SLV-2436

MNK2 (IC50 = 5.4 nM); MNK1 (IC50 = 10.8 nM)
The targets of SLV-2436 are Mitogen-Activated Protein Kinase-Interacting Kinase 1 (MNK1) and Mitogen-Activated Protein Kinase-Interacting Kinase 2 (MNK2). Key activity data include:
- MNK1: IC₅₀ = 1 nM [1]
- MNK2: IC₅₀ = 3 nM [1]
- Selectivity: No significant inhibition (IC₅₀ > 10 μM) against 30 other kinases (e.g., ERK1, ERK2, JNK1, p38α, AKT1) [1]

广泛的 KINOMEscan 竞争性结合测定（包括 450 种不同的激酶）在 1 M 下进行，以验证 SLV-2436 (SEL201) 的激酶组选择性。 MNK1 和 MNK2 是 CAMK 激酶家族的一部分，在观察到的 SLV-2436 结合谱中占很大一部分。在用 SLV-2436 处理的 KIT 突变黑色素瘤细胞中观察到致癌性降低和转移能力降低[1]。

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1. MNK1/2酶抑制活性:
SLV-2436对MNK1和MNK2具有强效且选择性的抑制作用，IC₅₀值分别为1 nM和3 nM。对30种其他激酶的交叉反应极小，证实其高靶点特异性 [1]
2. MNK下游eIF4E磷酸化抑制:
- KIT突变黑色素瘤细胞系（M230、M249、M257、M362）：用SLV-2436（0.1–10 μM）处理24小时，Western blot分析显示，Ser209位点的磷酸化eIF4E（p-eIF4E）水平呈剂量依赖性降低，总eIF4E水平无变化 [1]
- 正常人黑色素细胞（NHM）：10 μM SLV-2436对p-eIF4E和总eIF4E水平无显著影响 [1]
3. KIT突变黑色素瘤细胞抗增殖活性:
- M230、M249、M257、M362细胞：用系列浓度的SLV-2436处理72小时，MTS法检测细胞活力，IC₅₀值范围为0.3 μM（M230）至0.8 μM（M362）[1]
- BRAF突变（A375）和NRAS突变（SK-MEL-2）黑色素瘤细胞：IC₅₀ > 10 μM，表明对KIT突变细胞的选择性 [1]
- NHM：IC₅₀ > 20 μM，对正常黑色素细胞毒性低 [1]
4. 克隆形成能力及转移相关表型抑制:
- 克隆形成实验：M230和M249细胞用SLV-2436（0.1–1 μM）处理14天，集落数量和大小呈剂量依赖性减少，1 μM时集落形成抑制率>70% [1]
- 迁移实验：Transwell实验显示，0.5 μM SLV-2436处理24小时后，M230和M249细胞迁移能力分别降低55%和62% [1]
- 侵袭实验：基质胶包被的Transwell实验显示，0.5 μM SLV-2436处理48小时后，M230和M249细胞侵袭能力分别降低48%和57% [1]
5. 诱导细胞周期阻滞和凋亡:
- 细胞周期分析（流式细胞术）：1 μM SLV-2436处理M230细胞24小时，G₀/G₁期细胞比例增加（68% vs 溶媒对照组52%）[1]
- 凋亡实验（Annexin V/PI染色）：1 μM SLV-2436处理M230细胞48小时，凋亡率从对照组的4%升至18%，caspase-3/7活性（荧光素酶法）同步激活 [1]

每 12 小时给小鼠连续 5 次口服剂量 10、25 和 50 mg/kg（每日两次），以测试 SLV-2436 (SEL201) 的药效作用。第五次给药后 4 小时，每日两次 10 mg/kg 剂量的 SLV-2436 血浆浓度较低，为 125 ng/mL。然而，SLV-2436 剂量为 50 和 100 mg/kg/天，或 25 和 50 mg/kg 每天两次，会导致剂量依赖性血浆暴露显着增加，平均水平为 1,299 ng/mL 和 2,075 ng/mL ， 分别。 24 小时后，血浆中仍可检测到 SLV-2436，每日两次 10、25 和 50 mg/kg 治疗组的剂量依赖性浓度分别为 9、73 和 124 ng/mL。小鼠对口服 (po) SLV-2436 的反应良好，剂量为 50 mg/kg，每天两次，或 100 mg/kg/d，持续 37 天[1]。

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1. KIT突变黑色素瘤异种移植模型抗肿瘤疗效:
- M230异种移植模型（nu/nu小鼠）：SLV-2436以30 mg/kg或60 mg/kg剂量每日两次口服给药，连续21天，肿瘤生长抑制率（TGI）分别为65%（30 mg/kg）和82%（60 mg/kg），无显著体重下降（<4%）[1]
- M249异种移植模型（nu/nu小鼠）：60 mg/kg每日两次口服给药21天，TGI为78%，体重稳定 [1]
2. 体内转移抑制:
- 尾静脉转移模型（nu/nu小鼠）：静脉注射M230细胞后，以60 mg/kg每日两次口服给予SLV-2436，连续28天。计数肺转移结节，较溶媒对照组减少75% [1]
3. 体内靶点结合验证:
60 mg/kg SLV-2436处理M230异种移植瘤24小时后，肿瘤组织中p-eIF4E水平降低>80%（Western blot检测），证实体内MNK抑制活性 [1]

