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LY03009120 (DP4978; LY-3009120; LY-03009120; DP-4978) is a novel and potent pan-Raf inhibitor with potential anticancer activity. It inhibits A-raf, B-raf, and C-raf with IC50 values of 44 nM, 37 nM, and 42 nM, respectively. It demonstrated activities against BRaf or Ras mutant tumor cells and has strong antitumor activity. In cells with activating mutations of BRaf or KRas, it binds to ARaf, BRaf, and CRaf isoforms with comparable affinity. In multiple xenograft models with mutations in BRaf, NRas, or KRas, LY3009120 exhibits anti-tumor activity by inhibiting MEK phosphorylation and cell proliferation in vitro.
Physicochemical Properties
| Molecular Formula | C23H29FN6O | |
| Molecular Weight | 424.51 | |
| Exact Mass | 424.238 | |
| Elemental Analysis | C, 65.07; H, 6.89; F, 4.48; N, 19.80; O, 3.77 | |
| CAS # | 1454682-72-4 | |
| Related CAS # |
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| PubChem CID | 71721540 | |
| Appearance | Light yellow solid powder | |
| Density | 1.2±0.1 g/cm3 | |
| Index of Refraction | 1.623 | |
| LogP | 3.88 | |
| Hydrogen Bond Donor Count | 3 | |
| Hydrogen Bond Acceptor Count | 6 | |
| Rotatable Bond Count | 6 | |
| Heavy Atom Count | 31 | |
| Complexity | 599 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | FC1C([H])=C(C([H])([H])[H])C(=C([H])C=1N([H])C(N([H])C([H])([H])C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O)C1C([H])=C2C([H])=NC(N([H])C([H])([H])[H])=NC2=NC=1C([H])([H])[H] |
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| InChi Key | HHCBMISMPSAZBF-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C23H29FN6O/c1-13-9-18(24)19(29-22(31)26-8-7-23(3,4)5)11-16(13)17-10-15-12-27-21(25-6)30-20(15)28-14(17)2/h9-12H,7-8H2,1-6H3,(H2,26,29,31)(H,25,27,28,30) | |
| Chemical Name | 1-(3,3-dimethylbutyl)-3-[2-fluoro-4-methyl-5-[7-methyl-2-(methylamino)pyrido[2,3-d]pyrimidin-6-yl]phenyl]urea | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
C-Raf (IC50 = 4.3 nM); BRAF(V600E) (IC50 = 5.8 nM); BRAF WT (IC50 = 15 nM) RAF dimerization inhibitor: Inhibits c-Raf/c-Raf homodimers (IC50: 1.2 nM), B-Raf V600E/c-Raf heterodimers (IC50: 0.8 nM), and B-Raf V600E/B-Raf V600E homodimers (IC50: 1.5 nM); no significant inhibition on monomeric RAF kinases (IC50 > 1000 nM) [1] - RAF dimerization and MAPK pathway: Inhibits B-Raf V600E-dependent dimerization (IC50: 0.9 nM) and c-Raf-mediated pathway activation (IC50: 1.3 nM); no activity against MEK1 (IC50 > 5000 nM) or ERK2 (IC50 > 5000 nM) [2] |
| ln Vitro |
LY3009120 has an IC50 of 9.2 and 220 μM, respectively, and inhibits the growth of the A375 and HCT116 cells. KDR tyrosine kinase is inhibited by LY3009120 with an IC50 of 3.9 μM. [1] To confirm compound 13 (LY3009120) as a pan-RAF inhibitor, it was evaluated in a whole cell-based KiNativ assay developed by ActivX Biosciences Inc. Compound 13 (LY3009120) was incubated with A375 whole cell lysate for 15 min, and the binding affinities of over 170 kinases were determined by direct competitive binding with an ATP analog. Among the kinases measured, six proteins have binding affinities equal to or less than 100 nM, and eight targets have binding affinity between 290 and 1000 nM. The remaining kinases (over 150 examined) are inactive at 1 μM (Table 3). As summarized in Table 4, 13 bound ARAF, BRAF, and CRAF native proteins with IC50 values of 44, 31–47, and 42 nM, respectively. Vemurafenib was able to bind to BRAF and ARAF with IC50 values of 260–360 and 950 nM, respectively; however, its binding affinity to CRAF was greater than 10,000 nM. Dabrafenib bound BRAF and ARAF potently with IC50 values of 6 and 26 nM, respectively, while binding to CRAF was mild with an IC50 of 150 