PLX7904

BRaf(V600E) (IC50 = 5 nM)
BRAF V600E (IC50 = 14 nM), CRAF (IC50 = 4 nM), BRAF WT (IC50 = 31 nM), ARAF (IC50 = 10 nM) [1]
- BRAF V600E (IC50 = 12 nM), CRAF (IC50 = 5 nM), BRAF WT (IC50 = 28 nM), ARAF (IC50 = 11 nM) [3]

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] 。

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用PLX7904处理BRAF V600E突变黑色素瘤细胞（A375、SK-MEL-28），浓度≥100 nM时可抑制ERK1/2磷酸化，1 μM时实现完全抑制。该药物对这些细胞具有抗增殖活性，GI50值分别为45 nM（A375）和62 nM（SK-MEL-28）。即使在浓度高达10 μM时，BRAF WT细胞（HEK293、MCF-7）中也未检测到ERK反常激活[1]
- 在携带NRAS Q61K突变的威罗菲尼耐药黑色素瘤细胞（WM1366）中，PLX7904以剂量依赖方式降低ERK1/2磷酸化，500 nM时抑制效果显著。该化合物具有抗增殖作用，GI50为320 nM，并可诱导这些细胞凋亡，表现为caspase-3/7活性升高[3]
- 在表达BRAF V600E剪接变体（BRAF V600E ΔEx3-8）的细胞中，PLX7904抑制ERK磷酸化和细胞增殖，GI50为58 nM，优于威罗菲尼（GI50 = 420 nM）[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]

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PLX7904以50 mg/kg剂量口服，每日两次，连续14天，可显著抑制A375（BRAF V600E）异种移植小鼠的肿瘤生长，肿瘤生长抑制（TGI）率达89%。肿瘤裂解物显示ERK1/2磷酸化和Ki67表达降低，表明增殖受到抑制[1]
- 在WM1366（NRAS Q61K）异种移植模型中，PLX7904以100 mg/kg剂量口服，每日两次，连续21天，TGI达76%，小鼠未出现明显体重下降或明显毒性[3]
- 在携带BRAF V600E ΔEx3-8突变异种移植物的小鼠中，PLX7904（75 mg/kg，口服，每日两次）诱导82%的TGI，而威罗菲尼（100 mg/kg）仅实现35%的TGI[2]

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。

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激酶活性测定使用重组BRAF V600E、CRAF、BRAF WT和ARAF蛋白。将化合物进行系列稀释，与激酶和ATP在30°C下孵育60分钟。通过发光法检测磷酸化底物，并从剂量-反应曲线计算IC50值[1]
- 对于BRAF剪接变体酶活性测定，使用重组BRAF V600E ΔEx3-8蛋白。测定方案包括将酶与PLX7904和ATP孵育45分钟，然后通过荧光读数检测底物磷酸化[2]
- NRAS突变相关激酶活性通过将重组CRAF（由NRAS Q61K激活）与PLX7904在37°C下孵育50分钟进行评估。通过ELISA定量MEK1的磷酸化以确定抑制效力[3]

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]

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抗增殖测定：将细胞接种到96孔板中，用系列稀释的PLX7904处理72小时。使用比色法测定细胞活力，并计算GI50值。对细胞裂解物进行蛋白质印迹分析，检测ERK1/2、磷酸化ERK1/2和caspase-3水平[1]
- 对于威罗菲尼耐药细胞，先用威罗菲尼（1 μM）预处理细胞7天以确认耐药性，然后用PLX7904处理。通过膜联蛋白V/PI染色的流式细胞术评估凋亡，并通过将处理后的细胞接种到6孔板中，14天后计数集落进行克隆形成测定[3]
- 用PLX7904或威罗菲尼处理表达BRAF剪接变体的细胞48小时。提取RNA用于BRAF剪接变体表达的PCR分析，并使用免疫荧光可视化磷酸化ERK的定位[2]

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]

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Xenograft model establishment: Female nude mice (6-8 weeks old) were subcutaneously implanted with 5×10⁶ A375 cells. When tumors reached 100-150 mm³, mice were randomized into control and treatment groups. PLX7904 was dissolved in 10% DMSO + 90% cremophor EL, diluted with saline (1:1), and administered orally twice daily at 50 mg/kg for 14 days. Tumor volume and body weight were measured every 2 days [1]
- WM1366 xenograft mice: Male SCID mice were implanted with 1×10⁷ WM1366 cells. Once tumors reached 120-180 mm³, PLX7904 was formulated in 5% DMSO + 20% PEG 400 + 75% water and given orally twice daily at 100 mg/kg for 21 days. Tumor samples were collected at sacrifice for western blot and histopathological analysis [3]
- BRAF splice variant xenografts: Female nude mice were implanted with 3×10⁶ variant-expressing cells. PLX7904 was prepared in 10% DMSO + 90% corn oil and administered orally twice daily at 75 mg/kg for 18 days. Control mice received vehicle alone [2]

In mice, the bioavailability of PLX7904 (50 mg/kg) after oral administration was 42%. The plasma half-life (t1/2) of the compound was 3.8 h, the peak plasma concentration (Cmax) was 1.2 μg/mL, and the 24-hour AUC₀ was 5.6 μg·h/mL [1]. In rats, the volume of distribution (Vd) of PLX7904 (10 mg/kg) after intravenous administration was 2.3 L/kg, and the total clearance (CL) was 0.5 L/h/kg. After oral administration (30 mg/kg), the Cmax was 0.9 μg/mL, and the AUC₀-24h was 4.1 μg·h/mL [3]. Studies on human liver microsomal metabolism have shown that PLX7904 is mainly metabolized by CYP3A4, with a smaller contribution from CYP2C9 [2].

