PLX7904

BRaf(V600E) (IC50 = 5 nM)

PLX7940 能够有效抑制突变 BRAF 黑色素瘤细胞中 ERK1/2 的激活，但不会过度激活突变 RAS 表达细胞中的 ERK1/2。在突变型 N-RAS 介导的维莫非尼耐药细胞中，PLX7904 促进细胞凋亡并抑制进入 S 期以及贴壁依赖性生长，这与 ERK1/2 重新激活驱动恶性特性的重新获得一致。此外，PLX7904 正在人 SCC 细胞系 A431 和人乳腺癌细胞系 SKBR3 中进行测试，因为这些细胞通过馈入 RAS 的上游信号（分别通过 EGFR 和 HER2 受体的过表达）激活 MAPK 通路[1][2] 。

PLX7904 抑制每组 8 只小鼠的 COLO205 异种移植物生长[1]。在体内试验中，相同剂量的vemurafenib可加速皮下b9肿瘤的生长，而同样有效的BRAFV600E抑制剂PLX7904则不能。[１]

PLX7904（也称为 PB04）是一种新型有效、选择性悖论破坏者 B-Raf 抑制剂，在突变 RAS 表达细胞中针对 BRAFV600E 的 IC50 约为 5 nM。

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生化分析和kinome选择性分析。[1]
体外RAF激酶活性是通过测量生物素化底物肽的磷酸化来确定的，如前所述25。PLX7904也在浓度为1 μM的287个激酶组中进行了测试。对抑制50%以上的激酶进行IC50测定。287个激酶代表了kinome系统发育树的所有主要分支。287种激酶的抑制筛选是根据合同在CRO公司的激酶热点服务中作为补充面板进行的。

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Phospho-ERK alphasgreen试验。[1]
为了确定化合物处理对ERK1/2磷酸化的影响，将细胞置于96孔板中，在裂解前37℃用8点化合物滴定1小时。为了检测pERK，将细胞裂解液与链霉亲和素包被的alphasgreen供体珠、抗小鼠IgG alphasgreen受体珠、生物素化的抗ERK1/2兔抗体和仅在Thr202和Tyr204磷酸化时才能识别ERK1/2的小鼠抗体一起孵育。生物素化的ERK1/2抗体与链霉亲和素包被的alphasgreen供体微球和ERK1/2结合（无论其磷酸化状态如何），磷酸化的ERK1/2抗体与受体微球和Thr202/Tyr204磷酸化的ERK1/2结合。ERK1/2 Thr202/Tyr204磷酸化的增加使供体和受体alphasgreen小珠接近，产生一个可以在EnVision读取器上量化的信号。与DMSO对照相比，抑制ERK磷酸化导致信号丢失。

对于 MTT 测定，将 2×103 个细胞一式三份接种到 96 孔常规培养基（其中含有用于 PRT 系的 PLX4720）中。第二天用 PBS 洗涤细胞两次后，用指定的 RAF 抑制剂更换培养基。 48小时后更换培养基，再过48小时后，向孔中加入10μL 5mg/mL MTT试剂并孵育3小时。然后，用 0.1 M 甘氨酸（pH 10.5）在 DMSO 中的 1:10 稀释液过夜溶解甲臜晶体。然后，使用 Multiskan® Spectrum 分光光度计，在 450 nM 下对孔进行评估。显示的结果是三个独立实验的综合结果，并已标准化为 DMSO 条件。显示的误差线准确表示平均值的标准误差。

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安克雷奇独立生长试验。[1]
在六孔板的每孔中镀25,000个B9细胞，底层为1%，顶层为0.4%的低熔点琼脂，含有含有10% FBS的RPMI1640培养基。对于RAF抑制剂的研究，在软琼脂中生长的B9细胞用vemurafenib、PLX4720或PLX7904（指定浓度）或0.2%终浓度的DMSO处理3周。在EGFR配体研究中，在软琼脂中生长的B9细胞分别用AREG、TGF-α或HB-EGF按指定浓度处理3周。对于vemurafinib和erlotinib联合研究，在软琼脂中生长的B9细胞用vemurafenib、erlotinib或两种化合物的组合（指定浓度）或DMSO处理3周。使用AxioVision Rel 4.8软件对≥100 μm的锚定无关菌落进行评分。[１]

