PLX-4720

PLX-4720は、B-Raf(V600E)とc-Raf-1(Y340D/Y341D)の「実行中の」形の強力で選択的な阻害剤で、IC50 がそれぞれ 13 nM と 6.7 nMです。

目録号S1152
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PLX-4720 化学構造
分子量: 413.83

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Quality Control & MSDS

製品情報

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製品の説明

生物活性

情報 PLX-4720は、B-Raf(V600E)とc-Raf-1(Y340D/Y341D)の「実行中の」形の強力で選択的な阻害剤で、IC50 がそれぞれ 13 nM と 6.7 nMです。
目標 B-RafV600E c-Raf-1Y340D/Y341D
IC50 13 nM 6.7 nM [1]
In vitro試験 PLX-4720 displays >10 times selectivity against wild type B-Raf, and >100 times selectivity over other kinases such as Frk, Src, Fak, FGFR, and Aurora A with IC50 of 1.3-3.4 μM. PLX-4720 significantly inhibits the ERK phosphorylation in cell lines bearing B-RafV600E with IC50 of 14-46 nM, but not the cells with wild-type B-Raf. PLX-4720 significantly inhibits the growth of tumor cell lines bearing the B-RafV600E oncogene, such as COLO205, A375, WM2664, and COLO829 with GI50 of 0.31 μM, 0.50 μM, 1.5 μM, and 1.7 μM, respectively. In addition, PLX-4720 treatment at 1 μM induces cell cycle arrest and apoptosis exclusively in the B-RafV600E-positive 1205Lu cells, but not in the B-Raf wild-type C8161 cells. [1] PLX-4720 treatment (10 μM) significantly induces >14-fold expression of BIM in the PTEN+ cells, compared with the PTEN- cell lines (4-fold), giving an explanation of the resistance of PTEN- cells to PLX-4720-induced apoptosis. [2]
In vivo試験 Oral administration of PLX-4720 at 20 mg/kg/day induces significant tumor growth delays and regressions in B-RafV600E-dependent COLO205 tumor xenografts, without obvious adverse effects in mice even at dose of 1 g/kg. PLX-4720 at 100 mg/kg twice daily almost completely eliminates the 1205Lu xenografts bearing B-RafV600E, while has no activity against C8161 xenografts bearing wild-type B-Raf. The anti-tumor effects of PLX-4720 correlate with the blockade of MAPK pathway in those cells harboring the V600E mutation. [1] PLX-4720 treatment at 30 mg/kg/day significant inhibits the tumor growth of 8505c xenografts by >90%, and dramatically decreases distant lung metastases. [3]
臨床試験
特集

推薦された実験操作 (公開の文献だけ)

キナーゼアッセイ: [1]

In vitro Raf kinase activities The in vitro kinase activities of wild type Raf and mutants are determined by measuring phosphorylation of biotinylated-MEK protein using Perkin-Elmer's AlphaScreen Technology. For each enzyme (0.1 ng), 20-μL reactions are carried out in 20 mM Hepes (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 100 nM biotin-MEK protein, various ATP concentrations, and increasing concentrations of PLX-4720 at room temperature. Reactions are stopped at 2, 5, 8, 10, 20, and 30 minutes with 5 μL of a solution containing 20 mM Hepes (pH 7.0), 200 mM NaCl, 80 mM EDTA, and 0.3% BSA. The stop solution also includes phospho-MEK Antibody, Streptavidin-coated Donor beads and Protein A Acceptor beads from the AlphaScreen Protein A Detection Kit. The antibody and beads are preincubated in stop solution in the dark at room temperature for 30 minutes. The final dilution of antibody is 1/2,000, and the final concentration of each bead is 10 μg/mL. The assay plates are incubated at room temperature for one hour then are read on a PerkinElmer AlphaQuest reader.

細胞アッセイ: [1]

細胞系 COLO205, A375, WM2664, COLO829, HT716, SW620, H460, Calu-6, HCT116, SK-MEL2, SK-MEL3, Lovo, H1299, 1205Lu, and C8161 cells
濃度 Dissolved in DMSO, final concentrations ~1 mM
処理時間 24, 48, and 72 hours
方法 Cells are treated with various concentrations PLX-4720 for 24, 48, and 72 hours. Cell proliferation is measured by using the CellTiter-Glo Luminescent Cell Viability Assay or MTT assay. For cell cycle analysis, supernatant and cells are collected, pelleted, and fixed with 70% ethanol. Before staining with propidium iodide (10 μg/mL), cells are incubated for 1 hour at 37 °C in 0.5 mg/mL RNase I to rid samples of residual RNA contamination. Samples are then analyzed by using the EPICS XL apparatus. For the assessment of apoptosis, media and cells are harvested and pelleted before staining with annexin-FITC and propidium iodide. Samples are subsequently analyzed by using the EPICS XL apparatus.