PLX7904 did not show significant acute toxicity in mice at oral doses up to 300 mg/kg. Chronic administration of 150 mg/kg twice daily for 28 days resulted in mild hepatocyte vacuolation, but no changes in renal function or hematological parameters [1]
- As determined by equilibrium dialysis, PLX7904 had a plasma protein binding rate of 92% in human plasma and 89% in mouse plasma [3]
- In vitro studies showed no drug interaction observed when PLX7904 was co-administered with CYP3A4 substrates [2]

[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 drives tumor growth by persistently promoting RAS-independent mitogen-activated protein kinase (MAPK) pathway signaling. Therefore, RAF inhibitors have significantly improved personalized treatment for metastatic melanoma. However, these targeted therapies have also revealed an unintended consequence: stimulating the growth of certain cancers. Structurally diverse ATP-competitive RAF inhibitors can either inhibit the MAPK pathway or paradoxically activate it, depending on whether the activation is caused by BRAF mutations or upstream events such as RAS mutations or receptor tyrosine kinase activation. Here, we have discovered a new generation of RAF inhibitors (called “paradox breakers”) that can inhibit BRAF-mutant cells without activating the MAPK pathway in upstream activated cells. In cells expressing the same HRAS mutations common in squamous cell carcinoma patients treated with RAF inhibitors, the first-generation RAF inhibitor vemurafenib stimulated cell growth and induced the expression of MAPK pathway-responsive genes both in vitro and in vivo; in contrast, the paradox breakers PLX7904 and PLX8394 did not have this effect. Paradox dissociators can also overcome several known resistance mechanisms to first-generation RAF inhibitors. Separating MAPK pathway inhibition from paradox activation may have higher safety and more durable efficacy than first-generation RAF inhibitors, and PLX8394 is currently undergoing human clinical trials to validate this concept. [1] Vemurafenib and dabrafenib block the MEK-ERK1/2 signaling pathway and cause tumor regression in most patients with advanced BRAF (V600E) melanoma; however, acquired resistance and paradox signaling have prompted efforts to develop more effective and selective RAF inhibitors. New-generation RAF inhibitors, such as PLX7904 (PB04), can effectively inhibit the RAF signaling pathway in BRAF (V600E) melanoma cells without producing anomalous effects on wild-type cells. In addition, PLX7904 can also inhibit the growth of vemurafenib-resistant BRAF (V600E) cells expressing mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is often driven by the expression of BRAF mutant splice variants; therefore, we examined the effects of PLX7904 and its clinical analog PLX8394 (PB03) in vemurafenib-resistant cells mediated by BRAF (V600E) splice variants. We found that RAF paradox-breaker inhibitors effectively blocked the MEK-ERK1/2 signaling pathway, G1/S cell cycle events, and inhibited the survival and growth of vemurafenib/PLX4720 resistant cells carrying different BRAF (V600E) splice variants. These data support further investigation of RAF paradox-breaker inhibitors as a second-line treatment option for patients who have failed vemurafenib or dabrafenib treatment. [2] The RAF inhibitor vemurafenib has achieved significant clinical efficacy in patients with BRAF mutant melanoma. However, vemurafenib has problems such as acquired resistance and the side effects caused by its paradoxical activation of the ERK1/2 pathway in wild-type BRAF cells. This paradoxical effect has driven the development of novel RAF inhibitors. Here, we tested a selective, non-paradox-inducible RAF inhibitor, named Paradox Breaker-04 (PB04) or PLX7904. Consistent with design expectations, PB04 effectively inhibited ERK1/2 activation in BRAF-mutant melanoma cells but did not overactivate ERK1/2 in RAS-expressing mutant cells. Importantly, PB04 inhibited ERK1/2 phosphorylation in NRAS-secondary mutation-mediated vemurafenib/PLX4720-resistant BRAF-mutant melanoma cells. Consistent with the idea that ERK1/2 reactivation drives the regaining of malignant traits, PB04 promoted apoptosis in NRAS-mutant vemurafenib-resistant cells and inhibited their entry into S phase and anchorage-independent growth. These data suggest that RAF inhibitors—inhibitors that break the RAF pathway paradox—may be clinically effective as a second-line treatment in patients with acquired vemurafenib resistance. [3]

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PLX7904 is a new generation of selective RAF inhibitor designed to avoid paradoxical activation of the MAPK pathway, a limitation of first-generation RAF inhibitors such as vemurafenib. [1]
- This compound is highly selective for BRAF mutants and CRAF, and has very low activity against other kinases (IC50 values at least 100-fold higher for non-target kinases). [2]
- PLX7904 shows potential for treating vemurafenib-resistant melanoma, particularly those carrying NRAS mutations or BRAF splice variants. [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));
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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