B-Raf (Kd = 2.4 nM); B-Raf (IC50 = 10 nM)

L-779450 (L-779,450) shows a high degree of specificity towards Raf. Only one other tested kinase, p38MAPK, which shares a kinase domain with Raf structurally, was inhibited. At doses ranging from 0.3 to 2 μM, L-779450 inhibits the anchorage-independent growth of human tumor lines[2]. The effects of L-779450 (L-779,450) on TRAIL sensitivity are investigated here in melanoma cell lines with high TRAIL sensitivity (A-375 and SK-Mel-147), moderate sensitivity (Mel-HO, SK-Mel-13, and SK-Mel-28), and permanent resistance (MeWo, Mel-2a, and SK-Mel-103), as well as in TRAIL-selected cell lines with acquired resistance (A-375-TS and Mel-HO-TS). L-779450 has only mild direct effects on apoptosis, but it significantly increases TRAIL-induced apoptosis in melanoma cells that are susceptible to it and overcomes TRAIL resistance in Mel-2a, SK-Mel-103, A-375-TS, and Mel-HO-TS. 16–35% of cells induce apoptosis at 24 hours[3].

Western blot analysis of ERK1 and ERK2 activation[2]
Cells were deprived of either 4HT for 24 h in phenol-red-free medium that contained 5% charcoal-stripped FCS and then treated with DMSO (0.13%), L-779450 (10 μ M) or U0126 (2.3 μ M) for 1 h. Cells were washed and resuspended in serum-free DMEM. A volume of 1 ml, containing 1.25 × 106 cells, was added to microfuge tubes. Then the cells were pulsed with DMSO, 4HT or the positive control 20 nM PMA for ½ h at 37°C. Following stimulation, the tubes were centrifuged in a microcentrifuge for 30 s, the supernatants were removed, cell pellets were resuspended in 110 μl of cold lysis buffer (25 mM Tris-HCl, pH 7.4; 50 mM NaCl; 0.5% sodium deoxycholate; 2% NP-40; 0.2% SDS; 1 mM phenylmethylsulfonyl fluoride; 50 μg/ml aprotinin, 50 μM leupeptin; 0.5 mM Na3VO4) and placed on ice for 15 min. Lysates were centrifuged for 15 min at 14 000 rpm in an Eppendorf microcentrifuge, supernatants (98 μl) were removed and mixed with 42 μl of 3.3 × sample buffer (200 mM Tris-HCl, pH 6.8; 33% glycerol; 6.6% SDS; 16.6% β-mercaptoethanol; 0.04% bromophenol blue). Samples were boiled (5 min) and frozen. A measure of 15 μl of prepared samples were electrophoresed through a 10% SDS-PAGE gel, and proteins were electrophoretically transferred to PVDF membranes. Membranes were incubated overnight at 4°C in blocking buffer (25 mM Tris-HCl, pH 8.0; 125 mM NaCl; 0.1% Tween-20; 1% BSA; 0.1% sodium azide). Membranes were then incubated for 2 h with the primary antibody diluted in blocking buffer (anti-active ERK, 1:20 000 or anti-p90Rsk (1:10 000). The blots were washed twice in TBST (25 mM Tris-HCl, pH 8.0; 125 mM NaCl; 0.025% Tween-20) and incubated with alkaline phosphatase (AP)-conjugated goat anti-rabbit Ig or goat-anti mouse Ig (1:10 000 in TBST) for 1 h at room temperature. The blots were washed twice in TBST and developed with the colorogenic substrates BCIP and NBT. This blot system cannot be readily ‘stripped and reprobed’, that is why parallel gels were run to detect total ERK2 as an additional loading control. The data shown are representative of at least two independently performed experiments.

