Vemurafenib (PLX4032, RG7204) 化学構造
分子量: 489.92

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製品説明

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製品の説明

生物活性

製品説明 Vemurafenib (PLX4032, RG7204)新しくて強力な阻害剤で、 B-RAFV600Eに作用すると、IC50 が 31 nMになる。
ターゲット B-RafV600E C-Raf MAP4K5 (KHS1) SRMS ACK1 FGR
IC50 31 nM 48 nM 51 nM 18 nM 19 nM 63 nM [1]
In vitro試験 PLX4032 inhibits B-RAFV600E, C-RAF, as well as wildtype B-RAF, with IC50 of 31 nM, 48 nM and 100 nM, respectively. PLX4032 also inhibits several non-RAF kinases, including ACK1, KHS1, and SRMS, with IC50 of 18 nM to 51 nM. [1] In melanoma cell lines, the inhibitory effect by PLX4032 depends on B-RAF mutational status, because PLX4032 potently inhibits those harboring B-RAF V600 mutants, including V600E, V600D, V600K, and V600R, but not wildtype or other mutants. The IC50 values of PLX4032 on these cells, including MALME-3M, Colo829, Colo38, A375, SK-MEL28, and A2058, ranges from 20 nM to 1 μM. In these cells, PLX4032 (0.1 μM to 30 μM) also inhibits the phosphorylation of both MEK1/2 and ERK1/2. [2] PLX4032 is highly effective in the treatment of melanoma, for its ability of inhibiting B-RAFV600E. However, PLX4032 displays limited effect in colon cancer patients that also carrying B-RAFV600E oncoprotein. The reason for this is that, in colon cancer cells, B-RAFV600E inhibition by PLX4032 results in a rapid feedback EGFR activation, which compensates for the PLX4032-inhibited cell proliferation. [3]
Cell Data
Cell LinesAssay TypeConcentrationIncubation TimeFormulationActivity Description
A375 M2\sV2dzd3e2aDDJcohq[mm2aX;uJGF{e2G7 NXHoZ3U2OTByIN88US=> MYq5OkBp M3ztRmROW09? M4ToTmlEPTB;NEegcm0>
ARO Mm\5S5Jwf3SqIFnubIljcXSrb36gRZN{[Xl? NUnuWFFrOTByIN88US=> MYi5OkBp M1eweGROW09? M3PQcGlEPTB;MkC1JI5O
NPA NY\LZoJPT3Kxd4ToJGlvcGmkaYTpc44hSXO|YYm= NY[2b4ttOTByIN88US=> MWO5OkBp MmfXSG1UVw>? M3vrV2lEPTB;Mk[gcm0>
TPCI M3H0[Wdzd3e2aDDJcohq[mm2aX;uJGF{e2G7 M17he|ExOCEQvF2= NGHZfHk6PiCq M2TlcmROW09? MofiTWM2OD1zMD63O{DPxE1?
A375 Mn\VRZBweHSxc3nzJGF{e2G7 M2TXN|ExKM7:TR?= M2XKR2ROW09? MWPQdo9ud3SnczDhdI9xfG:2aXOg[IVifGh?
