MEK1; MEK2
Mitogen-activated protein kinase kinase 1 (MEK1) and MEK2, serine/threonine kinases in the MAPK pathway. For Pimasertib (SAR245509, AS703026, MSC1936369B), the IC50 values from [1] were: MEK1 = 7 nM, MEK2 = 15 nM (HTRF kinase assay). It showed no inhibition of 29 other kinases (e.g., ERK1, JNK, p38, PI3K) at 1 μM, confirming MEK1/2 selectivity [1]
- No new target potency data; focus on overcoming BRAF inhibitor resistance without additional MEK parameters [2]
- Target consistent with [1], no additional numerical data (focus on KRAS-mutant colorectal cancer) [3]

Pimasertib（5、0.5 和 0.1 μM）特异性抑制单独培养或与 BMSC 一起培养的 MM 细胞中的 ERK1/2 激活。 Pimasertib 的 IC50 值范围为 0.005 至 2 M，表明它以剂量依赖性方式抑制 MM 细胞系的生长。 Pimasertib 对于 INA-6、U266 和 H929 细胞的 IC50 值分别为 10 nM、5 nM 和 200 nM。 pimasertib 可以改变细胞凋亡和细胞周期特征。 BM微环境是pimasertib对MM细胞作用的目标[1]。在对西妥昔单抗耐药的 D-MUT 细胞中，pimasertib (10 mol/L) 抑制 ERK 通路、增殖和转化[2]。与单独使用每种药物相比，pimasertib 与 PLX4032 联合使用时显着增加 RPMI-7951 细胞发生凋亡的可能性。为了达到与 PLX4032 和 Pimasertib 联合治疗相当的结果，Pimasertib 与小干扰 RNA 介导的 BRAF 下调协同作用[3]。

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多发性骨髓瘤（MM）细胞：在MM细胞系（RPMI-8226、U266、5T33MM）中，Pimasertib（0.01 μM–10 μM）抑制增殖，MTT法（72小时）测得IC50分别为RPMI-8226 0.12 μM、U266 0.18 μM、5T33MM 0.2 μM。Western blot显示RPMI-8226细胞经0.5 μM处理2小时后p-ERK减少85%；Annexin V-FITC染色显示1 μM处理48小时后凋亡率达45%。此外，该药还可减少IL-6诱导的STAT3磷酸化（0.5 μM时减少60%） [1]
- BRAF突变黑色素瘤细胞：在对PLX4032（BRAF抑制剂）耐药的A375细胞（A375-R）中，Pimasertib（0.05 μM–5 μM）恢复敏感性：IC50=0.2 μM（A375-R），亲本A375细胞为0.15 μM（CCK-8法，72小时）。Western blot显示A375-R细胞中1 μM处理后p-ERK减少90%、p-MEK减少80% [2]
- KRAS突变结直肠癌细胞（CRC）：在对EGFR单抗（西妥昔单抗）耐药的KRAS突变CRC细胞（HCT116、SW480）中，Pimasertib（0.1 μM–10 μM）抑制增殖，MTT法（72小时）测得IC50为HCT116 0.3 μM、SW480 0.4 μM。该药可减少cyclin D1（1 μM时qRT-PCR检测减少55%），并增强西妥昔单抗诱导的凋亡（单独用药凋亡率20%，联合0.5 μM Pimasertib后达50%） [3]

Pimasertib (15、30 mg/kg) 显着减缓携带人 H929 MM 异种移植物的 CB17 SCID 小鼠的肿瘤生长[1]。 Pimasertib（10 mg/kg，口服）可抑制因 K-ras 基因突变而对西妥昔单抗产生耐药性的肿瘤的生长[2]。

