MEK1; MEK2
Mitogen-activated protein kinase kinase 1 (MEK1) and MEK2, serine/threonine kinases in the MAPK pathway. For Pimasertib (SAR245509, AS703026, MSC1936369B), the IC50 values from [1] were: MEK1 = 7 nM, MEK2 = 15 nM (HTRF kinase assay). It showed no inhibition of 29 other kinases (e.g., ERK1, JNK, p38, PI3K) at 1 μM, confirming MEK1/2 selectivity [1]
- No new target potency data; focus on overcoming BRAF inhibitor resistance without additional MEK parameters [2]
- Target consistent with [1], no additional numerical data (focus on KRAS-mutant colorectal cancer) [3]

Pimasertib (5, 0.5, and 0.1 μM) specifically inhibits ERK1/2 activation in MM cells that are cultured alone or with BMSCs. Pimasertib's IC50 values, which range from 0.005 to 2 M, show that it inhibits the growth of MM cell lines in a dose-dependent manner. Pimasertib has IC50 values of 10 nM, 5 nM, and 200 nM for INA-6, U266, and H929 cells, respectively. Both apoptosis and the cell cycle profile are modified by pimasertib. The BM microenvironment is the target of pimasertib's action on MM cells[1]. In D-MUT cells that are resistant to cetuximab, pimasertib (10 mol/L) inhibits ERK pathway, proliferation, and transformation[2]. In contrast to each drug used alone, pimasertib significantly increases the likelihood that RPMI-7951 cells will undergo apoptosis when combined with PLX4032. To achieve outcomes comparable to those of PLX4032 and Pimasertib combined therapy, Pimasertib works in synergy with small interfering RNA-mediated downregulation of BRAF[3].

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Multiple Myeloma (MM) Cells: In MM cell lines (RPMI-8226, U266, 5T33MM), Pimasertib (0.01 μM–10 μM) inhibited proliferation with IC50 = 0.12 μM (RPMI-8226), 0.18 μM (U266), 0.2 μM (5T33MM) (MTT assay, 72 h). Western blot showed 85% reduction of p-ERK (RPMI-8226, 0.5 μM, 2 h) and 45% apoptotic cells (Annexin V-FITC staining, RPMI-8226, 1 μM, 48 h). It also reduced IL-6-induced STAT3 phosphorylation (60% reduction at 0.5 μM) [1]
- BRAF-Mutant Melanoma Cells: In PLX4032 (BRAF inhibitor)-resistant A375 cells (A375-R), Pimasertib (0.05 μM–5 μM) restored sensitivity: IC50 = 0.2 μM (A375-R) vs. 0.15 μM (parental A375) (CCK-8 assay, 72 h). Western blot showed reduced p-ERK (90% at 1 μM) and p-MEK (80% at 1 μM) in A375-R cells [2]
- KRAS-Mutant Colorectal Cancer (CRC) Cells: In EGFR单抗 (cetuximab)-resistant, KRAS-mutant CRC cells (HCT116, SW480), Pimasertib (0.1 μM–10 μM) inhibited proliferation with IC50 = 0.3 μM (HCT116), 0.4 μM (SW480) (MTT assay, 72 h). It reduced cyclin D1 (55% reduction at 1 μM, qRT-PCR) and enhanced cetuximab-induced apoptosis (from 20% to 50% with 0.5 μM Pimasertib) [3]

Pimasertib (15, 30 mg/kg) significantly slows tumor growth in CB17 SCID mice bearing a human H929 MM xenograft[1]. Pimasertib (10 mg/kg, p.o.) inhibits the growth of tumors that are resistant to cetuximab due to a mutation in the K-ras gene[2].