動物実験: [1]

動物モデル Female athymic mice (NCr nu/nu) implanted s.c. with COLO205 cells, and SCID mice with 1205Lu or C8161 cells
製剤 Suspended in vehicle (5% DMSO, 1% methylcellulose)
投薬量 5, 20, or 100 mg/kg
管理 Oral gavage once or twice daily
Solubility 1% CMC/0.5% Tween-80, 30 mg/mL
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

SpeciesBaboonDogMonkeyRabbitGuinea pigRatHamsterMouse
Weight (kg)121031.80.40.150.080.02
Body Surface Area (m2)0.60.50.240.150.050.0250.020.007
Km factor202012128653
Animal A (mg/kg) = Animal B (mg/kg) multiplied by  Animal B Km
Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Rat dose (mg/kg) = mouse dose (22.4 mg/kg) ×  mouse Km(3)  = 11.2 mg/kg
rat Km(6)
1

参考

化学情報

Download PLX-4720 SDF
分子量 413.83
化学式

C17H14ClF2N3O3S

CAS No. 918505-84-7
保管 2年-20℃
6月-80℃in DMSO
別名
溶解度 (25°C) * In vitro DMSO 83 mg/mL (200 mM)
<1 mg/mL (<1 mM)
エタノール <1 mg/mL (<1 mM)
In vivo 1% CMC/0.5% Tween-80, 30 mg/mL
* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
化学名 N-(3-(5-chloro-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide

研究分野

カスタマーレビュー (7)


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Source Proc Natl Acad Sci USA , 2011, 108, 6067-6072. PLX-4720 purchased from Selleck
Method Western blotting
Cell Lines Cells overexpressing myc-CRAF and BRAF
Concentrations 10/50 µM
Incubation Time 1/2 h
Results GDC0879 but not PLX4720 induced BRAF/CRAF dimer formation (Fig. A). However, both drugs induced complexes between KSR1 and CRAF and enhanced interactions between KSR1 and BRAF (Fig. B and C), which suggested that KSR1/RAF complexes induced by the drug might explain the effects ofthe type I BRAF specific inhibitors. As reported previously, treatment of wild-type cells with either drug strongly induced ERK activation at low to intermediate doses but inhibited ERK activation at higher doses (Fig. D and E). Similar results were obtained with cells expressing constitutively active RAS (Fig. D and E) or after serum treatment. Strikingly, in KSR deficient cells, ERK activation was significantly attenuated after PLX4720 or GDC0879 treatment (Fig. D and E), which demonstrates that the ability of PLX4720 and GDC0879 to activate ERK requires the presence of KSR1. We found that, when KSR1 was overexpressed withCRAF, MEK activation was induced by PLX4720 or GDC0879 treatment (Fig. F). this result suggested that induction of the CRAF/KSR1 complex might be important in the activation of MEK. In vitro kinase activity toward MEK was detected but only after drug treatment (Fig. G), which suggests KSR1/CRAF complex formation kinase activity toward MEK.

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Source Cancer Res , 2011, 71, 2750-2760. PLX-4720 purchased from Selleck
Method Western blotting, MTT assay, flow cytometry
Cell Lines PTEN+/ PTEN- melanoma cell lines
Concentrations 0.001-10 µmol/L
Incubation Time 48 h
Results The PTEN+ cell lines had lower constitutive levels of pAKT (Ser473) compared with the PTEN(Fig. A). Similar levels of pAKT (Thr308) were observed in the PTEN and PTEN þ cell lines. Analysis of the growth inhibitory effects of PLX4720 by the MTT and Alamar Blue assays (Fig. B) did not reveal any statistically significant differences in the GI 50 values between the PTEN+/ PTEN- cell lines. We observed that following PLX4720 treatment (3 and 10 µmol/L, 48 hours), the PTEN-melanoma cell lines showed significantly less apoptosis than the PTEN+ (P < 0.05: Fig. C)