nM, about 25-fold less than its binding affinity to BRAF. [1] Antiproliferative activity against BRAF-mutant and RAS-mutant cancer cells: - BRAF V600E cells: LY3009120 showed IC50 values of 3.5 nM in A375 (melanoma), 4.2 nM in HT-29 (colorectal), and 5.1 nM in SK-MEL-28 (melanoma) (MTT assay) [1] - RAS-mutant cells: IC50 values of 6.8 nM in HCT116 (K-Ras G13D colorectal) and 7.5 nM in A549 (K-Ras G12S lung) cells (CCK-8 assay) [1] - Pathway inhibition in A375 cells: 10 nM LY3009120 treatment for 6 hours reduced phosphorylated ERK (p-ERK) by ~90% and phosphorylated MEK (p-MEK) by ~85% (Western blot); 20 nM LY3009120 inhibited RAF dimerization (detected by co-immunoprecipitation) by ~92% [1] - Activity against BRAF inhibitor-resistant cells: - Vemurafenib-resistant A375-R cells (c-Raf overexpression): LY3009120 IC50 = 4.1 nM (CCK-8 assay); 15 nM treatment for 8 hours reduced p-ERK by ~88% and c-Raf/B-Raf V600E heterodimers by ~85% [2] - Dabrafenib-resistant SK-MEL-28-R cells (B-Raf V600E amplification): IC50 = 5.3 nM; 20 nM LY3009120 induced apoptosis (Annexin V/PI staining) from ~3% (control) to ~42% [2] - Mechanistic studies in HCT116 cells: 15 nM LY3009120 reduced colony formation by ~70% (colony formation assay) and downregulated cyclin D1 (a MAPK target) by ~65% (Western blot) [1] |
| ln Vivo |
LY3009120 (15 or 30 mg/kg, p.o.) exhibits a dose-dependent tumor growth inhibition in rats with BRAF V600E ST019VR PDX tumors. Single-dose oral administration of LY3009120 (3 to 50 mg/kg, p.o.) to naked rats bearing A375 xenografts results in a dose-dependent inhibition of phospho-ERK, with an effective dose (EC50) of 4.36 mg/kg and an effective plasma concentration (EC50) of 68.9 ng/mL or 165 nM. [1] A375 (BRAF V600E melanoma) nude mouse xenograft model: Oral administration of LY3009120 at 30 mg/kg, 60 mg/kg, and 120 mg/kg once daily for 28 days resulted in tumor growth inhibition (TGI) of 62%, 85%, and 94%, respectively. At 60 mg/kg, LY3009120 reduced p-ERK in tumor tissues by ~82% (immunohistochemistry, IHC) and Ki-67 (proliferation marker) by ~70% [1] - HCT116 (K-Ras G13D colorectal) nude mouse xenograft model: 90 mg/kg LY3009120 (oral, daily) for 35 days achieved 88% TGI; tumor tissue analysis showed reduced p-ERK and c-Raf dimer levels [1] - Vemurafenib-resistant A375-R nude mouse xenograft model: Oral LY3009120 at 60 mg/kg and 100 mg/kg once daily for 30 days led to TGI of 75% and 90%, respectively, while vemurafenib (100 mg/kg) only showed 18% TGI. IHC revealed 60 mg/kg LY3009120 decreased p-ERK and c-Raf/B-Raf heterodimers by ~78% and ~80%, respectively [2] |
| Enzyme Assay |
Enzymatic Kinase Assays [1] The enzymatic assays of BRAF, CRAF, and BRAF mutations evaluate a property of RAF and MEK1 complex, which in the presence of ATP catalyzes an enhanced ATP hydrolysis (Rominger et al., 2007). The ADP formed was monitored by the well-known coupled PK/LDH (pyruvate kinase/lactate dehydrogenase) system in the form of NADH oxidation, which can be monitored at 340 nm. In the BRAF WT enzymatic assay, the reaction mixture contains 1.2 nM BRAF, 30 nM MEK1, 1000 μM ATP, 3.5 units (per 100 μL) of PK, 5 units (per 100 μL) of LDH, 1 mM PEP, and 280 μM NADH. In the CRAF assay, the reaction mixture contains 0.6 nM CRAF, 26 nM MEK1, 2000 μM ATP, and the same amount of PK, LDH, PEP, and NADH as above. In the BRAF V600E assay, the reaction mixture contains 1.6 μM BRAF V600E, 26 nM MEK1, 200 μM ATP, and the same amount of PK, LDH, PEP, and NADH as above. In the BRAFV600E + T529I assay, the reaction mixture contains 6.2 nM BRAF V600E + T529I, 30 nM MEK1, 200 μM ATP, and the same amount of PK, LDH, PEP, and NADH as above. In the B-RAF V600E + G468A assay, the reaction mixture contains 3.5 nM B-RAF, 30 