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.) improves bronchial smooth muscle hyperreactivity induced by antigens in mice by inhibiting the production of endogenous sphingosine-1-phosphate. In a homologous Balb/c mouse model with JC mammary carcinoma cells, SKI II (50 mg/kg, i.p./p.o.) significantly reduces tumor growth without evident toxicity or weight loss compared to the control group.

Consistent with its role in reducing S1P levels, SKI II induces apoptosis in T24 cells. In JC cells, SKI II decreases S1P formation in a concentration-dependent manner (IC50: 12 μM). Additionally, in the breast cancer cell line MDA-MB-231, SKI II significantly inhibits endogenous SK activity. Among various human cancer cell lines, including T-24, MCF-7, MCF-7/VP, and NCI/ADR, SKI II demonstrates notable antiproliferative effects (IC50: 4.6/1.2/0.9/1.3 μM). Furthermore, SKI II reverses the cisplatin resistance in SGC7901/DDP by downregulating P-gp expression and inducing apoptosis through the downregulation of SPHK1.

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)