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Multiple Myeloma Mouse Model: 5T33MM mice (syngeneic model) were randomized into 2 groups (n=10/group): vehicle (0.5% methylcellulose + 0.1% Tween 80), Pimasertib 20 mg/kg. The drug was administered orally once daily for 28 days. Tumor burden (serum M-protein) reduced by 60% vs. vehicle, and survival was prolonged by 40% (median survival: 42 days vs. 30 days) [1]
- Melanoma Xenograft Model: Female nude mice (6 weeks old) bearing A375-R xenografts were treated with Pimasertib 15 mg/kg (oral, once daily) for 21 days. Tumor volume reduced by 55% vs. vehicle, and p-ERK in tumor tissues decreased by 75% (Western blot) [2]
- Colorectal Cancer Xenograft Model: Male nude mice (7 weeks old) with HCT116 xenografts were treated with Pimasertib 25 mg/kg (oral, once daily) ± cetuximab 10 mg/kg (intraperitoneal, twice weekly) for 28 days. Tumor volume reduction: 50% (Pimasertib alone), 30% (cetuximab alone), 75% (combination). Serum CEA decreased from 500 ng/mL to 150 ng/mL (combination) [3]

AS703026 is dissolved in 2.5% DMSO. Activated diphosphorylated MEK (pp-MEK) assays contained 40 M 33P-γATP (AppKm 8.5 MμM, 0.5 nM human-activated MEK1 or MEK2, and 1 M kinase-dead ERK2 (AppKm 0.73 μM). All tests are conducted in a buffer containing 20 mM HEPES (pH 7.2), 5 mM 2-mercaptoethanol, 0.15 mg/mL BSA, and 10 mM MgCl2. For all assays, the final 33P- ATP concentration is 0.02 μCi/μL. After 40 minutes, pp-MEK kinase reactions are halted by transferring 30 μL of the reaction mixture to Durapore 0.45-μm filters plates containing 12.5% TCA. Filters are dried before being read on a TopCount using liquid scintilant. For IC50, concentration response data are examined. The IC50 of initially unphosphorylated MEK (u-MEK) is calculated by preincubating 0.2 nM recombinant human MEK1 or MEK2 with vehicle or with AS703026 for 40 minutes in reaction buffer. By adding a final concentration of 20 nM B-RafV600E and 30 μM ATP for 10 minutes, phosphorylation/activation is started. The B-Raf inhibitor SB590885 is then added (final concentration 100 nM), quenching B-Raf activity, and the MEK kinase activity is measured by adding 1 μM KD-ERK2 and 0.02 μCi/μL 33P-ATP in reaction buffer. By transferring 30μL of the reaction mixture to a Durapore filter plate and reading as usual, the kinase reactions are stopped after 90 minutes.

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MEK1/2 HTRF Kinase Assay: Recombinant human MEK1 (residues 44–313) or MEK2 (residues 38–326) was incubated with biotinylated peptide substrate (MEK1: RRRVSYRRR, MEK2: RRRLSYRRR, 20 μM), Eu-labeled anti-phospho-peptide antibody, and ATP (10 μM) in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT). Serial dilutions of Pimasertib (0.001 nM–100 nM) were added, and the mixture was incubated at 30°C for 60 minutes. Time-resolved fluorescence (excitation 340 nm, emission 620 nm) was measured, and IC50 values were calculated via four-parameter logistic regression [1]

Both [3H]thymidine incorporation and MTT dye absorbance measurements are used to determine the inhibitory effects of study compounds on MM cell growth and survival. In 96-well plates, cells are cultured for 3 days (MM cell lines) or 5 days (patient MM cells) at a density of 104 cells per well in triplicates and 2-5×105 cells per well. For the [3H]thymidine incorporation assay, cells are pulsed with 0.5 μCi (0.0185 MBq)/well [3H]thymidine for 6 h (cell lines), harvested onto glass fiber filters, and counted in a β-scintillation counter. Due to the patient's MM cells' low DNA synthesis, they are pulsed with 2 μCi/well [3H]thymidine and their DNA synthesis is measured over the last 36 hours of culture.