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微阵列基因表达分析。[1]
将B9细胞分别置于1µM vemurafenib、1µM PLX7904或0.2% DMSO对照中孵育17小时。收获细胞，分离总RNA，按照制造商的说明使用Affymetrix Mouse420_2芯片检测基因表达。Vemurafenib应答基因的鉴定要求处理样品与对照样品的比值大于1.9（上调）或小于0.54（下调）。[１]

Bovine insulin is added to DMEM with 10% FBS, 1% penicillin/streptomycin, and 1% bovine insulin during the culture of COLO205 tumor cells at 37°C. Female Balb/C nude mice, 6–8 weeks old, weighing about 18–22 g, are subcutaneously injected at the right flank with COLO205 tumor cells (5×106) in 0.1 mL of PBS mixed with matrigel (50:50) to test the development of tumors. Eight mice are randomly assigned to each treatment group so that the mean weight and tumor size are balanced when the mean tumor size reaches about 100 mm3. DMEM 10% FBS 1% penicillin/streptomycin is used to grow B9 cells. The cells are trypsinized, washed three times with 20 mL RPMI, and then re-suspended, counted, and volume-adjusted to a final concentration of 5×107 cells per milliliter before final centrifugation. In 6- to 7-week-old female nude Balb/c mice, 5×106 cells are subcutaneously injected to begin B9 xenografts. When the average tumor size reaches 50–70 mm3, compound dosing begins. To maintain a balance between the average tumor size and body weight, animals are evenly distributed among treatment groups (n=10). Animals are given vehicle, vemurafenib 50 mg per kg, or PLX7904 50 mg per kg twice daily for days 1 through 14 and once daily for days 15 through 28. In weeks three and four, 2 g of 12-O-tetradecanoylphorbol-13-acetate (TPA) in 200 l of acetone is applied to the skin of every mouse.

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COLO205 tumour cells were cultured in DMEM 10% FBS 1% penicillin/streptomycin supplemented with bovine insulin, at 37 °C. Balb/C nude mice, female, 6–8 weeks old, weighing approximately 18–22 g, were inoculated subcutaneously at the right flank with COLO205 tumour cells (5 × 106) in 0.1 ml of PBS mixed with matrigel (50:50) for tumour development. The treatment was started when mean tumour size reached approximately 100 mm3, with eight mice in each treatment group randomized to balance the average weight and tumour size. B9 cells were expanded in DMEM 10% FBS 1% penicillin/streptomycin. Upon trypsinization the cells were washed three times with 20 ml RPMI, and after the final centrifugation were re-suspended, counted, and adjusted by volume to a final concentration of 5 × 107 cells per millilitre. B9 xenografts were started by injection of 5 × 106 cells subcutaneously in 6- to 7-week-old female nude Balb/c mice. Compound dosing started when the average size of tumours reached 50–70 mm3. Animals were equally distributed over treatment groups (n = 10) to balance the average tumour size and body weight. Animals were dosed orally for days 1–14 twice daily and days 15–28 once daily with vehicle, vemurafenib 50 mg per kg, or PLX7904 50 mg per kg. 12-O-tetradecanoylphorbol-13-acetate (TPA) was put on the skin of all mice twice a week during weeks 3 and 4 at a dose of 2 µg in 200 µl acetone. [1]

[1]. RAF inhibitors that evade paradoxical MAPK pathway activation. Nature. 2015 Oct 22;526(7574):583-586.

[2]. Inhibition of mutant BRAF splice variant signaling by next-generation, selective RAF inhibitors. Pigment Cell Melanoma Res. 2014 May;27(3):479-84.

[3]. Selective RAF inhibitor impairs ERK1/2 phosphorylation and growth in mutant NRAS, vemurafenib-resistant melanoma cells. Pigment Cell Melanoma Res. 2013 Jul;26(4):509-17.