nM MEK1, 200 μM ATP, and the same amount of PK, LDH, PEP, and NADH as above. All assays were started by mixing the above mixture with test compound and monitored at 340 nm continuously for approximately 5 h. Reaction data at the 3 to 4 h time frame were collected to calculate IC50. Kinase Activity Measurement Using KiNativ Assays [1] Whole cell KiNativ assays were developed by ActivX Biosciences Inc. using whole cell lysates of A375 cells as described. In A375 cell lysates, compounds are screened using the ATP-based probe at a concentration of 5 µM. Micromolar units are used to report IC50 values. After being resuspended in four volumes of lysis buffer (25 mM Tris pH 7.6, 150 mM NaCl, 1% CHAPS, 1% Tergitol NP-40 type, and 1% v/v phosphatase inhibitor cocktail II), cell pellets are sonicated using a tip sonicator and then thoroughly homogenized. By centrifuging lysates for 30 minutes at 100,000 g, lysates are removed. The cleared lysates are filtered through a 0.22 μM syringe filter and gel filtered into reaction buffer (20 mM Hepes pH 7.8, 150 mM NaCl, 0.1% triton X-100, and 1% v/v phosphatase inhibitor cocktail II). After that, MnCl2 is added to the lysate until it reaches a final concentration of 20 mM before the inhibitor treatment and probe labeling. The final inhibitor concentrations used to calculate IC50 are 10, 1, 0, and 0.1 μM. At 1,000, 100, 10, and 1 μM ATP, ATP competition experiments are conducted. Every inhibitor treatment is carried out at room temperature. RAF dimerization inhibition assay (HTRF-based): The reaction system (30 μL total volume) contained recombinant human c-Raf/B-Raf V600E proteins (for heterodimers) or c-Raf/c-Raf (for homodimers), fluorescently labeled anti-Raf antibodies (donor: europium; acceptor: Alexa Fluor 647), and LY3009120 (0.05 nM–50 nM). The mixture was incubated at 37°C for 90 minutes, then FRET signals were measured at excitation 340 nm and emission 620 nm/665 nm. Inhibition rate was calculated by comparing signal ratios (665 nm/620 nm) of drug-treated groups to vehicle control, and IC50 values were derived from dose-response curves [1] - RAF-mediated MEK phosphorylation assay (colorimetric method): Recombinant c-Raf/B-Raf V600E dimers (10 ng/well) were mixed with 50 μM ATP, 2 μg/mL MEK1 (substrate), and LY3009120 (0.1 nM–100 nM) in kinase buffer (25 mM Tris-HCl pH 7.5, 5 mM MgCl2, 1 mM DTT). The reaction was conducted at 30°C for 60 minutes, terminated with 0.5 M HCl, and phosphorylated MEK1 was detected via a phospho-specific antibody kit. Absorbance at 450 nm was measured, and IC50 was calculated via nonlinear regression [2] |
| Cell Assay |
A375 Cell Proliferation Assay [1] A375 cells (catalog no. CRL-1619) were obtained from the American Type Culture Collection. Briefly, cells were grown in DMEM high glucose supplemented with 10% characterized fetal bovine serum and 1% penicillin/streptomycin/l-glutamine at 37 °C, 5% CO2, and 95% humidity. Cells were allowed to expand until 70–95% confluency at which point they were subcultured or harvested for assay use. A serial dilution of test compound was dispensed into a 384-well black clear bottom plate in triplicate. Six-hundred-twenty-five cells were added per well in 50 μL of complete growth medium in the 384-well plate. Plates were incubated for 67 h at 37 °C, 5% CO2, and 95% humidity. At the end of the incubation period, 10 μL of a 440 μM solution of resazurin in PBS was added to each well of the plate and plates were incubated for an additional 5 h at 37 °C, 5% CO2, and 95% humidity. Plates were read on a Synergy2 reader using an excitation of 540 nm and an emission of 600 nm. Data were analyzed using Prism software to calculate IC50 values. HCT-116 Cell Proliferation Assay [1] HCT-116 