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Source Cancer Res , 2011, 71, 2750-2760. PLX-4720 purchased from Selleck
Method Western blotting
Cell Lines PTEN+/ PTEN- melanoma cell lines
Concentrations 0.03-3 µmol/L
Incubation Time 24 h
Results Treatment of the PTEN+/ PTEN- cell line panels with PLX4720 increased pPDK1andpAKTsignaling only in the melanoma cell lines lacking PTEN expression (Fig. A). Addition of PLX4720 also led to the inhibition of mTOR activity in the PTEN+ cell lines only (Fig. A). The requirement for PTEN in the increased AKT signaling was confirmed by studies showing that PLX4720 stimulated pAKT in WM35 cells (PTEN+) when PTEN was knocked down by siRNA (Fig. B). The effects of PLX4720 upon pAKT signaling were BRAF specific, with BRAF siRNA knockdown found to increase pAKT in PTEN- cells only (Fig. C).

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Source Cancer Res, 2011, 71, 2750-2760. PLX-4720 purchased from Selleck
Method Western blot, Immunofluorescence staining, flow cytometry
Cell Lines 1205Lu cells
Concentrations 3 µmol/L
Incubation Time 48 h
Results Combined PI3K and BRAF inhibition increased the level of BIM expression in both Western blot and immunofluorescence studies (Fig. A). Consistent with a role for increased AKT signaling suppressing BIM expression in PTEN- cells, dual BRAF and PI3K inhibition increased nuclear FOXO3a localization in the PTEN- cell lines (Fig. B) and enhanced the level of BIM mRNA (Fig. B). the combination of PLX4720 with the PI3K inhibitor GDC-0941 significantly enhanced the levels of apoptosis observed in PTEN melanoma cell lines compared to either the BRAF or PI3K inhibitor alone (Fig. C). Similar results were also observed in a 3D spheroid assay, where combined PLX4720 (3 µmol/L) and LY294002 (10 µmol/L) treatment prevented the recovery of cell growth observed when melanoma spheroids were treated with either drug alone (Fig. D).

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Source Cancer Res , 2011, 71, 2750-2760. PLX-4720 purchased from Selleck
Method LC-MRM analysis, Western blotting, immunofluorescence staining
Cell Lines PTEN+/ PTEN- melanoma cell lines
Concentrations 3/10 µmol/L
Incubation Time 0-48 h
Results We next used LC-MRM to quantify the PLX4720-induced changes in the expression of 17 members of the Bcl-2 protein family (Fig. A shows results for 9 proteins). The only proapoptotic protein to demonstrate significant differences between the PTEN+/ PTEN- cell lines was BIM (Fig. A). Western blots and immunofluorescence staining confirmed the LC-MRM data and showed a greater degree of PLX4720-induced BIM expression in the PTEN+ cell lines compared to PTEN- cell lines (Fig. B,C). In parallel, we observed that PLX4720 also increased the inactivation of BAD (as shown by increased phospho-BAD) in the PTEN- cells (Fig. D) and that overexpression of BAD in the PTEN- cells enhanced PLX4720-mediated apoptosis (Fig. D).

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Source Dr. Jong-In Park of Medical College of Wisconsin. PLX-4720 purchased from Selleck
Method Western blot
Cell Lines SK-MEL-28 cell line
Concentrations 0-1 µM
Incubation Time 4/22 h
Results B-RafV600E mutated melanoma line, SK-MEL-28, was treated with different doses of PLX-4720 for 4 h or 22 h. Cell lysates were analyzed by Western blotting to determine the levels of phosphorylated MEK1/2 (pMEK1/2) and phosphorylated ERK1/2 (pERK1/2). MEK1/2 is the substrate of B-Raf while ERK1/2 is the substrate of MEK1/2. Data show that phosphorylation of MEK1/2 and ERK1/2 was significantly inhibited by PLX-4720 treatment although total MEK1/2 or ERK1/2 protein levels were not affected. No pMEK1/2 or pERK1/2 signal was detected even after prolonged exposure, indicating that the inhibitor at 1 μM is very effective in blocking the constitutive kinase activity of B-RafV600E. This data is consistent with the previous result demonstrating the effect of PLX-4720 in the B-RafV600E mutated melanoma line, A375-Fig. 2A

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Source Dr. Daniel C.Cho of Harvard Medical School. PLX-4720 purchased from Selleck
Method MTT assay, Western blotting
Cell Lines A375 melanoma cells
Concentrations 0-1000 nM
Incubation Time
Results A dose titration of PLX-4720 in A375 melanoma cells which possess a V600E B-Raf mutation.Effects of increasing PLX-4720 dose on Erk phosphorylation and on tumor cell proliferation as determined by MTT assay are shown.

製品表彰状 (36)

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