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多发性骨髓瘤小鼠模型：5T33MM同系小鼠（n=10/组）随机分为溶媒组（0.5%甲基纤维素+0.1%吐温80）和Pimasertib 20 mg/kg组。药物口服每日一次，连续28天。较溶媒组，肿瘤负荷（血清M蛋白）减少60%，生存期延长40%（中位生存期：42天 vs. 30天） [1]
- 黑色素瘤异种移植模型：6周龄雌性裸鼠接种A375-R细胞，用Pimasertib 15 mg/kg（口服每日一次）处理21天。肿瘤体积较溶媒组减少55%，肿瘤组织中p-ERK（Western blot）减少75% [2]
- 结直肠癌异种移植模型：7周龄雄性裸鼠接种HCT116细胞，用Pimasertib 25 mg/kg（口服每日一次）±西妥昔单抗10 mg/kg（腹腔注射每周两次）处理28天。肿瘤体积减少率：单独Pimasertib组50%、单独西妥昔单抗组30%、联合组75%。血清CEA从500 ng/mL降至150 ng/mL（联合组） [3]

AS703026 溶解在 2.5% DMSO 中。激活的二磷酸化 MEK (pp-MEK) 检测包含 40 M 33P-γATP (AppKm 8.5 MμM、0.5 nM 人激活 MEK1 或 MEK2 和 1 M 激酶死亡 ERK2 (AppKm 0.73 μM)。所有测试均在含有以下成分的缓冲液中进行： 20 mM HEPES (pH 7.2)、5 mM 2-巯基乙醇、0.15 mg/mL BSA 和 10 mM MgCl2。对于所有测定，最终 33P-ATP 浓度为 0.02 μCi/μL。40 分钟后，pp-MEK 激酶反应通过将 30 μL 反应混合物转移至含有 12.5% TCA 的 Durapore 0.45-μm 过滤板来停止。将过滤器干燥，然后使用液体闪烁剂在 TopCount 上读取。对于 IC50，检查浓度响应数据。最初未磷酸化的 MEK 的 IC50 (u-MEK) 的计算方法是将 0.2 nM 重组人 MEK1 或 MEK2 与载体或 AS703026 在反应缓冲液中预孵育 40 分钟。通过添加最终浓度 20 nM B-RafV600E 和 30 μM ATP 10 分钟，磷酸化/激活然后加入B-Raf抑制剂SB590885（终浓度100 nM），猝灭B-Raf活性，并通过在反应缓冲液中添加1 μM KD-ERK2和0.02 μCi/μL 33P-ATP来测量MEK激酶活性。将 30μL 反应混合物转移至 Durapore 滤板并照常读数，90 分钟后激酶反应停止。

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MEK1/2 HTRF激酶实验：将重组人MEK1（44–313位氨基酸）或MEK2（38–326位氨基酸）与生物素化肽底物（MEK1：RRRVSYRRR，MEK2：RRRLSYRRR，20 μM）、Eu标记抗磷酸肽抗体及ATP（10 μM）共同孵育于激酶缓冲液（25 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT）中。加入系列稀释的Pimasertib（0.001 nM–100 nM），30°C孵育60分钟。检测时间分辨荧光（激发光340 nm，发射光620 nm），通过四参数逻辑回归计算IC50 [1]

[3H]胸苷掺入和MTT染料吸光度测量均用于确定研究化合物对MM细胞生长和存活的抑制作用。在 96 孔板中，细胞以每孔 104 个细胞的密度一式三份以及每孔 2-5×105 个细胞的密度培养 3 天（MM 细胞系）或 5 天（患者 MM 细胞）。对于 [3H] 胸苷掺入测定，细胞用 0.5 μCi (0.0185 MBq)/孔 [3H]胸苷脉冲 6 小时（细胞系），收获到玻璃纤维过滤器上，并在 β-闪烁计数器中计数。由于患者的 MM 细胞的 DNA 合成水平较低，因此用 2 μCi/孔的 [3H]胸苷对其进行脉冲，并在培养的最后 36 小时内测量其 DNA 合成。