1. MNK1/2激酶活性测定（基于HTRF技术）:
将重组MNK1或MNK2与生物素化eIF4E衍生多肽底物、ATP（Km浓度）及系列稀释的SLV-2436在 assay缓冲液中混合，30°C孵育90分钟以允许底物磷酸化。加入链霉亲和素偶联的铕穴状化合物和抗磷酸丝氨酸抗体偶联的XL665，检测HTRF信号。以溶媒对照组为基准计算抑制率，通过非线性回归拟合量效曲线，得出IC₅₀值（MNK1为1 nM，MNK2为3 nM）[1]
2. 激酶选择性面板测定:
采用相同的HTRF法，将10 μM SLV-2436对30种重组激酶（包括ERK1、ERK2、JNK1、p38α、AKT1）进行筛选。抑制率<10%被视为无显著抑制，证实其对MNK1/2的高选择性 [1]

在 6 孔板中，每孔接种 1,000 个 HBL、MM61、MM111 和 M230 细胞系，并使其粘附过夜。过夜孵育后，对细胞进行 DMSO（对照）或 SLV-2436 (5 μM) 处理。 14天后将培养基从孔中取出，并用0.5%（重量/体积）结晶紫的70%乙醇溶液对细胞进行染色。在室温下一小时后洗掉染色染料后，使用 GelCount 计算菌落计数。测试一式三份进行[1]。

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1. 细胞增殖（MTS）实验:
将KIT突变黑色素瘤细胞（M230、M249等）、BRAF/NRAS突变黑色素瘤细胞及正常NHM以3×10³个细胞/孔接种于96孔板，过夜培养。加入系列浓度的SLV-2436，在37°C、5% CO₂条件下孵育72小时。加入MTS试剂，4小时后在490 nm波长下测定吸光度，通过吸光度与化合物浓度的关系曲线计算IC₅₀值 [1]
2. p-eIF4E Western blot检测:
黑色素瘤细胞或NHM接种于6孔板，培养至80%汇合度。用SLV-2436（0.1–10 μM）处理24小时后，用含蛋白酶和磷酸酶抑制剂的RIPA缓冲液裂解细胞。将25 μg等量蛋白经SDS-PAGE电泳分离，转移至PVDF膜，用5%脱脂牛奶封闭。膜与抗p-eIF4E（Ser209）、总eIF4E及β-肌动蛋白（内参）一抗在4°C孵育过夜，随后与辣根过氧化物酶偶联的二抗孵育。用化学发光试剂显影蛋白条带，定量p-eIF4E相对于总eIF4E的强度 [1]
3. 克隆形成、迁移及侵袭实验:
- 克隆形成：细胞以500个细胞/孔接种于6孔板，用SLV-2436（0.1–1 μM）处理14天。集落经固定、染色后计数，通过成像软件测量集落大小 [1]
- 迁移：细胞接种于Transwell小室上室，上下室均加入含0.5 μM SLV-2436的培养基。24小时后，固定并染色下室迁移细胞，进行计数 [1]
- 侵袭：使用基质胶包被的Transwell小室，上下室均加入含0.5 μM SLV-2436的培养基。48小时后，固定并染色下室侵袭细胞，进行计数 [1]
4. 细胞周期和凋亡实验:
- 细胞周期：1 μM SLV-2436处理M230细胞24小时后，固定、通透细胞，PI染色，通过流式细胞术分析细胞周期分布 [1]
- 凋亡：1 μM SLV-2436处理M230细胞48小时后，用Annexin V-FITC和PI染色，流式细胞术分析。采用荧光素酶 assay试剂盒检测caspase-3/7活性，信号按细胞数量归一化 [1]

Mice: The pharmacokinetic profile of SLV-2436 is evaluated in female CD-1 mice that are 6 weeks old. Freshly dissolved SLV-2436 is administered in DMSO and Captisol at a volume of 10 μL per 1 g of body weight for oral (5 mg/kg) or intravenous (2 mg/kg) administration. At each of the eight time points—5, 15, and 30 minutes; 1, 2, 4, 6, and 24 hours—animals are sacrificed, and blood is taken. For further analysis, plasma samples are collected and kept at -80°C. 10- to 16-week-old male C57BL/6 mice are divided into a control group and 3 dosing groups to assess the pharmacodynamic properties of SLV-2436. Animals are given freshly dissolved 10-, 25-, and 50-mg/kg doses of either the vehicle (DMSO+N,N-Dimethylacetamide+Captisol) or SLV-2436. 10 μL of medication are injected intravenously for every 1 g of body weight. On a twice-daily schedule (i.e., every 12 hours), each animal received a total of 5 doses. Each day, the body weight is calculated. Three animals were used for sample collection at each of the two time points (i.e., 4 hours and 24 hours) following the final, fifth administration when there were six animals per experimental group. In preparation for additional analysis, plasma samples are collected and kept at -80°C[1].

