ERK1; ERK2
CC-90003 was discovered to strongly inhibit the kinase activities of ERK1 and ERK2 with IC50s in the range of 10 to 20 nmol/L and had good kinase selectivity in biochemical, cellular, and mass spectrometry assays of 347 kinases. A 258-kinase biochemical assay panel revealed that CC-90003 significantly inhibited 213 kinases (50% inhibition), moderately inhibited 28 kinases (50%-80% inhibition), and significantly inhibited 17 kinases (>80% inhibition). A375 BRAF V600E-mutant melanoma cell line was used in an ActivX cellular kinase screening, and only 5 of 194 kinases (ERK1, ERK2, MKK4, MKK6, and FAK) were inhibited by >80% at 1 mmol/L of CC-90003. In a Cerep panel of 40 nonkinase enzymes and receptors, no significant inhibition (14%) was observed at the same concentration. Only 3 kinases, KDR, FLT3, and PDGFRa, were inhibited in cells at biologically significant concentrations through our iterative analyses, in addition to ERK1/2. CC-90003 was particularly effective against tumors with BRAF mutations. Many times, but not always, CC-90003 had cytotoxic effects on PDAC, lung cancer, and colorectal cancer cell lines that were KRAS-mutant. CC-90003 does not significantly reduce the proliferation of healthy bronchial epithelial cells or lung fibroblasts[1].

CC-90003 was discovered to strongly inhibit the kinase activities of ERK1 and ERK2 with IC50s in the range of 10 to 20 nmol/L and had good kinase selectivity in biochemical, cellular, and mass spectrometry assays of 347 kinases. A 258-kinase biochemical assay panel revealed that CC-90003 significantly inhibited 213 kinases (50% inhibition), moderately inhibited 28 kinases (50%-80% inhibition), and significantly inhibited 17 kinases (>80% inhibition). A375 BRAF V600E-mutant melanoma cell line was used in an ActivX cellular kinase screening, and only 5 of 194 kinases (ERK1, ERK2, MKK4, MKK6, and FAK) were inhibited by >80% at 1 mmol/L of CC-90003. In a Cerep panel of 40 nonkinase enzymes and receptors, no significant inhibition (14%) was observed at the same concentration. Only 3 kinases, KDR, FLT3, and PDGFRa, were inhibited in cells at biologically significant concentrations through our iterative analyses, in addition to ERK1/2. CC-90003 was particularly effective against tumors with BRAF mutations. Many times, but not always, CC-90003 had cytotoxic effects on PDAC, lung cancer, and colorectal cancer cell lines that were KRAS-mutant. CC-90003 does not significantly reduce the proliferation of healthy bronchial epithelial cells or lung fibroblasts[1].

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CC-90003 demonstrated potent antiproliferative activity across a panel of 240 cancer cell lines in a 3-day viability assay. Among 27 BRAF-mutant cell lines, 25 (93%) were sensitive (Gl₅₀ < 1 µM). Among 37 KRAS-mutant cell lines, 28 (76%) were sensitive. In many KRAS-mutant pancreatic, lung, and colorectal cancer cell lines, CC-90003 induced cytotoxic effects, as indicated by a decrease in cell number below the starting point and Caspase 3/7 activation. [1]
In the KRAS C13D-mutant colorectal cancer cell line HCT-116, CC-90003 was more potent than the ERK inhibitor GDC-0994 in decreasing cell growth and induced cell death starting at 1 µM, while GDC-0994 up to 10 µM did not. [1]
CC-90003 potently inhibited downstream MAPK pathway signaling. A single administration potently reduced DUSP4 levels and inhibited ERK phosphorylation for 24 hours in A375 (BRAF mutant) and HCT-116 (KRAS mutant) cell lines. [1]
In an ex vivo 3D soft-agar colony formation assay using 84 KRAS-mutant patient-derived xenograft (PDX) models, 18 models showed sensitivity to CC-90003 with absolute IC₅₀ values below 1 µM. [1]

Although CC-90003 was well tolerated at a variety of doses (12.5 mg bi-daily to 100 mg thrice-daily) in in vivo studies using an HCT-116 xenograft model, doses of 50 mpk bi-daily and 75 mpk bi-daily caused mortality by study days 6 to 18. Inhibition of tumor growth occurs with both dosing regimens (qd and b.i.d.). CC-90003 inhibits the development of three PDX models with KRAS mutations in vivo[1].

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In an HCT-116 xenograft model in nude mice, CC-90003 administered orally once daily (qd) or twice daily (b.i.d.) led to dose-dependent tumor growth inhibition (TGI). The minimally efficacious and tolerated dose was 50 mg/kg qd, achieving 65% TGI. Higher doses (50 and 75 mg/kg b.i.d.) caused mortality. [1]
In three sensitive KRAS-mutant PDX models (lung cancer LXFA-983, pancreatic cancer PAXF-2059, and colorectal cancer CXF-243), CC-90003 (50 mg/kg qd) caused tumor stasis in the lung and pancreatic models. The colorectal model (CXF-243) was less responsive, providing a model to study resistance. [1]
In a KRAS-mutant lung cancer PDX model (LXFL-1674), continuous co-administration of CC-90003 (50 mg/kg qd, p.o.) with docetaxel (15 mg/kg, i.v., on days 0 and 7) induced complete tumor regression by day 18 with no tumor regrowth observed up to day 52 after treatment cessation. An intermittent dosing schedule of CC-90003 (4 days on/3 days off) combined with docetaxel (days 5 and 12) was less effective, inducing tumor stasis and partial regression, followed by regrowth after treatment stopped. [1]