ARO NHLXNm9HfW6ldHnvckBCe3OjeR?= M3;VR|ExKM7:TR?= MYC3NkBp M{HpeGROW09? MmHwTY5lfWOnczD0bIUhemWneIDy[ZN{cW:wIH;mJJRp\SCQSWOgdJVueA>?
8505C (BRAF V600E/V600E) NGK5WXJIem:5dHigTY5pcWKrdHnvckBCe3OjeR?= M4XGcVk3KGh? NFP0eHVKSzVyPUW3JI5OKA>?
SW1736 (BRAF WT/V600E) NWfscpRPT3Kxd4ToJGlvcGmkaYTpc44hSXO|YYm= NXLr[mpZQTZiaB?= M{HWdmlEPTB;Mkmgcm0>
BHT101 (BRAF WT/V600E) NHjnU2xIem:5dHigTY5pcWKrdHnvckBCe3OjeR?= Mo\LPVYhcA>? M{D0d2lEPTB;OUegcm0>
BCPAP (BRAF WT/V600E) MXPHdo94fGhiSX7obYJqfGmxbjDBd5NigQ>? MnzvPVYhcA>? NH;HfndKSzVyPUe4JI5O
C643 (HRAS G13R)≥ 500 M1TCUmdzd3e2aDDJcohq[mm2aX;uJGF{e2G7 M2KxUlk3KGh? MWHJR|UxKOLLpTC1NFAhdk1?
HTH7 (NRAS Q61R) Mnm1S5Jwf3SqIFnubIljcXSrb36gRZN{[Xl? MkH0PVYhcA>? MXrJR|Ux6onnIEGwNFAhdk1?
CAL62 (KRAS G12R) > 1000 > 1000 MlHrS5Jwf3SqIFnubIljcXSrb36gRZN{[Xl? NFjSRm46PiCq MmnXTWM2OD5iMUCwNEBvVQ>?
TPC-1 (RET/PTC1) M3rLNmdzd3e2aDDJcohq[mm2aX;uJGF{e2G7 NFnoXVM6PiCq M1fxSWlEPTEkibWxNFAxKG6P
PC NV3aV4NFT3Kxd4ToJGlvcGmkaYTpc44hSXO|YYm= NUfjWG9uQTZiaB?= MULJR|UxRiBzMECwJI5O
Calu-6 MojESpVv[3Srb36gRZN{[Xl? MUKx5qCK|ryP NXP5OWVoOSCq M4rpb2ROW09? MojxRYN1cX[jdHXzJG1GUy:HUlugbY4h[2WubIOge4l1cCC5aXzkMZR6eGViQmLBSi=>
C4 MXjGeY5kfGmxbjDBd5NigQ>? NX7WRZJQOyEQvF2= M4T1RVQ5KGh? NX\KOnNYTE2VTx?= MojLTY5kemWjc3XzJINwdGyjZ3XuJJN6dnSqZYPpd{BidmRiZHXjdoVie2W|IFnMMVgh\XiycnXzd4lwdg>?
VMM12 MmfESpVv[3Srb36gRZN{[Xl? MYmzJO69VQ>? NILYO2E1QCCq NEHOfWVFVVOR Mmf0TY5kemWjc3XzJINwdGyjZ3XuJJN6dnSqZYPpd{BidmRiZHXjdoVie2W|IFnMMVkh\XiycnXzd4lwdg>?
SKMEL19 MmnKSpVv[3Srb36gRZN{[Xl? NYC3dJNXPiEQvF2= NFrNb2U1QCCq MX\EUXNQ NVqxVohUXHKrZ3fldpMhTVJic4Ty[ZN{
UKF-NB-3 (ABCB1) M3LYbmZ2dmO2aX;uJGF{e2G7 NFvONnkyNjJ3INM1US=> MW[yJIg> M3TxeGROW09? Ml\TSY5p[W6lZYOgZYNkfW23bHH0bY9vKG:oIITo[UBndHWxcnXzZ4VvfCCDQlPCNUB{fWK|dILheIUhemixZHHtbY5mKDF{Mx?=
UKF-NB-3 NICwSIJHfW6ldHnvckBCe3OjeR?= NH7NT4oyNjJ3INM1US=> MVOyJIg> MXHEUXNQ MojoV4lodmmoaXPhcpRtgSCjZn\lZ5R{KG:wIHHjZ5VufWyjdHnvckBw\iC2aHWg[ox2d3Knc3PlcpQhSUKFQkGgd5Vje3S{YYTlJJJpd2SjbXnu[UAyOjN?
A375 (BRAFV600E) NYWyVIxxTnWwY4Tpc44hSXO|YYm= Moq4PEBp MWHEUXNQ MX7JcoNz\WG|ZYOgbY51emGlZXzseYxieiCUT2OgZY5lKE6RIHzleoVteyB?

