SLV-2436 (SEL201-88) is a novel effective and ATP-competitive inhibitor of MNK1 and MNK2 (IC50: 10.8/5.4 nM).

MNK1:10.8 nM, MNK2:5.4 nM

在体外激酶实验中，使用重组的MNK1和MNK2蛋白以及逐渐增加的SLV-2436浓度，结果表明SLV-2436具有高度活性，其IC50分别为MNK1的10.8纳摩尔和MNK2的5.4纳摩尔。为了确认SLV-2436的激酶选择性，进行了1微摩尔浓度的KINOMEscan（DiscoverX）竞争性结合实验，该实验包含了450种不同的激酶（32）。观察到SLV-2436的结合谱显著集中在包含MNK1和MNK2的CAMK激酶家族。

为探究SLV-2436的药效学特性，对小鼠连续口服给药5次，剂量分别为10、25和50 mg/kg，每12小时一次(即每天两次)。在10 mg/kg的两次日剂量下，第五次给药后4小时，测定得到SLV-2436的低血浆浓度为125 ng/ml。然而，以25和50 mg/kg的剂量每天两次给药，相当于每天SLV-2436的剂量为50和100 mg/kg，显著增加了剂量依赖性的血浆暴露量，分别达到平均水平1,299 ng/ml和2,075 ng/ml。在24小时时间点，SLV-2436在血浆中仍可检测到，三个剂量组（10、25和50 mg/kg每天两次给药）的剂量依赖性浓度分别为9、73和124 ng/ml。口服(p.o.)给药SLV-2436，剂量为每天两次的50 mg/kg，即每天100 mg/kg，连续37天，小鼠能够良好耐受。此外，连续给药37天，SLV-2436的剂量为每天100 mg/kg，未引起任何明显的临床毒性迹象。在研究终点进行了包括血液学和生化参数在内的血液化学检查，并确认了SLV-2436以每天100 mg/kg的剂量进行多次给药是安全的。尽管如此，SLV-2436表现出了良好的口服生物利用度，口服给药后0.25小时达到最大血浆浓度1,078 ng/ml。

Western blot analysis. Cells were treated with dasatinib, imatinib, or SEL201 at the indicated times, and pellets were harvested to obtain protein extracts. Briefly, cell pellets were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 [NP-40], 0.5% sodium deoxycholate, and 0.1% SDS). After sonication, cell lysates were centrifuged at 15,871 g for 15 minutes. The supernatants were collected, and protein concentrations were quantified. Equal amounts of protein were loaded and separated on a 10% SDS-PAGE. After transferring to a nitrocellulose membrane (Bio-Rad), 5% milk/TBS was used to block for 1 hour, and then probed for target antibodies overnight at 4°C. After incubation with HRP-conjugated secondary antibodies for 1 hour at room temperature, the signals of targeted protein were developed with chemiluminescence substrate ECL Western blotting detection reagent.

All animals were handled in strict accordance with the good animal practice and maintained according to the standards of pathogen-free conditions. The pharmacokinetic profile of SEL201 was assessed in 6-week-old female CD-1 mice (3 animals per time point). SEL201 was freshly dissolved in DMSO and then diluted in Captisol (Ligand) for administration with a volume of 10 μl per 1 g of body weight via the oral (p.o.; 5 mg/kg) or i.v. (2 mg/kg) route. Animals were sacrificed at 8 time points (5, 15, and 30 minutes and 1, 2, 4, 6, and 24 hours) and blood samples harvested. Plasma samples were collected and stored at –80°C for further analysis. To evaluate the pharmacodynamic properties of SEL201, 10- to 16-week-old male C57BL/6 mice (stock 000664, The Jackson Laboratory) were divided into a control group and 3 dosing groups. Animals were given either vehicle (DMSO + N,N-Dimethylacetamide + Captisol) or SEL201 at 10-, 25-, and 50-mg/kg doses (freshly dissolved). Drugs were administered p.o. in a volume of 10 μl per 1 g of body weight. Each animal received a total of 5 doses with the twice-daily schedule (i.e., every 12 hours). Body weight was assessed once daily. Six animals per experimental group supported sample collection at 2-time points (i.e., 4 hours and 24 hours) after the last, fifth administration, with 3 animals per time point. Plasma samples were collected and stored at –80°C for further analysis. For safety assessment of SEL201, 7- to 8-week-old tumor-bearing female Hsd: Athymic Nude-Foxn1nu mice (strain code 069, Envigo) were used. Before use, SEL201 was freshly dissolved, and doses of 50 mg/kg were administered twice daily p.o. in a volume of 10 μl per 1 g of body weight. Body weight was assessed every day. At the end of the experiment on day 37, mice were anesthetized and blood samples for total cell counts and biochemistry were obtained.