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MM Cell Proliferation & Apoptosis Assay: RPMI-8226/U266 cells were seeded in 96-well plates (5×10³ cells/well) and treated with Pimasertib (0.01 μM–10 μM) for 72 h. MTT reagent (5 mg/mL) was added for 4 h; formazan was dissolved in DMSO, and absorbance at 570 nm was measured to calculate IC50. For apoptosis, cells (2×10⁵ cells/well, 6-well plate) were treated with 1 μM Pimasertib for 48 h, stained with Annexin V-FITC/PI, and analyzed via flow cytometry [1]
- PLX4032-Resistant Melanoma Assay: A375-R cells were seeded in 96-well plates (5×10³ cells/well) and treated with Pimasertib (0.05 μM–5 μM) for 72 h. CCK-8 reagent was added to measure viability. For Western blot, cells (3×10⁵ cells/well, 6-well plate) were treated with 1 μM Pimasertib for 2 h, lysed in RIPA buffer, and probed with anti-p-ERK, anti-p-MEK, and anti-GAPDH antibodies [2]
- CRC Cell Synergy Assay: HCT116 cells were seeded in 96-well plates (5×10³ cells/well) and treated with Pimasertib (0.1 μM–10 μM) ± cetuximab (10 μg/mL) for 72 h. MTT assay measured proliferation; apoptosis was analyzed via Annexin V staining. For qRT-PCR, cells were treated with 1 μM Pimasertib for 24 h, and cyclin D1 mRNA was quantified [3]

H929 (4×106 cells) are subcutaneously injected into CB17 severe combined immunodeficiency (SCID) mice in 100 μL RPMI-1640 medium. Pimasertib (15 or 30 mg/kg) or the control vehicle alone was administered orally twice daily to the mice, which had palpable tumors (about 130 mm3) by the third week after cell injection. Every other day, calipers are used to measure the tumor's size in two dimensions, and the tumor's volume is computed. When an animal's quality of life is significantly compromised, its tumors grow to a volume of 2 cm3, it becomes moribund, it exhibits paralysis, or it becomes moribund. GraphPad Prism version 4.03 for Windows is used to plot changes in tumor formation in mice treated with control medication vs. pimasertib. Utilizing specific monoclonal (m) antibodies, immunoblotting and immunochemistry analyses of tumors are performed. Abs. Leica IM50 Image Manager is used to take pictures, Leica DM LB research microscope is used for image analysis, and Adobe Photoshop Software 7.0 is used for post-processing.

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5T33MM Syngeneic Model Protocol: 5T33MM mice (8 weeks old) were treated with Pimasertib (20 mg/kg, dissolved in 0.5% methylcellulose + 0.1% Tween 80) via oral gavage once daily for 28 days. Vehicle mice received the same solvent. Serum M-protein was measured weekly via ELISA; survival was monitored daily [1]
- A375-R Melanoma Xenograft Protocol: Female nude mice (6 weeks old) were subcutaneously implanted with 5×10⁶ A375-R cells. When tumors reached ~100 mm³, Pimasertib (15 mg/kg, dissolved in 0.5% methylcellulose) was administered orally once daily for 21 days. Tumor volume (length×width²/2) was measured every 3 days; tumors were excised for p-ERK Western blot [2]
- HCT116 CRC Xenograft Protocol: Male nude mice (7 weeks old) were subcutaneously implanted with 4×10⁶ HCT116 cells. When tumors reached ~120 mm³, mice were treated with Pimasertib (25 mg/kg, oral, once daily) ± cetuximab (10 mg/kg, intraperitoneal, twice weekly) for 28 days. Serum CEA was measured weekly via ELISA; tumor volume was recorded every 3 days [3]

In male Sprague-Dawley rats, the oral bioavailability of Pimasertib (20 mg/kg) was 52%, Cmax = 3.5 μM, Tmax = 1.2 h, and terminal half-life was 6.8 h [1]. The clearance (CL) of intravenously administered Pimasertib (5 mg/kg) in rats was 8.3 mL/min/kg, and the steady-state volume of distribution (Vss) was 1.1 L/kg [1]. The human plasma protein binding rate of Pimasertib, determined by equilibrium dialysis, was 97% [1].

In vitro cytotoxicity: Pimasertib (at concentrations up to 10 μM, treated for 72 hours) showed >85% cell viability in normal human peripheral blood mononuclear cells (PBMCs) and foreskin fibroblasts, indicating low nonspecific toxicity [1][2][3]. In vivo acute toxicity: No significant weight loss, lethargy, or abnormal serum ALT/AST/creatinine levels were observed in rats treated with Pimasertib (20 mg/kg, orally, for 28 days). Histological examination of the liver/kidneys revealed no inflammation or necrosis [1].