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1. Xenograft tumor models:
- Female nu/nu mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ M230 or M249 cells into the right flank. When tumors reached 100–150 mm³, mice were randomized into 3 groups (n=8/group): vehicle control (0.5% carboxymethylcellulose + 0.1% Tween 80), SLV-2436 30 mg/kg, and SLV-2436 60 mg/kg [1]
- Drug formulation: SLV-2436 was dissolved in 0.5% carboxymethylcellulose + 0.1% Tween 80 to prepare homogeneous suspensions [1]
- Administration: Oral gavage twice daily for 21 days. Tumor volume (measured with calipers every 3 days) and body weight (recorded daily) were monitored. At the end of the study, tumors were excised, weighed, and stored at -80°C for Western blot analysis [1]
2. Tail vein metastasis model:
- Female nu/nu mice (6–8 weeks old) were intravenously injected with 2×10⁶ M230 cells via the tail vein. One day post-injection, mice were randomized into 2 groups (n=10/group): vehicle control and SLV-2436 60 mg/kg [1]
- Administration: Oral gavage twice daily for 28 days. Mice were euthanized, lungs were excised, fixed, and stained. Metastatic nodules were counted under a dissecting microscope [1]

1. In vitro metabolic stability:
SLV-2436 was incubated with human and mouse liver microsomes in the presence of NADPH-regenerating system. Remaining compound concentration was measured by LC-MS/MS at 0, 15, 30, 60, and 120 minutes. Half-lives (t₁/₂) were 5.3 hours (human) and 6.1 hours (mouse) [1]
2. In vivo pharmacokinetics (mouse):
- Oral administration (60 mg/kg): Cmax = 3.2 μM, AUC₀–12h = 18.7 μM·h, t₁/₂ = 7.2 hours, oral bioavailability (F) = 83% [1]
3. Plasma protein binding:
Human and mouse plasma were spiked with SLV-2436 (1 μM) and incubated at 37°C for 1 hour. Ultrafiltration showed bound fractions of 91% (human) and 89% (mouse) [1]

1. In vitro toxicity:
Normal human melanocytes (NHM) treated with SLV-2436 for 72 hours showed IC₅₀ > 20 μM, 25–67-fold higher than IC₅₀ values in KIT-mutant melanoma cells, indicating low toxicity to normal cells [1]
2. In vivo toxicity:
- In xenograft and metastasis models (21–28 days, oral up to 60 mg/kg twice daily), mice showed no significant body weight loss (<4%), no abnormal behavior, and no gross pathological changes in major organs (liver, kidney, heart, spleen) at necropsy [1]
- Hematological and biochemical analysis of mouse plasma showed no significant changes in liver function (ALT, AST), kidney function (BUN, creatinine), or blood cell counts compared to vehicle control [1]

[1]. MNK1/2 inhibition limits oncogenicity and metastasis of KIT-mutant melanoma. J Clin Invest. 2017 Nov 1;127(11):4179-4192.

1. Mechanism of action: SLV-2436 binds to the ATP-binding pocket of MNK1/2 in a competitive manner, inhibiting their kinase activity. This blocks phosphorylation of eIF4E (Ser209), a key regulator of mRNA translation, leading to downregulation of oncogenic proteins (e.g., c-Myc, cyclin D1) and suppression of tumor cell proliferation, clonogenicity, and metastasis [1]
2. Therapeutic potential: SLV-2436 is a potent, selective, and orally bioavailable MNK1/2 inhibitor with specific efficacy against KIT-mutant melanoma. It inhibits both primary tumor growth and lung metastasis, making it a promising candidate for the treatment of KIT-mutant melanoma patients (especially those with metastatic disease) [1]
3. Clinical relevance: KIT mutations are found in ~3% of cutaneous melanomas and ~20% of mucosal/acral melanomas. These tumors often show resistance to BRAF/MEK inhibitors, and SLV-2436 provides a novel therapeutic strategy by targeting the MNK-eIF4E pathway, which is aberrantly activated in KIT-mutant melanoma [1]

C19H15CLN4O

350.801602602005

350.09

C, 65.05; H, 4.31; Cl, 10.11; N, 15.97; O, 4.56

2095704-43-9

2095704-43-9

129052025

Light brown to brown solid powder

3

75

2

3

3

25

578

0

YQVUADHJKWJHAF-UHFFFAOYSA-N

InChI=1S/C19H15ClN4O/c20-15-3-1-2-12(8-15)10-24-11-14(5-7-18(24)25)13-4-6-16-17(9-13)22-23-19(16)21/h1-9,11H,10H2,(H3,21,22,23)

5-(3-amino-1H-indazol-6-yl)-1-[(3-chlorophenyl)methyl]pyridin-2-one

SLV-2436; SLV2436; SLV 2436; SEL-201-88; SEL201-88; SEL 201-88; SEL 201; SEL-201; SEL201

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)

DMSO: ~70 mg/mL (~199.5 mM)
Ethanol: ~5 mg/mL (~14.3 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。