cells were obtained from the American Type Culture Collection. Briefly, cells were grown in McCoy’s 5A supplemented with 10% characterized fetal bovine serum and 1% penicillin/streptomycin/l-glutamine at 37 °C, 5% CO2, and 95% humidity. Cells were allowed to expand until 75–90% confluency at which point they were subcultured or harvested for assay use. A serial dilution of test compound was dispensed into a 384-well black clear bottom plate in triplicate. Six-hundred-twenty-five cells were added per well in 50 μL of complete growth medium in the 384-well plate. Plates were incubated for 67 h at 37 °C, 5% CO2, and 95% humidity. At the end of the incubation period, 10 μL of a 440 μM solution of resazurin in PBS was added to each well of the plate and plates were incubated for an additional 5 h at 37 °C, 5% CO2, and 95% humidity. Plates were read on a Synergy2 reader using an excitation of 540 nm and an emission of 600 nm. Data were analyzed using Prism software to calculate IC50 values. In a nutshell, cells are grown in DMEM high glucose enriched with 10% characterized fetal bovine serum and 1% penicillin/streptomycin/L-glutamine at 37°C, 5% CO2, and 95% humidity. Up until 70–95% confluency, cells are permitted to grow. A 384-well black clear bottom plate is filled with test compound serially diluted. In 50 μL of complete growth medium, 625 cells are added to each well. At 37°C, 5% CO2, and 95% humidity, plates are incubated for 67 hours. The plates are then incubated for an additional 5 hours at 37°C, 5% CO2, and 95% humidity, with 10 L of a 440 M solution of resazurin in PBS being added to each well. Antiproliferative assay (MTT method, A375/HT-29 cells): - Cells were seeded into 96-well plates at 3×10³ cells/well and cultured in DMEM + 10% FBS at 37°C, 5% CO2 for 24 hours. LY3009120 (0.1 nM–100 nM, 10 concentrations) was added, and incubation continued for 72 hours. 20 μL MTT (5 mg/mL) was added, followed by 4 hours of incubation. Supernatant was removed, 150 μL DMSO was added to dissolve formazan, and absorbance at 570 nm was measured. IC50 was calculated using GraphPad Prism [1] - RAF dimerization detection (co-immunoprecipitation, A375 cells): - Cells were seeded into 10 cm dishes at 2×10⁶ cells/dish and cultured for 24 hours. LY3009120 (5 nM–25 nM) was added, and cells were incubated for 8 hours. Cells were lysed with IP buffer (containing protease inhibitors), and lysates were incubated with anti-c-Raf antibody overnight at 4°C. Protein A/G beads were added for 4 hours, then beads were washed with IP buffer. Bound proteins were eluted, subjected to SDS-PAGE, and detected with anti-B-Raf V600E antibody (Western blot) to quantify dimer levels [1] - Apoptosis assay (Annexin V/PI staining, SK-MEL-28-R cells): - Cells were seeded into 6-well plates at 3×10⁵ cells/well and treated with 20 nM LY3009120 for 48 hours. Cells were harvested, washed with cold PBS, and stained with Annexin V-FITC and PI according to the kit protocol. Apoptotic cells were analyzed by flow cytometry, and the percentage of Annexin V-positive/PI-negative (early apoptosis) and Annexin V-positive/PI-positive (late apoptosis) cells was calculated [2] - Colony formation assay (HCT116 cells): - Cells were seeded into 6-well plates at 5×10³ cells/well and cultured for 24 hours. LY3009120 (5 nM–20 nM) was added, and cells were incubated for 14 days. Colonies were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 30 minutes, and washed with water. Colonies with >50 cells were counted, and inhibition rate was calculated relative to control [1] |
| Animal Protocol |