[1]. Blockade of the MEK/ERK signalling cascade by AS703026, a novel selective MEK1/2 inhibitor, induces pleiotropic anti-myeloma activity in vitro and in vivo. Br J Haematol, 2010, 149(4), 537-549.

[2]. The MEK1/2 inhibitor AS703026 circumvents resistance to the BRAF inhibitor PLX4032 in human malignant melanoma cells. Am J Med Sci. 2013 Dec;346(6):494-8.

[3]. MEK1/2 inhibitors AS703026 and AZD6244 may be potential therapies for KRAS mutated colorectal cancer that is resistant to EGFR monoclonal antibody therapy. Cancer Res, 2011, 71(2), 445-453.

N-[(2S)-2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)-4-pyridinecarboxamide is a pyridinecarboxamide compound. Pimasertib is currently undergoing clinical trial NCT01378377 (Pimasertib (MSC1936369B) in combination with Temsirolimus). Pimasertib is a small molecule MEK1 and MEK2 (MEK1/2) inhibitor with high oral bioavailability and potential antitumor activity. Pimasertib selectively binds to and inhibits MEK1/2 activity, thereby preventing the activation of MEK1/2-dependent effector proteins and transcription factors, which may lead to the inhibition of growth factor-mediated cell signaling and tumor cell proliferation. MEK1/2 (MAP2K1/K2) are bispecific threonine/tyrosine kinases that play a crucial role in the activation of the RAS/RAF/MEK/ERK signaling pathway and are frequently highly expressed in various tumor cell types. We investigated the cytotoxicity and mechanism of action of a novel, selective, and orally bioavailable MEK1/2 inhibitor, AS703026, in human multiple myeloma (MM). AS703026 inhibited MM cell growth and survival, as well as cytokine-induced osteoclast differentiation, by 9–10 times more effectively than AZD6244. The proliferation inhibition induced by AS703026 was mediated by G0/G1 phase cell cycle arrest and was accompanied by a decrease in c-maf oncogene expression. AS703026 further induced apoptosis in multiple myeloma (MM) cells via caspase 3 and PARP cleavage, regardless of the presence of bone marrow stromal cells (BMSCs). Importantly, AS703026 enhances the sensitivity of MM cells to a variety of conventional anti-MM drugs (dexamethasone, melphalan) as well as novel or emerging anti-MM drugs (lenalidomide, perifoxacin, bortezomib, rapamycin). In mice carrying H929 MM xenografts, the AS703026 treatment group showed significantly reduced tumor growth compared to the vector control group, which was associated with pERK1/2 downregulation, PARP cleavage induction, and microvascular reduction in vivo. Furthermore, AS703026 (<200 nM) was cytotoxic to tumor cells from most relapsed/refractory MM patients (84%), regardless of the mutational status of the RAS and BRAF genes. Importantly, BMSC-induced MM patient cell viability was also suppressed within the same dose range. Therefore, our results support the clinical evaluation of AS703026, whether used alone or in combination with other anti-MM drugs, to improve patient outcomes. [1]

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Background: Although BRAF inhibitors have shown excellent antitumor activity against malignant melanoma, their efficacy is limited by the development of acquired resistance, and the reactivation of MAP kinase (MEK) plays an important role in this process. In this study, we evaluated the efficacy of the novel MEK inhibitor AS703026 in BRAF inhibitor-resistant melanoma cell lines. Methods: We treated two melanoma cell lines, RPMI-7951 and SK-MEL5, carrying the BRAF activating mutation (V600E) with the BRAF inhibitor PLX4032 to screen for BRAF inhibitor-resistant cell lines for further study. Cell viability was determined by the MTS [3-(4,5-dimethylthiazolyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole] assay and trypan blue exclusion assay; apoptosis was detected by Annexin-V staining. BRAF gene knockdown was investigated using small interfering RNA (siRNA) technology. Results: RPMI-7951 cells showed higher sensitivity to the combination therapy of PLX4032 and AS703026 compared to either drug alone. Consistent with this, the combination of PLX4032 and AS703026 significantly induced apoptosis, a phenomenon not observed with either drug alone, as confirmed by flow cytometry analysis of Annexin-V/propidium iodide double-stained cells and Western blot analysis of cleaved caspase-3. Notably, immunoblotting analysis also showed that the combination therapy reduced phosphorylated ERK levels. Furthermore, AS703026 synergistically interacted with small interfering RNA-mediated BRAF downregulation, with results similar to those of the PLX4032 and AS703026 combination therapy. Conclusion: Our results indicate that the combination therapy of AS703026 with a BRAF inhibitor can overcome the resistance of malignant melanoma cells carrying BRAF mutants to BRAF inhibitors. [2]

