cancer Discov. 2013 Jul;3(7):742-50.

2. Chaikuad A, et al. A unique inhibitor binding site in ERK1/2 is associated with slow binding kinetics. Nat Chem Biol. 2014 Oct;10(10):853-60.

3. Jiang S, Wang Z, Zhu T, et al. The Downregulation of EIF3a Contributes to Vemurafenib Resistance in Melanoma By Activating ERK Via PPP2R1B[J]. 2021

4. Lang J Y, Wang X, Xie Q, et al. Targeting KRAS-mutant stomach/colorectal tumours by disrupting the ERK2-p53 complex[J]. bioRxiv. 2020

5. Chang G, Xiao W, Xu Z, et al. Pterostilbene Induces Cell Apoptosis and Cell Cycle Arrest in T-Cell Leukemia/Lymphoma by Suppressing the ERK1/2 Pathway[J]. BioMed Research International. 2017;2017:9872073

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7. Bin Y F, Ma N, Lu Y X, et al. Erythromycin reverses cigarette smoke extract-induced corticosteroid insensitivity by inhibition of the JNK/c-Jun pathway[J]. Free Radical Biology and Medicine. 2020, 152: 494-503.

8. Xie D, Ge X, Ma Y, et al. Clemastine improves hypomyelination in rats with hypoxic–ischemic brain injury by reducing microglia-derived IL-1β via P38 signaling pathway[J]. Journal of neuroinflammation. 2020, 17(1): 1-17.

9. Zhao Z, Xue F, Gu Y, et al. Crosstalk between the muscular estrogen receptor α and BDNF/TrkB signaling alleviates metabolic syndrome via 7, 8-dihydroxyflavone in female mice[J]. Molecular Metabolism. 2020: 101149.

10. Caiola E, Iezzi A, Tomanelli M, et al. LKB1 deficiency renders non-small-cell lung cancer cells sensitive to ERK inhibitors.: ERK inhibitors in LKB1 mutated NSCLC[J]. Journal of Thoracic Oncology. 2019.