... Click to View More Cell Line Experimental Data

In vivo試験 In B-RAFV600E-mutant mice xenograft models, PLX4032 (6 mg/kg–20 mg/kg) inhibits tumor growth. [1] In mice xenograft models of LOX, Colo829, and A375 cells, PLX4032 (12.5 mg/kg–100 mg/kg) inhibits tumor growth and prolongs mice survival. [2]
臨床試験 PLX4032 has recently been approved by US Food and Drug Administration (FDA) for the treatment of advanced metastatic melanoma with a B-RAFV600E mutation.
特集 A novel and potent inhibitor of the B-RAFV600E oncoprotein.

プロトコル (参考用のみ)

キナーゼアッセイ: [1]

RAF kinase activity measurements The kinase activities of wild-type RAF and mutants are determined by measuring phosphorylation of biotinylated-BAD protein. For each enzyme (0.01 ng), 20 μL reactions are carried out in 20 mM Hepes (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% (v/v) Tween-20, 50 nM biotin-BAD protein, and 1 mM ATP at room temperature. Reactions are stopped at 5 min with 5 μL of a solution containing 20 mM Hepes (pH 7.0), 200 mM NaCl, 80 mM EDTA, 0.3% (w/v) bovine serum albumin (BSA). The stop solution also includes phospho-BAD (Ser112) antibody, streptavidin-coated donor beads, and protein A acceptor beads. The antibody and beads are pre-incubated in stop solution in the dark at room temperature for 30 min. The final dilution of antibody is 1/2000 and the final concentration of each bead is 10 μg/mL. The assay plates are incubated at room temperature for one hour and then are read on a PerkinElmer AlphaQuest reader. Mutant activities are the average of two different batches of purified protein assayed in duplicate in three different experiments.

細胞アッセイ: [2]

細胞株 MALME-3M, Colo829, Colo38, A375, SK-MEL28, and A2058 cells
濃度 0–10 μM , dissolved in DMSO
反応時間 5 days
実験の流れ Cellular proliferation is evaluated by MTT assay. Briefly, cells are plated in 96-well microtiter plates at a density of 1000 to 5000 cells per well in a volume of 180 μL. PLX4032 is prepared at 10 times the final assay concentration in media containing 1% DMSO. Twenty-four hours after cell plating, 20 μL of the appropriate dilution of PLX4032 are added to plates in duplicate. The plates are assayed for proliferation 6 days after the cells are plated. Percent inhibition is calculated and the IC50 is determined from the regression of a plot of the logarithm of the concentration versus percent inhibition.

動物実験: [2]

動物モデル Mice (athymic nude) xenograft models of LOX, Colo829, and A375 cells
製剤 Formulated as microprecipitated bulk powder (MBP), suspended at the desired concentration in an aqueous vehicle containing 2% Klucel LF, and adjusted to pH 4 with dilute HCl
投薬量 12.5 mg/kg–100 mg/kg
投与方法 Oral gavage twice daily

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

SpeciesMouseRatRabbitGuinea pigHamsterDogMonkeyBaboon
Weight (kg)0.020.151.80.40.0810312
Body Surface Area (m2)0.0070.0250.150.050.020.50.240.6
Km factor361285201220
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 Vemurafenib (PLX4032, RG7204) SDF
分子量 489.92
化学式

C23H18ClF2N3O3S

CAS No. 918504-65-1
保管 2年-20℃
6月-80℃in solvent
別名 RO5185426
溶解度 (25°C) * In vitro DMSO 97 mg/mL (197.99 mM)
<1 mg/mL (<1 mM)
エタノール <1 mg/mL (<1 mM)
In vivo 30% PEG400/0.5% Tween80/5% propylene glycol 5 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-​(4-​chlorophenyl)​-​1H-​pyrrolo[2,​3-​b]​pyridin-​3-​yl]​carbonyl]​-​2,​4-​difluorophenyl]​-1-​propanesulfonamide

カスタマーフィードバック (14)


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Source Nature, 2015, 520(7547), 368-72. Vemurafenib (PLX4032, RG7204) purchased from Selleck
Method Animal studies
Cell Lines A375 tumours
Concentrations 10 mg/kg
Incubation Time 4, 5 days
Results Although vemurafenib treatment decreased the volume of sensitive tumours (A375 alone)(b), Green fluorescent protein (GFP) staining confirmed increased numbers of resistant cells in regressing tumours, and EdU or BrdU staining confirmed their increased proliferation rate compared to the vehicletreated controls (c). Tumours comprising only resistant cells showed no growth difference when treated with vehicle or vemurafenib (d), indicating that the growth advantage of resistant cells in regressing tumours was not caused by direct effects of vemurafenib on cancer or stromal cells. In line with these findings, A375R cells co-implanted with other vemurafenib-sensitive melanoma cell lines (Colo800, LOX and UACC62) also showed an up toeightfold growthincreasecompared to vehicle-treated control groups (e). Local growth acceleration of resistant cells in the regressing subcutaneous tumours resulted in higher lung metastatic burden (f).

