SKI II (SphK-I2) is a highly selective and non-ATP-competitive S1P receptor inhibitor (IC50: 0.5 μM) while exhibiting no inhibitory action on other kinases including PKCα, PI3K, and ERK2.

SPHK:0.5 μM

SKI II（50 mg/kg,i.p.）通过抑制小鼠内源性1-磷酸鞘氨醇生成,可改善抗原诱导的支气管平滑肌高反应性.在携带JC乳腺癌细胞的同源Balb/c小鼠实体瘤模型中, SKI II（50 mg/kg,i.p./p.o.）可显著降低肿瘤生长,与对照组相比,无明显的毒性或体重减轻.

与降低S1P水平一致,SKI II可诱导T24细胞凋亡。在JC细胞中,SKI II可浓度依赖性降低S1P形成（IC50：12 μM）。在乳腺癌细胞系MDA-MB-231中,SKI II可明显抑制内源性SK活性。在人类癌细胞系,包括T-24,MCF-7,MCF-7/VP,NCI/ADR中,SKI II均有显著的抗增殖效果（IC50：4.6/1.2/0.9/1.3 μM）。此外,通过下调P-gp的表达,及通过下调SPHK1而上调凋亡,SKI II可逆转SGC7901/DDP对顺铂的耐药性。

SK Assay: A medium-throughput assay suitable for screening for inhibitors of recombinant human SK has been established. Briefly, 5 μg of purified GST-SK fusion protein are combined with 12 nM sphingosine, which contains a 100-fold dilution of [3-3H]sphingosine (20 Ci/mmol), 1 mM ATP, 1 mM magnesium chloride, and 200 μL of assay buffer [20 mM Tris HCl (pH 7.4), 20% glycerol, 1 mM beta-mercaptoethanol, 1 mM EDTA, 20 mM zinc chloride, 1 mM sodium orthovanadate, 15 mM sodium fluoride, and 0.5 mM 4-deoxypyridoxine]. Assays are run for 30 min at 25°C with shaking and contains either 1% DMSO or 5 g/mL test compound, which corresponds to concentrations of 10–25 μM. The reactions are terminated with 50 μL of concentrated ammonium hydroxide, followed by extraction of the assay mixture with chloroform:methanol (2:1). The aqueous portion is transferred to scintillation vials and radioactivity is quantified as a measure of [3H]]S1P formation using a Beckman LS 3801 Scintillation Counter. The intra-assay coefficient of variation is ~10%, whereas interassay variation is ~20%.

T24, MCF-7, MCF-7/VP, and NCI/ADR cells are plated into 96-well tissue culture plates at ~15% confluency. After 24 hours, cells are treated with various concentrations of inhibitors. After an additional 48 hours, cell survival is assayed using the sulforhodamine B assay.(Only for Reference)