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Effects of signal transduction inhibitors on proliferation[2]
Either the signal transduction inhibitors or the solvent control DMSO were added to the top rows of 96-well plates and serially diluted threefold with media containing 5% FCS. The following inhibitors were used, a Raf inhibitor L-779450 that competes with ATP for binding to the Raf catalytic site, the MEK inhibitors PD98059 and U0126, and the PI3K inhibitor LY294002. The structures of these inhibitors are presented in Figure 2. After the serial dilutions were made, a constant amount of IL-3 (2.5% WEHI-3B supernatant) or 4HT (125 nM) was added to the wells. The cytokine-dependent, ΔRaf:ER- or ΔMEK1:ER-responsive cells were washed twice with PBS and then seeded at 10 000 cells/well in 96-well plates and incubated for 18 h. [3H]thymidine was added for the last 4–6 h, and then the plates were harvested and the amount of [3H]thymidine incorporation was determined on a Wallac 1450 Microbeta plus liquid scintillation counter. The fold inhibition was determined by dividing the average amount of [3H]thymidine incorporated with the vehicle DMSO by the average of [3H]thymidine incorporated in the presence of the drug at the same concentrations of DMSO. Each experiment was performed four to six times with two to five different clones from each type of cell line.

TRAIL (20 ng/mL), the pan-RAF inhibitor L-779450 (0.1–50 μM), the MEK inhibitor U0126 (20 M), and the selective BRAF(V600E) inhibitor Vemurafenib/PLX4032 are all used to induce apoptosis. The xCELLigence system is used for ongoing cell growth monitoring. Cell numbers that are attached correspond to relative cell indices. Analyses of the cell cycle are carried out to quantify apoptosis and cell cycle arrest. Propidium iodide (200 mg/mL) is used to stain trypsinized cells for 1 hour, and flow cytometry is used to quantify sub-G1 fractions, which represent cells with DNA fragments.

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Cell transfection with siRNA[3] Transient cell transfection was performed in 6-well plates at 24 hours after seeding (70% confluence). Treatment with L-779,450/TRAIL followed after another 24 hours. Amounts of 20 pmol siRNA and 4 μl TurboFect were used per well. The siRNAs for Smac (sc-36505), Bax (sc-29212), Bak (sc-29786), Bim (sc-29802), and the scrambled control (sc-37007) were used.

[1]. The identification of potent, selective and CNS penetrant furan-based inhibitors of B-Raf kinase. Bioorg Med Chem Lett. 2008 Aug 1;18(15):4373-6.

[2]. Differential effects of kinase cascade inhibitors on neoplastic and cytokine-mediated cell proliferation. Leukemia. 2003 Sep;17(9):1765-82.

[3]. RAF inhibition overcomes resistance to TRAIL-induced apoptosis in melanoma cells. J Invest Dermatol. 2014 Feb;134(2):430-440.

2-Chloro-5-(2-phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)phenol belongs to the imidazole class of compounds. The Raf/MEK/ERK and PI3K/Akt signaling pathways regulate cell proliferation and inhibit apoptosis. Due to the high mutation frequency of upstream regulators, aberrant expression of these pathways is prevalent in human cancers. This study examined the effects of Raf, MEK, and PI3K inhibitors on conditionally transformed hematopoietic cells to determine whether they exhibit cytotoxic differences in cytokine- and oncogene-mediated proliferation, and whether simultaneous inhibition of both pathways is a more effective method for inducing apoptosis. In the hematopoietic model system used, cell proliferation was conditional, occurring only upon the presentation of interleukin-3 (IL-3) or the estrogen receptor antagonist 4-hydroxytamoxifen (4HT), the latter of which activates the conditional oncoprotein (DeltaRaf:ER). Therefore, by adding signal transduction inhibitors and IL-3 or 4HT, the effects of these drugs were tested in the same cells under cytokine- and oncoprotein-mediated growth conditions, thus avoiding heterogeneity at genetic and differentiation stages. At concentrations close to the IC50 values of the previously reported Raf inhibitor L-779,450, this inhibitor inhibited DNA synthesis and induced apoptosis in hematopoietic FDC-P1 cells. These cells, after transformation, exhibited proliferative responses to Raf-1 or A-Raf (FD/ΔRaf-1:ER and FD/ΔA-Raf:ER). However, when cells were cultured in IL-3, the inhibitor had a smaller effect on DNA synthesis and apoptosis. The Raf inhibitor showed weak inhibitory activity against B-Raf or MEK1-responsive cells, indicating drug specificity. The MEK inhibitor also inhibited DNA synthesis and induced apoptosis in Raf-responsive cells, with a more significant inhibitory effect on Raf-responsive cells than on cytokine-mediated proliferation. The PI3K inhibitor LY294002 inhibits Raf-mediated proliferation, indicating that some of the long-term proliferative effects mediated by Raf depend on PI3K. Simultaneous inhibition of the Raf/MEK/ERK and PI3K/Akt pathways has been shown to be a more effective way to inhibit DNA synthesis and induce apoptosis, and requires lower drug concentrations. [1]

