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Purity: ≥98%
Ro 5126766 (also known as Ro-5126766; CH5126766) is a novel, potent, first-in-class dual inhibitor of MEK/RAF mitogen-activated protein kinases (MAPKs) with anticancer activity. With IC50 values of 8.2, 56, 160 nM, and 190 nM, respectively, it allosterically inhibits BRAFV600E, CRAF, MEK, and BRAF. RO5126766 may have anti-neoplastic effects because it is a protein kinase inhibitor with a focus on the Raf and MEK kinases. The target gene transcription that encourages the malignant transformation of cells was inhibited by RO5126766's inhibition of the kinase activities of Raf and MEK. Both Raf and MEK are serine/threonine-specific kinases that react to extracellular stimuli like mitogens and participate in the control of cellular processes like gene expression, mitosis, differentiation, and apoptosis.
| Targets |
MEK (IC50 = 160 nM); BRAF V600E (IC50 = 8.2 nM); Braf (IC50 = 190 nM); CRAF (IC50 = 56 nM)
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| ln Vitro |
Avutometinib (Ro 5126766), an allosteric inhibitor, binds to MEK directly and prevents RAF from phosphorylating it by creating a stable RAF-MEK complex. Ro 5126766 blocks both RAF's phosphorylation of MEK and MEK's activation of ERK. Avutometinib effectively prevents ERK2 activation by MEK1 in cell-free MEK and RAF kinase assays with an IC50 of 160 nM (SD=±0.043) and prevents BRAF, BRAFV600E, and CRAF from phosphorylating MEK1 protein (IC50=190 nM, SD=±0.003, IC50=8.2 nM, SD=±0.0015), and CRAF (IC50=56 nM, SD=±0.016) respectively. In a variety of human tumor cell lines, including KRAS/HRAS and BRAF mutant cell lines and KRAS/HRAS and BRAF wild-type cells, avatometinib efficiently inhibits both MEK and ERK phosphorylation[1]. Human breast cancer MDA-MB-231 cells with KRAS and BRAF mutations are treated with avatometinib, with or without statins, inhibiting the rate-limiting enzyme in the mevalonate pathway. This is done to determine whether the mevalonate pathway affects the sensitivity to MEK inhibitors. In a dose-dependent manner, the combined treatment of avutometinib and XU 62-320 shows a more notable reduction in cell growth than the individual treatment of avutometinib alone. The significant combined effects of avutometinib at a concentration of 40 nM and XU 62-320 at a concentration of 0.3 μM are also confirmed to have suppressed cell colony formation[2].
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| ln Vivo |
Avutometinib (Ro 5126766) inhibits growth and induces tumor regressions in KRAS-mutant xenograft models more efficiently than PD0325901, another allosteric MEK inhibitor. Ro 5126766 has an ED50 of 0.03 to 0.23 mg/kg and an ED90 of 0.15 to 1.56 mg/kg, according to preclinical data from a number of mouse xenograft models with human tumors. Target trough concentrations of 17 to 133 ng/L and 87 to 901 ng/mL, respectively, are linked to these effective doses. [1]. The HCT116 model is used in this experiment to administer Avutometinib or PD0325901 at their respective maximum tolerated doses (MTDs) of 1.5 and 25 mg/kg, respectively. In the tumors from the drug-treated mice, these doses inhibit pERK and ERK signaling output to comparable degrees 4 hours after the initial drug administration. Additionally, the ED50 for avutometinib and PD0325901 in HCT116 models are 0.056 and 0.80 mg/kg, respectively. As a result, the doses used in this experiment are 26.8 and 31.3 times higher than the doses with a 50% efficacy, respectively. Each of these tumors significantly regresses when either medication is taken orally daily. However, while tumor models receiving PD0325901 become refractory after 10 days of treatment, tumor models receiving avutometinib lose their ability to inhibit tumor growth for the full 28-day treatment period[3].