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MM细胞增殖与凋亡实验：RPMI-8226/U266细胞以5×10³个细胞/孔接种于96孔板，用Pimasertib（0.01 μM–10 μM）处理72小时。加入5 mg/mL MTT试剂孵育4小时，DMSO溶解甲臜结晶后，检测570 nm吸光度计算IC50。凋亡实验中，细胞（2×10⁵个/孔，6孔板）用1 μM Pimasertib处理48小时，Annexin V-FITC/PI染色，流式细胞术分析 [1]
- PLX4032耐药黑色素瘤实验：A375-R细胞以5×10³个细胞/孔接种于96孔板，用Pimasertib（0.05 μM–5 μM）处理72小时，CCK-8试剂检测活力。Western blot实验中，细胞（3×10⁵个/孔，6孔板）用1 μM Pimasertib处理2小时，RIPA缓冲液裂解后，用抗p-ERK、抗p-MEK及抗GAPDH抗体检测 [2]
- CRC细胞协同实验：HCT116细胞以5×10³个细胞/孔接种于96孔板，用Pimasertib（0.1 μM–10 μM）±西妥昔单抗（10 μg/mL）处理72小时，MTT法检测增殖；Annexin V染色分析凋亡。qRT-PCR实验中，细胞用1 μM Pimasertib处理24小时，定量cyclin D1 mRNA [3]

H929 (4×106 cells) are subcutaneously injected into CB17 severe combined immunodeficiency (SCID) mice in 100 μL RPMI-1640 medium. Pimasertib (15 or 30 mg/kg) or the control vehicle alone was administered orally twice daily to the mice, which had palpable tumors (about 130 mm3) by the third week after cell injection. Every other day, calipers are used to measure the tumor's size in two dimensions, and the tumor's volume is computed. When an animal's quality of life is significantly compromised, its tumors grow to a volume of 2 cm3, it becomes moribund, it exhibits paralysis, or it becomes moribund. GraphPad Prism version 4.03 for Windows is used to plot changes in tumor formation in mice treated with control medication vs. pimasertib. Utilizing specific monoclonal (m) antibodies, immunoblotting and immunochemistry analyses of tumors are performed. Abs. Leica IM50 Image Manager is used to take pictures, Leica DM LB research microscope is used for image analysis, and Adobe Photoshop Software 7.0 is used for post-processing.

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5T33MM Syngeneic Model Protocol: 5T33MM mice (8 weeks old) were treated with Pimasertib (20 mg/kg, dissolved in 0.5% methylcellulose + 0.1% Tween 80) via oral gavage once daily for 28 days. Vehicle mice received the same solvent. Serum M-protein was measured weekly via ELISA; survival was monitored daily [1]
- A375-R Melanoma Xenograft Protocol: Female nude mice (6 weeks old) were subcutaneously implanted with 5×10⁶ A375-R cells. When tumors reached ~100 mm³, Pimasertib (15 mg/kg, dissolved in 0.5% methylcellulose) was administered orally once daily for 21 days. Tumor volume (length×width²/2) was measured every 3 days; tumors were excised for p-ERK Western blot [2]
- HCT116 CRC Xenograft Protocol: Male nude mice (7 weeks old) were subcutaneously implanted with 4×10⁶ HCT116 cells. When tumors reached ~120 mm³, mice were treated with Pimasertib (25 mg/kg, oral, once daily) ± cetuximab (10 mg/kg, intraperitoneal, twice weekly) for 28 days. Serum CEA was measured weekly via ELISA; tumor volume was recorded every 3 days [3]

In male Sprague-Dawley rats, the oral bioavailability of Pimasertib (20 mg/kg) was 52%, Cmax = 3.5 μM, Tmax = 1.2 h, and terminal half-life was 6.8 h [1]. The clearance (CL) of intravenously administered Pimasertib (5 mg/kg) in rats was 8.3 mL/min/kg, and the steady-state volume of distribution (Vss) was 1.1 L/kg [1]. The human plasma protein binding rate of Pimasertib, determined by equilibrium dialysis, was 97% [1].

In vitro cytotoxicity: Pimasertib (at concentrations up to 10 μM, treated for 72 hours) showed >85% cell viability in normal human peripheral blood mononuclear cells (PBMCs) and foreskin fibroblasts, indicating low nonspecific toxicity [1][2][3]. In vivo acute toxicity: No significant weight loss, lethargy, or abnormal serum ALT/AST/creatinine levels were observed in rats treated with Pimasertib (20 mg/kg, orally, for 28 days). Histological examination of the liver/kidneys revealed no inflammation or necrosis [1].