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Mutant BRAF is a key oncogene in melanoma, and selective inhibitors have been approved for the treatment of melanoma. However, the molecular mechanism of RAF inhibition in melanoma cells remains unclear. This study investigated the effect of the pan-RAF inhibitor L-779,450, which inhibits the proliferation of BRAF mutant and wild-type melanoma cell lines. In addition, the combination of L-779,450 with the death ligand tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) enhanced apoptosis and overcame TRAIL resistance in melanoma cells. Enhanced apoptosis occurred simultaneously with mitochondrial pathway activation, manifested as loss of mitochondrial membrane potential and release of cytochrome c, Smac (a second mitochondrial-derived caspase activator), and apoptosis-inducing factor (AIF). Subsequently, caspase-9 and caspase-3 were activated. L-779,450/TRAIL-induced apoptosis could be inhibited by Bcl-2 overexpression, and was Bax-dependent. Therefore, L-779,450 activation of Bax alone was confirmed by conformational changes in Bax, while Bak remained unactivated. Furthermore, the BH3-domain-only protein Bim was upregulated under the influence of L-779,450. The important roles of Smac, Bax, and Bim in this process were confirmed by small interfering RNA (siRNA)-mediated gene knockdown experiments. L-779,450 also led to morphological changes indicating autophagy, which was confirmed by the autophagy marker light chain 3-II (LC3-II). The pro-apoptotic effect of L-779,450 may explain the anti-tumor effects of RAF inhibitors, which can be considered when evaluating RAF inhibitors for the treatment of melanoma. [2]

C20H14CLN3O

347.8

347.083

C, 69.07; H, 4.06; Cl, 10.19; N, 12.08; O, 4.60

303727-31-3

303727-31-3

9950176

Light yellow to yellow solid powder

1.335g/cm3

579.783ºC at 760 mmHg

304.442ºC

1.671

5.164

2

3

3

25

425

0

OC1=CC(C2=C(C3=CC=NC=C3)N=C(C4=CC=CC=C4)N2)=CC=C1Cl

WXJLXRNWMLWVFB-UHFFFAOYSA-N

InChI=1S/C20H14ClN3O/c21-16-7-6-15(12-17(16)25)19-18(13-8-10-22-11-9-13)23-20(24-19)14-4-2-1-3-5-14/h1-12,25H,(H,23,24)

2-chloro-5-(2-phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)phenol

L779450; L 779450; L-779450; L 779,450; 2-chloro-5-(2-phenyl-5-(pyridin-4-yl)-1H-imidazol-4-yl)phenol; L-779,450; 2-chloro-5-(2-phenyl-5-pyridin-4-yl-1H-imidazol-4-yl)phenol; L779450; CHEMBL373011; Phenol, 2-chloro-5-[2-phenyl-4-(4-pyridinyl)-1H-imidazol-5-yl]-; L-779,450; L779,450

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)

DMSO: 70~100 mg/mL (201.3~287.5 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.19 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 (7.19 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 (7.19 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.)