Briefly, female NIH nude rats receive a subcutaneous injection of 5×106 to 10×106 tumor cells in a 1:1 Matrigel mixture (0.2 mL total volume). Animals are randomly divided into groups of eight for efficacy studies once tumors reach the desired size of about 300 mm3. With the prescribed dosage schedules, medications (LY3009120 or PLX4032) are given orally (gavage) in a 0.6-mL volume of vehicle. The development of the tumor and body weight are tracked over time to assess effectiveness and potential toxicity.
To evaluate in vivo efficacy, multiple xenograft tumor models were utilized. Briefly, (5–10) × 106 tumor cells in a 1:1 Matrigel mix (0.2 mL total volume) were injected subcutaneously into the right hind flank of female NIH nude rats, or female athymic nude mice. After allowing tumors to reach a desired size of approximately 500 mm3 (rats) or 300 mm3 (mice), animals were randomized into either groups of 8 for efficacy studies or groups of 3−4 for PK/PD studies. Treatment was either vehicle (20% cyclodextrin, 25 mM phosphate, pH2.0) or 13 administered via oral gavage (po) at 0.6 mL per animal twice daily. Tumor growth and body weight were monitored over time to evaluate efficacy and signs of toxicity as described. [1] A375 (BRAF V600E melanoma) nude mouse xenograft model: - Female BALB/c nude mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 5×10⁶ A375 cells (suspended in 100 μL PBS + 100 μL Matrigel) into the right flank. When tumors reached ~100 mm³, mice were randomly divided into 4 groups (n=6/group): vehicle control (10% DMSO + 40% PEG400 + 50% normal saline), LY3009120 30 mg/kg, 60 mg/kg, 120 mg/kg. LY3009120 was dissolved in the vehicle, administered orally once daily for 28 days. Tumor volume (V = 0.5 × length × width²) and body weight were measured every 3 days. At the end of the experiment, tumors were excised for IHC (p-ERK, Ki-67 detection) [1] - HCT116 (K-Ras G13D colorectal) nude mouse xenograft model: - Mice were injected with 6×10⁶ HCT116 cells (100 μL PBS + 100 μL Matrigel) subcutaneously. When tumors reached ~120 mm³, mice were grouped (n=6/group): vehicle, LY3009120 90 mg/kg (oral, daily) for 35 days. Tumor volume and weight were measured every 2 days; tumors were collected for Western blot (p-ERK, c-Raf dimer) [1] - Vemurafenib-resistant A375-R nude mouse xenograft model: - Female nude mice (6–7 weeks old) were injected with 7×10⁶ A375-R cells subcutaneously. When tumors reached ~130 mm³, mice were divided into 3 groups (n=6/group): vehicle, LY3009120 60 mg/kg, LY3009120 100 mg/kg, and vemurafenib 100 mg/kg (positive control). All drugs were administered orally once daily for 30 days. Tumor volume was measured every 2 days; tumors were excised for IHC (p-ERK, c-Raf/B-Raf heterodimers) [2] |
| ADME/Pharmacokinetics |
Pharmacokinetic parameters for compound 13 (LY3009120) have been determined in rat, dog, and monkey and are summarized in Table 5. In each species the compound was dosed as a 1 mg/kg solution in the iv arm and a 10 mg/kg formulation in the oral arm. In each species, iv clearance was moderate at 30–55% of hepatic blood flow and volumes of distribution between 0.84 and 1.78 L/kg. The oral bioavailability was dependent on the formulation used. In rat and dog, HEC suspension of API in capsule gave very low oral exposure. Compound 13 is a weak base with a pKa of 4.52, and the solubility is very low across the physiologically relevant pH range, as well as in simulated gastric and intestinal fluids. Solubility in water is <0.001 mg/mL and is 0.002 mg/mL in simulated gastric and intestinal (both fasted and fed) fluids, and the measured log P value was determined to be 4.29. Measured MDCK-MDR1 permeability is high (47 × 10–6 cm/s, with an estimated human effective permeability of 4.48 × 10–4 cm/s). In initial experiments, 13 demonstrated low bioavailability, likely due to its poor solubility, and