Oncogenic activation of BRAF fuels cancer growth by constitutively promoting RAS-independent mitogen-activated protein kinase (MAPK) pathway signalling. Accordingly, RAF inhibitors have brought substantially improved personalized treatment of metastatic melanoma. However, these targeted agents have also revealed an unexpected consequence: stimulated growth of certain cancers. Structurally diverse ATP-competitive RAF inhibitors can either inhibit or paradoxically activate the MAPK pathway, depending whether activation is by BRAF mutation or by an upstream event, such as RAS mutation or receptor tyrosine kinase activation. Here we have identified next-generation RAF inhibitors (dubbed 'paradox breakers') that suppress mutant BRAF cells without activating the MAPK pathway in cells bearing upstream activation. In cells that express the same HRAS mutation prevalent in squamous tumours from patients treated with RAF inhibitors, the first-generation RAF inhibitor vemurafenib stimulated in vitro and in vivo growth and induced expression of MAPK pathway response genes; by contrast the paradox breakers PLX7904 and PLX8394 had no effect. Paradox breakers also overcame several known mechanisms of resistance to first-generation RAF inhibitors. Dissociating MAPK pathway inhibition from paradoxical activation might yield both improved safety and more durable efficacy than first-generation RAF inhibitors, a concept currently undergoing human clinical evaluation with PLX8394. [1]

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Vemurafenib and dabrafenib block MEK-ERK1/2 signaling and cause tumor regression in the majority of advanced-stage BRAF(V600E) melanoma patients; however, acquired resistance and paradoxical signaling have driven efforts for more potent and selective RAF inhibitors. Next-generation RAF inhibitors, such as PLX7904 (PB04), effectively inhibit RAF signaling in BRAF(V600E) melanoma cells without paradoxical effects in wild-type cells. Furthermore, PLX7904 blocks the growth of vemurafenib-resistant BRAF(V600E) cells that express mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is also frequently driven by expression of mutation BRAF splice variants; thus, we tested the effects of PLX7904 and its clinical analog, PLX8394 (PB03), in BRAF(V600E) splice variant-mediated vemurafenib-resistant cells. We show that paradox-breaker RAF inhibitors potently block MEK-ERK1/2 signaling, G1/S cell cycle events, survival and growth of vemurafenib/PLX4720-resistant cells harboring distinct BRAF(V600E) splice variants. These data support the further investigation of paradox-breaker RAF inhibitors as a second-line treatment option for patients failing on vemurafenib or dabrafenib. [2]

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The RAF inhibitor vemurafenib achieves remarkable clinical responses in mutant BRAF melanoma patients. However, vemurafenib is burdened by acquired drug resistance and by the side effects associated with its paradoxical activation of the ERK1/2 pathway in wild-type BRAF cells. This paradoxical effect has driven the development of a new class of RAF inhibitors. Here, we tested one of these selective, non-paradox-inducing RAF inhibitors termed paradox-breaker-04 (PB04) or PLX7904. Consistent with its design, PB04 is able to efficiently inhibit activation of ERK1/2 in mutant BRAF melanoma cells but does not hyperactivate ERK1/2 in mutant RAS-expressing cells. Importantly, PB04 inhibited ERK1/2 phosphorylation in mutant BRAF melanoma cells with acquired resistance to vemurafenib/PLX4720 that is mediated by a secondary mutation in NRAS. Consistent with ERK1/2 reactivation driving the re-acquisition of malignant properties, PB04 promoted apoptosis and inhibited entry into S phase and anchorage-independent growth in mutant N-RAS-mediated vemurafenib-resistant cells. These data indicate that paradox-breaker RAF inhibitors may be clinically effective as a second-line option in a cohort of acquired vemurafenib-resistant patients. [3]

C24H22F2N6O3S

512.53

512.144

C, 56.24; H, 4.33; F, 7.41; N, 16.40; O, 9.36; S, 6.26

1393465-84-3

90116945

White to light brown solid powder

1.5±0.1 g/cm3

1.672

1.58

129

2

10

8

36

895

0

S(N([H])C1C([H])=C([H])C(=C(C=1F)C(C1=C([H])N([H])C2=C1C([H])=C(C([H])=N2)C1C([H])=NC(C2([H])C([H])([H])C2([H])[H])=NC=1[H])=O)F)(N(C([H])([H])[H])C([H])([H])C([H])([H])[H])(=O)=O

DKNZQPXIIHLUHU-UHFFFAOYSA-N

InChI=1S/C24H22F2N6O3S/c1-3-32(2)36(34,35)31-19-7-6-18(25)20(21(19)26)22(33)17-12-30-24-16(17)8-14(9-29-24)15-10-27-23(28-11-15)13-4-5-13/h6-13,31H,3-5H2,1-2H3,(H,29,30)

5-(2-cyclopropylpyrimidin-5-yl)-3-[3-[[ethyl(methyl)sulfamoyl]amino]-2,6-difluorobenzoyl]-1H-pyrrolo[2,3-b]pyridine

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.88 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80+，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。