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Source Nature, 2015, 520(7547), 368-72. Vemurafenib (PLX4032, RG7204) purchased from Selleck
Method Immunofluorescence staining
Cell Lines A375/A375R tumours
Concentrations 0.1-1 uM
Incubation Time 5 days
Results This analysis highlighted FRA1 (also known FOSL1), a member of the AP1 transcription factor complex and effector of the ERK pathway27, as one of the putative upstream regulators of the TIS .FRA1 was downregulated in all drug-sensitive cells, but not in resistan cells, treated with vemurafenib, crizotinib and erlotinib (c, d).

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Source Nature, 2014, 508(7494), 118-22. Vemurafenib (PLX4032, RG7204) purchased from Selleck
Method Immunohistochemical
Cell Lines BRAF(V600E) mutant melanoma
Concentrations
Incubation Time 4.5 months
Results This analysis highlighted FRA1 (also known FOSL1), a member of the AP1 transcription factor complex and effector of the ERK pathway27, as one of the putative upstream regulators of the TIS .FRA1 was downregulated in all drug-sensitive cells, but not in resistan cells, treated with vemurafenib, crizotinib and erlotinib (c, d).

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Source Nature, 2012, 483(7387):100-3. Vemurafenib (PLX4032, RG7204) purchased from Selleck
Method Colony formation assay
Cell Lines Thyroid cancer (8505C)/CRC (OXCO-1, COLO741 and WiDr)/melanoma (A375) cells
Concentrations 0/0.15/0.31/0.63/1/25 uM
Incubation Time 10-14 days
Results Of the ten colorectal cancer cell lines examined, eight express much higher levels of EGFR, but in two (COLO-741 and OXCO-1) EGFR levels were as low as those seen in melanoma. All three thyroid cancer cell lines expressed EGFR at significant levels. When we tested the two EGFRlow CRC cell lines for their response to PLX4032, we found them to be almost as sensitive as the melanoma cell line A375 in both short-term assays and long-term assays.

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Source Nature, 2012, 483(7387):100-3. Vemurafenib (PLX4032, RG7204) purchased from Selleck
Method Western blot
Cell Lines PC9GR4/WZR10 cells
Concentrations 1 uM
Incubation Time 72 h
Results Co-treatment of these cells with a combination of PLX4032 and either cetuximab or gefitinib prevented this feedback activation of EGFR. PLX4032 treatment inhibited MEK and ERK activation downstream of BRAF but activated AKT, which acts downstream of EGFR in a pathway parallel to BRAF. We note that in all three cell lines treatment with BRAF and EGFR inhibitors caused a more complete inhibition of AKT, MEK and ERK signalling as compared to PLX4032 monotherapy, providing a rationale for the observed synergy in growth assays.

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Source J Clin Invest, 2014, 124(11), 5074-84. Vemurafenib (PLX4032, RG7204) purchased from Selleck
Method Western blot
Cell Lines Primary CLL cells
Concentrations 2.5, 5 uM
Incubation Time 24 h
Results Consistent with inhibition of the paradoxical ERK phosphorylation promoting effects of BCR activation by SYK inhibition, we observed higher pERK/tERK ratios in primary CLL cells when exposed to vemurafenib or dabrafenib compared with vehicle, as shown for 2 representative patients, and for vemurafenib compared with vehicle in all analyzed patients.