Kinase selectivity profiling for CC-90003 was performed using multiple assays. Biochemical activity was assessed against a panel of 258 kinases at 1 µM CC-90003 concentration. Covalent modification was confirmed by incubating intact protein kinases with a 10-fold molar excess of CC-90003 at room temperature for 1 hour, followed by desalting and mass spectrometric analysis using MALDI-TOF/TOF to detect a mass shift corresponding to compound binding. Cellular kinase activity was evaluated using an active-site dependent competition binding assay (ActivX) in A375 BRAF V600E mutant melanoma cells treated with 1 µM CC-90003, measuring the reduction in binding of a broad-spectrum kinase probe to ~200 kinases. [1]

The cells were plated on 96-well clear bottom black-well plates at a density of 3,000 cells per well in 90 mL of growth medium, and they were then incubated overnight at 37 C with 5% CO2 under standard cell culture growth conditions. The next day, one plate per cell line was used for the "Day 0" cell growth control readout, and the remaining plates received treatment with DMSO as a control and 9-point 3-fold dilutions of one compound or a combination of compounds. Three duplicates of each concentration were tested. At 72 hours, cell viability was assessed.

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Cell Proliferation Assessment: Cells were plated at 3,000 cells/well in 96-well plates. After overnight incubation, cells were treated with 9-point 3-fold dilutions of CC-90003 or DMSO control in triplicate. Cell viability was measured 72 hours later using a luminescent cell viability assay reagent according to the manufacturer's instructions, and luminescence was read on a plate reader. [1]
PDX Ex Vivo 3D Clonogenic Assay: Dissociated patient-derived tumor cells were grown in multi-layer soft-agar. Twenty-four hours after seeding, test compounds were added at 10 concentrations ranging from 10 to 0 µM. Cultures were incubated for 8-13 days until colonies >50 µm in diameter formed. Colonies were then stained and counted using an automatic image analysis system. Sigmoidal concentration-response curves were fitted to the data. [1]
Mammocult Assay for Stem-like Cells: PDX-derived cells (150,000 cells/well) were plated in ultra-low attachment plates in MammoCult medium supplemented with hydrocortisone and heparin, with or without compounds. After tumor spheres formed (approximately 7 days), spheres were imaged, collected, dissociated with trypsin, and a portion was re-plated for secondary sphere formation. This passaging was repeated for up to 5 passages. RNA was extracted from cells for gene expression analysis. [1]

Female athymic nude mice inoculated subcutaneously with 5×106 HCT-116 cancer cells
100 mg/kg qd and 50 mg/kg qd; 25 mg/kg b.i.d. and 12.5 mg/kg b.i.d.
orally

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HCT-116 Xenograft Study: Female athymic nude mice were inoculated subcutaneously with 5 x 10⁶ HCT-116 cells. Twelve days post-implant, when tumors reached 108-126 mm³, mice were randomized and treated orally once or twice daily with vehicle (5% DMSO, 15% Solutol, 80% PBS) or CC-90003 (free base) at various doses (12.5-100 mg/kg). [1]
PDX In Vivo Studies: PDX models were grown in immunocompromised mice. For efficacy studies, mice bearing established tumors were randomized and treated orally with CC-90003 (salt formulation, bioequivalent to free base 50 or 100 mg/kg) once daily, or with vehicle. Tumor volumes were measured twice weekly. For the combination study with docetaxel, CC-90003 was administered orally at 50 mg/kg qd either continuously (days 0-27) or intermittently (4 days on/3 days off), and docetaxel was administered intravenously at 15 mg/kg on specified days (e.g., days 0 & 7, or days 5 & 12). [1]

In the HCT-116 xenograft study, CC-90003 was generally well tolerated at a dose of 100 mg/kg once daily. However, doses of 50 mg/kg twice daily and 75 mg/kg twice daily resulted in death during days 6 to 18 of the study. [1]

[1]. Mol Cancer Res . 2019 Feb;17(2):642-654.

CC-90003 is a covalent, potent, and selective ERK1/2 inhibitor designed to bind to specific cysteine residues at ATP binding sites. Its covalent mechanism enables durable target inhibition. [1]
This study revealed a potential resistance mechanism to ERK inhibition in a KRAS-mutant colorectal cancer PDX model (CXF-243), involving enhanced baseline MAPK pathway signaling and activation of parallel pathways (JNK/JUN, MSK), enabling tumor cells to survive even with significant ERK occupancy by the drug. [1]
In a KRAS-mutant lung cancer PDX model, the combination of CC-90003 and docetaxel not only significantly inhibited tumor growth but also modulated stem cell networks (e.g., downregulation of CCND2, Dkk1, CXCL12, ALDH1A1), suggesting its role in cancer stem cells, which may help prevent tumor recurrence. [1]

C22H21F3N6O2

458.44

458.17

C, 57.64; H, 4.62; F, 12.43; N, 18.33; O, 6.98

1621999-82-3

90331177

White to off-white solid powder

4.6

3

10

7

33

667

0

ILUKRINUNLAVMH-UHFFFAOYSA-N

InChI=1S/C22H21F3N6O2/c1-5-18(32)28-17-8-12(2)6-7-15(17)29-20-14(22(23,24)25)11-27-21(31-20)30-16-9-19(33-4)26-10-13(16)3/h5-11H,1H2,2-4H3,(H,28,32)(H2,26,27,29,30,31)

N-[2-[[2-[(2-methoxy-5-methylpyridin-4-yl)amino]-5-(trifluoromethyl)pyrimidin-4-yl]amino]-5-methylphenyl]prop-2-enamide

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: ≥ 2.5 mg/mL (5.45 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 (5.45 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 (5.45 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.)