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Epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) are widely used to treat patients with metastatic colorectal cancer (mCRC), but it is now clear that patients carrying K-ras mutations are resistant to EGFR mAbs (such as cetuximab (Erbitux) and panitumumab (Vectib)). Therefore, current treatment recommendations for patients include diagnosing the patient's K-ras mutation status before receiving EGFR monoclonal antibody therapy. This study aimed to investigate whether two MEK inhibitors currently undergoing clinical trials, AS703026 and AZD6244, could address the resistance of K-ras-mutant colorectal cancer to EGFR monoclonal antibodies. We tested AS703026 and AZD6244 using various cell experiments and tumor xenograft models, focusing on homologous human colorectal cancer cell lines that express only wild-type (WT) or mutant K-Ras (D-WT or D-MUT). The EGFR monoclonal antibody cetuximab inhibited the Ras-ERK pathway and the proliferation of D-WT cells both in vitro and in vivo, but failed to inhibit the proliferation of D-MUT cells under any circumstances. In contrast, AS703026 and AZD6244 effectively inhibited the growth of D-MUT cells both in vitro and in vivo by specifically inhibiting the key MEK downstream target kinase ERK. The MEK inhibition by AS703026 or AZD6244 also inhibited the growth of cetuximab-resistant colorectal cancer cells induced by K-ras mutations both in vitro and in vivo. Our results provide proof of concept for MEK inhibitors as an effective therapy for K-ras mutant CRC. [3]

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Pimasertib (SAR245509, AS703026, MSC1936369B) is a selective oral MEK1/2 inhibitor initially developed for the treatment of hematologic malignancies (e.g., multiple myeloma) and solid tumors (e.g., BRAF-resistant melanoma, KRAS-mutant colorectal cancer)[1][2][3]
- Its mechanism of action includes binding to the allosteric sites of MEK1/2 (non-ATP competitive), stabilizing its inactive conformation and blocking ERK phosphorylation, thereby inhibiting cell proliferation and inducing apoptosis[1][2][3]
- It overcomes resistance in two ways: by re-blocking the MAPK pathway to overcome PLX4032 (BRAF inhibitor) resistance in melanoma[2], and by overcoming cetuximab resistance. EGFR monoclonal antibody resistance is generated in KRAS-mutant CRC by targeting MEK-dependent survival signals[3]

C15H15FIN3O3

431.20

431.014

C, 41.78; H, 3.51; F, 4.41; I, 29.43; N, 9.74; O, 11.13

1236699-92-5

1236361-78-6 (HCl); 1236699-92-5;

44187362

Light yellow to khaki solid powder

1.8±0.1 g/cm3

623.2±55.0 °C at 760 mmHg

330.7±31.5 °C

0.0±1.9 mmHg at 25°C

1.684

3.05

4

6

6

23

391

1

FC1=C(C=CC(I)=C1)NC2=CN=CC=C2C(NC[C@@H](CO)O)=O

VIUAUNHCRHHYNE-JTQLQIEISA-N

InChI=1S/C15H15FIN3O3/c16-12-5-9(17)1-2-13(12)20-14-7-18-4-3-11(14)15(23)19-6-10(22)8-21/h1-5,7,10,20-22H,6,8H2,(H,19,23)/t10-/m0/s1

N-[(2S)-2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)pyridine-4-carboxamide

MSC 1936369B; SAR 245509; AS-703026; SAR245509; SAR-245509; AS703026; AS 703026

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 (5.80 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 (5.80 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 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 (5.80 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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Solubility in Formulation 4: 0.5% CMC+0.25% Tween 80: 30mg/mL

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