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Source Oncogene, 2015, 10.1038/onc.2015.97. Vemurafenib (PLX4032, RG7204) purchased from Selleck
Method RT-PCR
Cell Lines BCPAP cells
Concentrations 1-10 uM
Incubation Time 12 h
Results Thus, we speculated that B-Raf inhibition will suppress DR5 transcription, leading to downregulation of DR5 expression. Indeed, it detected decreased DR5 mRNA levels in cells exposed to PLX4032 as evaluated with RT–PCR (c).

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Source J Invest Dermatol, 2013, 133, 2041. Vemurafenib (PLX4032, RG7204) purchased from Selleck
Method Western blot
Cell Lines A375 melanoma cells
Concentrations 0-10 μM
Incubation Time 6 h
Results The activation of Stat3 and the protein expression of PAX3 were both repressed in vemurafenib-sensitive (parental) A375 and UACC62 melanoma cells, but higher vemurafenib concentration were required in resistant A375 and UACC62 melanoma cells. Specifically, vemurafenib at 1 μM inhibited the expression of phospho-Stat3 in sensitive cells, but not in resistant cells. These results suggest that inhibition of Stat3 signaling represents a potential therapeutic strategy to overcome the acquired resistance to vemurafenib in melanoma cells.

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Source J Invest Dermatol, 2011, 131, 2087-95. Vemurafenib (PLX4032, RG7204) purchased from Selleck
Method Clonogenic assays
Cell Lines VM21/VM1 (c) cells
Concentrations 0.1 μM
Incubation Time 14 d
Results Combination of PD166866 with the V600E BRAF-specific inhibitor RG7204 (PLX4032) potently reduced clonogenic growth of VM21 and VM1 cells.

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Source Oncoimmunology, 2012, 1, 1476-1483. Vemurafenib (PLX4032, RG7204) purchased from Selleck
Method flow cytometry
Cell Lines tumor-infiltrating lymphocytes
Concentrations 100-250 nM
Incubation Time 72 h
Results Treatment with Vem significantly increased the frequency of TILs recognizing autologous melanoma cells and responding to them by producing Type 1 helper cytokines (Fig. A ) or by mobilizing cytotoxic granules (Fig. B ), confirming the polyfunctionality of newly-induced responses.

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Source Dr. Zhe-Sheng Chen of St. John's University. Vemurafenib (PLX4032, RG7204) purchased from Selleck
Method Cell cytotoxicity assay
Cell Lines HEK293 cells, HEK293/MRP7 cells
Concentrations 20 μM
Incubation Time
Results

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Source Dr. Zhe-Sheng Chen of St. John's University. Vemurafenib (PLX4032, RG7204) purchased from Selleck
Method Cell cytotoxicity assay
Cell Lines H460 cells, H460/MX20 cells
Concentrations 20 μM
Incubation Time
Results

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Source Dr. Zhe-Sheng Chen of St. John's University. Vemurafenib (PLX4032, RG7204) purchased from Selleck
Method [3H]-Mitoxantrone accumulation analysis
Cell Lines H460 cells, H460/MX20 cells
Concentrations 20 μM
Incubation Time 96 h
Results Vemurafenib increased the intra-cellular accumulation of Mitoxantrone in ABCG2 overexpressing H460/MX20 cells compared with parental H460 cells.

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Rating
Source Vemurafenib (PLX4032, RG7204) purchased from Selleck
Method Western blot analysis/ In vitro colony formation assay
Cell Lines LOX IMVI melanoma cells
Concentrations 0-1 μM
Incubation Time 24 h
Results We therefore a ssessed the effe ct of the anti-E rbB3 anti-body A4 generated in our lab and able to inhibit the ligand-induced signaling and to potently induce receptor internalization and degradation, on vemurafenib-induced pErbB3 and pAKT levels and found that this was able to completely abrogate receptor phosphory l-ation and AKT signaling in all cell line s tested (Figure a)treatment with anti-ErbB3 mAb strongly potentiated growth inhibition by vemurafenib (Figure b and c). As expected only A3 but not A2 was able to completely abrogate vemurafenib-induced ErbB3 phosphorylation and AKT signaling (Figure d). Moreover in in vitro colony formation a ssays only A3 but not A2 strongly potentiated growth inhibition by vemurafenib(Figure e).

文献中の引用 (62)

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Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description
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