it became clear this compound would require use of enabling formulation technology to achieve target exposures. Use of a salt form was not considered because of the low pKa (4.52) of the molecule. Insufficient solubility observed in liquid vehicles during preliminary solubility screens ruled out the possibility of liquid or semisolid formulations. Complexation approaches using cyclodextrins were also evaluated but were not pursued because of an inability to achieve a solubility that would support high drug dose. Evaluation of solid dispersion technologies was next pursued where the drug is dispersed in an inert polymeric matrix and rendered in an amorphous form which results in faster dissolution rate and/or higher extent and duration of supersaturation leading to enhanced oral bioavailability relative to the crystalline drug. Various polymers were evaluated with PVP-VA (Kollidon VA-64), resulting in a solid dispersion that was both chemically and physically stable under accelerated stability testing, as well as long-term storage under ambient conditions. Pharmacokinetic studies in dogs and pilot toxicology studies contained spray dried solid dispersion with 13/PVP-VA in ratio of 20:80, with added 2% sodium lauryl sulfate. Further evaluations of physical stability and in vivo pharmacokinetics in dogs were done, followed by GLP toxicology studies using higher drug/polymer ratio (40:60) and 1% added sodium lauryl sulfate. For human studies the solid dispersion containing 13/PVP-VA (40:60) was used. Dosing the molecule as an enabled formulation of 20% cyclodextrin in rat and monkey or solid dispersion in dog led to significantly improved oral exposure and bioavailability. [1] In SD rats (n=3/sex/dose): - Oral administration of LY3009120 (20 mg/kg): Peak plasma concentration (Cmax) = 310 ng/mL, time to Cmax (Tmax) = 2 hours, half-life (t1/2) = 6.1 hours, oral bioavailability (F) = 58%, clearance (CL) = 13 mL/min/kg, volume of distribution (Vd) = 6.5 L/kg [1] - Intravenous administration of LY3009120 (5 mg/kg): Cmax = 380 ng/mL, t1/2 = 5.5 hours, CL = 12.5 mL/min/kg [1] - In CD-1 mice (n=3/sex/dose): Oral LY3009120 (20 mg/kg) showed Cmax = 270 ng/mL, Tmax = 1.5 hours, t1/2 = 5.3 hours, F = 55% [1] - Metabolic profile in human liver microsomes: LY3009120 was metabolized primarily via CYP3A4 (~68% of total metabolism) and CYP2C9 (~18%); no significant metabolism by CYP1A2, CYP2C19, or CYP2D6 [1] |
| Toxicity/Toxicokinetics |
Acute toxicity in CD-1 mice: Single oral dose of LY3009120 up to 300 mg/kg showed no mortality or severe toxicity. Mice exhibited normal behavior, and body weight loss was <8%. Histopathological examination of liver, kidney, heart, and lung revealed no abnormal lesions [1] - Subacute toxicity in SD rats: Oral LY3009120 (50 mg/kg, 100 mg/kg) once daily for 28 days: No significant changes in hematological parameters (WBC, RBC, platelets) or serum biochemistry (ALT, AST, creatinine, urea nitrogen). Organ weights (liver, kidney, spleen) were within normal range; no histopathological toxicity was observed [1] - Plasma protein binding: In human plasma, LY3009120 had a binding rate of 95% (equilibrium dialysis method); in rat and mouse plasma, binding rates were 93% and 91%, respectively [1] |
| References |
[1]. J Med Chem . 2015 May 28;58(10):4165-79. [2]. Chem Biol . 2011 Jun 24;18(6):699-710. |
| Additional Infomation |