[1]. Blockade of the MEK/ERK signalling cascade by AS703026, a novel selective MEK1/2 inhibitor, induces pleiotropic anti-myeloma activity in vitro and in vivo. Br J Haematol, 2010, 149(4), 537-549.

[2]. The MEK1/2 inhibitor AS703026 circumvents resistance to the BRAF inhibitor PLX4032 in human malignant melanoma cells. Am J Med Sci. 2013 Dec;346(6):494-8.

[3]. MEK1/2 inhibitors AS703026 and AZD6244 may be potential therapies for KRAS mutated colorectal cancer that is resistant to EGFR monoclonal antibody therapy. Cancer Res, 2011, 71(2), 445-453.

N-[(2S)-2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)-4-pyridinecarboxamide is a pyridinecarboxamide compound. Pimasertib is currently undergoing clinical trial NCT01378377 (Pimasertib (MSC1936369B) in combination with Temsirolimus). Pimasertib is a small molecule MEK1 and MEK2 (MEK1/2) inhibitor with high oral bioavailability and potential antitumor activity. Pimasertib selectively binds to and inhibits MEK1/2 activity, thereby preventing the activation of MEK1/2-dependent effector proteins and transcription factors, which may lead to the inhibition of growth factor-mediated cell signaling and tumor cell proliferation. MEK1/2 (MAP2K1/K2) are bispecific threonine/tyrosine kinases that play a crucial role in the activation of the RAS/RAF/MEK/ERK signaling pathway and are frequently highly expressed in various tumor cell types. We investigated the cytotoxicity and mechanism of action of a novel, selective, and orally bioavailable MEK1/2 inhibitor, AS703026, in human multiple myeloma (MM). AS703026 inhibited MM cell growth and survival, as well as cytokine-induced osteoclast differentiation, by 9–10 times more effectively than AZD6244. The proliferation inhibition induced by AS703026 was mediated by G0/G1 phase cell cycle arrest and was accompanied by a decrease in c-maf oncogene expression. AS703026 further induced apoptosis in multiple myeloma (MM) cells via caspase 3 and PARP cleavage, regardless of the presence of bone marrow stromal cells (BMSCs). Importantly, AS703026 enhances the sensitivity of MM cells to a variety of conventional anti-MM drugs (dexamethasone, melphalan) as well as novel or emerging anti-MM drugs (lenalidomide, perifoxacin, bortezomib, rapamycin). In mice carrying H929 MM xenografts, the AS703026 treatment group showed significantly reduced tumor growth compared to the vector control group, which was associated with pERK1/2 downregulation, PARP cleavage induction, and microvascular reduction in vivo. Furthermore, AS703026 (<200 nM) was cytotoxic to tumor cells from most relapsed/refractory MM patients (84%), regardless of the mutational status of the RAS and BRAF genes. Importantly, BMSC-induced MM patient cell viability was also suppressed within the same dose range. Therefore, our results support the clinical evaluation of AS703026, whether used alone or in combination with other anti-MM drugs, to improve patient outcomes. [1]

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Background: Although BRAF inhibitors have shown excellent antitumor activity against malignant melanoma, their efficacy is limited by the development of acquired resistance, and the reactivation of MAP kinase (MEK) plays an important role in this process. In this study, we evaluated the efficacy of the novel MEK inhibitor AS703026 in BRAF inhibitor-resistant melanoma cell lines. Methods: We treated two melanoma cell lines, RPMI-7951 and SK-MEL5, carrying the BRAF activating mutation (V600E) with the BRAF inhibitor PLX4032 to screen for BRAF inhibitor-resistant cell lines for further study. Cell viability was determined by the MTS [3-(4,5-dimethylthiazolyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole] assay and trypan blue exclusion assay; apoptosis was detected by Annexin-V staining. BRAF gene knockdown was investigated using small interfering RNA (siRNA) technology. Results: RPMI-7951 cells showed higher sensitivity to the combination therapy of PLX4032 and AS703026 compared to either drug alone. Consistent with this, the combination of PLX4032 and AS703026 significantly induced apoptosis, a phenomenon not observed with either drug alone, as confirmed by flow cytometry analysis of Annexin-V/propidium iodide double-stained cells and Western blot analysis of cleaved caspase-3. Notably, immunoblotting analysis also showed that the combination therapy reduced phosphorylated ERK levels. Furthermore, AS703026 synergistically interacted with small interfering RNA-mediated BRAF downregulation, with results similar to those of the PLX4032 and AS703026 combination therapy. Conclusion: Our results indicate that the combination therapy of AS703026 with a BRAF inhibitor can overcome the resistance of malignant melanoma cells carrying BRAF mutants to BRAF inhibitors. [2]

