YM201636 (IC50=33 nM), a specific PIKfyve inhibitor, is less effective to p110α and insensitive to Fabl, which is yeast orthologue.

PIKfyve:33 nM

在血清饥饿处理的NIH3T3细胞中, YM201636（0.8 μM）明显降低PtdIns(3,5)P2产量,降低达 80%。在3T3L1脂肪细胞中,YM-201636（IC50=54 nM）抑制2-脱氧葡萄糖吸收, 按160 nM剂量处理时,则能够完全抑制2-脱氧葡萄糖吸收。在MDCK细胞中,YM201636细胞间隙连接蛋白Claudin-1和Claudin-2的连续再循环,导致细胞内累积,也延迟上皮屏障的形成。

Following 3T3L1 adipocyte serum-starvation and insulin stimulation, cell lysates containing protease inhibitors are clarified and then subjected to immunoprecipitation with anti-PIKfyve antibodies. Washed beads are mixed with 100 μM PtdIns and preincubated for 15 min with YM-201636 (100 nM) or vehicle in the assay buffer (50 mM Tris-HCl, pH 7.5, 1 mM EGTA and 10 mM MgCl2). The kinase assay (50 μL final volume) is carried out for 15 min at 37 °C with 15 μM ATP and [γ-32P]ATP (30 μCi). Lipids are extracted, spotted on TLC glass plates (250 μm), resolved by a chloroform/methanol/water/ammonia solvent system and detected by autoradiography[2].

YM-201636 is dissolved in DMSO and diluted with DMEM and added to cells at a final concentration of 800 nM. Cells are treated with YM-201636 or a DMSO control for 2 h. For TER measurements cells are plated at confluency on Transwell permeable polyester filters (0.4 μM pore size) with surface area of 0.33 cm2. Media is changed ever 2-3 days and cells are grown for 7 days prior to TER measurements[4].