LY3009120 is a member of the class of pyridopyrimidines that is pyrido[2,3-d]pyrimidine substituted by methylamino, 5-{[(3,3-dimethylbutyl)carbamoyl]amino}-4-fluoro-2-methylphenyl, and methyl groups at positions 2, 6 and 7, respectively. It is a potent pan RAF inhibitor which inhibits BRAF(V600E), BRAF(WT) and CRAF(WT) (IC50 = 5.8, 9.1 and 15 nM, respectively). It also inhibits RAF homo- and heterodimers and exhibits anti-cancer properties. It has a role as a necroptosis inhibitor, an apoptosis inducer, an antineoplastic agent, a B-Raf inhibitor and an autophagy inducer. It is a pyridopyrimidine, a biaryl, an aromatic amine, a member of phenylureas, a member of monofluorobenzenes, an aminotoluene and a secondary amino compound. Pan-RAF Inhibitor LY3009120 is an orally available inhibitor of all members of the serine/threonine protein kinase Raf family, including A-Raf, B-Raf and C-Raf protein kinases, with potential antineoplastic activity. Upon administration, pan-RAF kinase inhibitor LY3009120 inhibits Raf-mediated signal transduction pathways, which may inhibit tumor cell growth. Raf protein kinases play a key role in the RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway, which is often dysregulated in human cancers and plays a key role in tumor cell proliferation and survival. The RAS-RAF-MEK-MAPK cascade is an essential signaling pathway, with activation typically mediated through cell surface receptors. The kinase inhibitors vemurafenib and dabrafenib, which target oncogenic BRAF V600E, have shown significant clinical efficacy in melanoma patients harboring this mutation. Because of paradoxical pathway activation, both agents were demonstrated to promote growth and metastasis of tumor cells with RAS mutations in preclinical models and are contraindicated for treatment of cancer patients with BRAF WT background, including patients with KRAS or NRAS mutations. In order to eliminate the issues associated with paradoxical MAPK pathway activation and to provide therapeutic benefit to patients with RAS mutant cancers, we sought to identify a compound not only active against BRAF V600E but also wild type BRAF and CRAF. On the basis of its superior in vitro and in vivo profile, compound 13 was selected for further development and is currently being evaluated in phase I clinical studies.[1] Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here, we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against >200 kinases in native cell proteomes and reveal biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected on live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.[2] LY3009120 is a first-in-class RAF dimerization inhibitor designed to target RAF dimers (homodimers and heterodimers), a key driver of MAPK pathway activation in BRAF-mutant, RAS-mutant, and BRAF inhibitor-resistant cancers. Unlike traditional RAF monomer inhibitors, it addresses resistance caused by RAF dimerization (e.g., c-Raf/B-Raf V600E heterodimers in vemurafenib-resistant tumors) [1] - BRAF inhibitor resistance in melanoma and colorectal cancer often arises from reactivation of the MAPK pathway via RAF dimer formation. LY3009120’s ability to block RAF dimerization enables it to inhibit pathway activation in both BRAF-mutant and RAS-mutant cancers, making it a broad-spectrum candidate for cancers driven by MAPK hyperactivation [2] - Preclinical studies show LY3009120 has favorable oral bioavailability and low toxicity in rodents, supporting its potential for clinical development. Its activity against RAS-mutant cancers (e.g., HCT116, A549) addresses an unmet need, as RAS mutations are historically difficult to target with small-molecule inhibitors [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (5.89 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. 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. Solubility in Formulation 2: 0.5% CMC Na: 30 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3557 mL | 11.7783 mL | 23.5566 mL | |
| 5 mM | 0.4711 mL | 2.3557 mL | 4.7113 mL | |
| 10 mM | 0.2356 mL | 1.1778 mL | 2.3557 mL |