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| Enzyme Assay |
The inhibitory activities against CRAF, BRAF, or BRAF V600E enzymes are measured by quantification of phosphorylation of inactive K97R MEK1 [MEK1] by recombinant RAF proteins [BRAF: B-RAF wt, BRAF V600E: B-RAF V600E or CRAF: Raf-1] with Europium-anti-MEK1/2 (pSer218/222) antibody and SureLight allophycocyanine-anti-6his antibody by measuring time-resolved fluorescence (TRF). Alternately, using the IMAP fluorescence polarization (FP) Screening Express Kit, the inhibitory activities against the RAF enzymes are quantified by quantifying the phosphorylation of a fluorescein-labeled peptide corresponding to human MEK1 212-224 and human MEK2 217-229 (5-Fl-SGQLIDSMANSFV-NH2, MEKtide). By using active MEK1 (MEK1 S218E/S222E) and inactive, dephosphorylated ERK2 (MAP kinase 2/Erk 2) in a coupled assay, the inhibition of MEK1 is assessed. The IMAP FP Screening Express Kit is used to measure how many times an fluorescently-labeled peptide substrate (FAM-Erktide, IPTTPITTTYFFFK-5FAM-COOH) is phosphorylated by ERK2.
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| Cell Assay |
The number of viable cells is assessed with a Cell Counting Kit-8 assay. Breast cancer in people Human melanoma SK-MEL-28 cells, MDA-MB-231 cells, and non-small cell lung cancer cells all A549 cells are plated in 96-well plates at a density of 2,000 cells per well and allowed to grow for 24 hours before being exposed to Ro 5126766 (10, 20, 40, and 80 nM) for 72 hours. The samples' absorbance at 450 nm is measured using a multi-plate reader after an additional 4 hours of kit reagent incubation[2].
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| Animal Protocol |
Mice: Female BALB-nu/nu mice (CAnN.Cg-Foxn1nu/CrlCrlj nu/nu) are provided with unlimited access to water and standard mouse food. The right flank of the 7- to 9-week-old mice is subcutaneously injected with a total of 5×106 (HCT116) or 1×107 (Calu-6 and COLO205) tumor cells per mouse. Avutometinib (1.5 mg/kg or 2.0 mg/kg), PD0325901 (25 mg/kg), or the vehicle [5% DMSO and 10% 2-hydroxypropyl-β-cyclodextrin (HPCD) solution in distilled water], is given orally once daily to the mice once the tumor volume reaches 200 mm3 (day 0). At the maximum tolerated dose (MTD), medications are administered. Calculated tumor growth inhibition (TGI). For each compound, the value of the 50% effective dose (ED50) is calculated[3].
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| References |
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| Molecular Formula |
C21H18N5O5FS
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|---|---|
| Molecular Weight |
471.46152
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| Exact Mass |
471.10
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| Elemental Analysis |
C, 53.50; H, 3.85; F, 4.03; N, 14.85; O, 16.97; S, 6.80
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| CAS # |
946128-88-7
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| Related CAS # |
946128-88-7
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| Appearance |
Solid powder
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| SMILES |
CC1=C(C(=O)OC2=C1C=CC(=C2)OC3=NC=CC=N3)CC4=C(C(=NC=C4)NS(=O)(=O)NC)F
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| InChi Key |
LMMJFBMMJUMSJS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H18FN5O5S/c1-12-15-5-4-14(31-21-25-7-3-8-26-21)11-17(15)32-20(28)16(12)10-13-6-9-24-19(18(13)22)27-33(29,30)23-2/h3-9,11,23H,10H2,1-2H3,(H,24,27)
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| Chemical Name |
3-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]-4-methyl-7-pyrimidin-2-yloxychromen-2-one
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| Synonyms |
Avutometinib; RO5126766; RO5126766; RO 5126766; CH5126766; CH 5126766; CH5126766
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 94~125 mg/mL (199.4~265.1 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.41 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 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.41 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 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.41 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 5% DMSO+45% PEG 300+ddH2O: 20mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1211 mL | 10.6054 mL | 21.2107 mL | |
| 5 mM | 0.4242 mL | 2.1211 mL | 4.2421 mL | |
| 10 mM | 0.2121 mL | 1.0605 mL | 2.1211 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT03681483 | Active Recruiting |
Drug: RO5126766 | Advanced Non-small Cell Lung Cancer |
Memorial Sloan Kettering Cancer Center |
October 31, 2018 | Early Phase 1 |
| NCT03875820 | Active Recruiting |
Drug: VS-6766 Drug: Defactinib |
NSCLC Pancreatic Cancer |
Institute of Cancer Research, United Kingdom |
December 12, 2017 | Phase 1 |
| NCT00773526 | Completed | Drug: RO5126766 | Neoplasms | Hoffmann-La Roche | November 2008 | Phase 1 |
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