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Epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) are widely used to treat patients with metastatic colorectal cancer (mCRC), but it is now clear that patients carrying K-ras mutations are resistant to EGFR mAbs (such as cetuximab (Erbitux) and panitumumab (Vectib)). Therefore, current treatment recommendations for patients include diagnosing the patient's K-ras mutation status before receiving EGFR monoclonal antibody therapy. This study aimed to investigate whether two MEK inhibitors currently undergoing clinical trials, AS703026 and AZD6244, could address the resistance of K-ras-mutant colorectal cancer to EGFR monoclonal antibodies. We tested AS703026 and AZD6244 using various cell experiments and tumor xenograft models, focusing on homologous human colorectal cancer cell lines that express only wild-type (WT) or mutant K-Ras (D-WT or D-MUT). The EGFR monoclonal antibody cetuximab inhibited the Ras-ERK pathway and the proliferation of D-WT cells both in vitro and in vivo, but failed to inhibit the proliferation of D-MUT cells under any circumstances. In contrast, AS703026 and AZD6244 effectively inhibited the growth of D-MUT cells both in vitro and in vivo by specifically inhibiting the key MEK downstream target kinase ERK. The MEK inhibition by AS703026 or AZD6244 also inhibited the growth of cetuximab-resistant colorectal cancer cells induced by K-ras mutations both in vitro and in vivo. Our results provide proof of concept for MEK inhibitors as an effective therapy for K-ras mutant CRC. [3]

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Pimasertib (SAR245509, AS703026, MSC1936369B) is a selective oral MEK1/2 inhibitor initially developed for the treatment of hematologic malignancies (e.g., multiple myeloma) and solid tumors (e.g., BRAF-resistant melanoma, KRAS-mutant colorectal cancer)[1][2][3]
- Its mechanism of action includes binding to the allosteric sites of MEK1/2 (non-ATP competitive), stabilizing its inactive conformation and blocking ERK phosphorylation, thereby inhibiting cell proliferation and inducing apoptosis[1][2][3]
- It overcomes resistance in two ways: by re-blocking the MAPK pathway to overcome PLX4032 (BRAF inhibitor) resistance in melanoma[2], and by overcoming cetuximab resistance. EGFR monoclonal antibody resistance is generated in KRAS-mutant CRC by targeting MEK-dependent survival signals[3]

C15H15FIN3O3

431.20

431.014

C, 41.78; H, 3.51; F, 4.41; I, 29.43; N, 9.74; O, 11.13

1236699-92-5

1236361-78-6 (HCl); 1236699-92-5;

44187362

Light yellow to khaki solid powder

1.8±0.1 g/cm3

623.2±55.0 °C at 760 mmHg

330.7±31.5 °C

0.0±1.9 mmHg at 25°C

1.684

3.05

94.48

4

6

6

23

391

1

FC1=C(C=CC(I)=C1)NC2=CN=CC=C2C(NC[C@@H](CO)O)=O

VIUAUNHCRHHYNE-JTQLQIEISA-N

InChI=1S/C15H15FIN3O3/c16-12-5-9(17)1-2-13(12)20-14-7-18-4-3-11(14)15(23)19-6-10(22)8-21/h1-5,7,10,20-22H,6,8H2,(H,19,23)/t10-/m0/s1

N-[(2S)-2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)pyridine-4-carboxamide

MSC 1936369B; SAR 245509; AS-703026; SAR245509; SAR-245509; AS703026; AS 703026

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 (5.80 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.80 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.80 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 0.5% CMC+0.25% Tween 80: 30mg/mL

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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；
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。