Opaganib (ABC294640)

The IC50 values for ABC294640 and DMS are determined by a newly developed HPLC-based SK activity assay. In brief, the test compounds are incubated with recombinant SK1 or SK2 and NBD-Sph in the isozyme-selective assay buffers detailed below with 1 mg/ml fatty acid-free bovine serum albumin, 100 μM ATP, and 400 μM MgCl₂. The product, i.e., NBD-S1P, is separated from NBD-Sph by HPLC as follows: Waters 2795 HPLC system with a Waters 2495 fluorescence detector, C8 Chromolith RP-8e column (100 × 4.6 mm), 1 ml/min mobile phase (acetonitrile/20 mM sodium phosphate buffer, pH2.5, at 45:55). Fluorescence is monitored with excitation at 465 nm and emission at 531 nm. The ratio of NBD-S1P/(NBD-Sph + NBD-S1P) is used as a measure of SK activity. SK-isozyme selective assay buffers each contained 20 mM Tris, pH7.4, 5 mM EDTA, 5 mM EGTA, 3 mM β-mercaptoethanol, 5% glycerol, 1× protease inhibitors and 1× phosphatase inhibitors. For the SK1 assay buffer, 0.25% (final) Triton X-100 is added; and for the SK2 buffer, 1 M (final) KCl is added. Assays are run for 2 h at room temperature, and then a 1.5 volume of methanol is added to terminate the kinase reaction. After 10 min, the samples are centrifuged at 20,000g to pellet the precipitated protein, and the supernatants are analyzed by HPLC. In experiments to determine the Ki for inhibition of SK2 by ABC294640, the ADP Quest assay system is used to measure kinase activity in the presence of varying concentrations of sphingosine and ABC294640. To determine the effects of ABC294640 on cellular SK activity, near-confluent MDA-MB-231 cells are serum-starved overnight, and then treated with varying concentrations of ABC294640. The cells are then incubated with [³H]sphingosine at a final concentration of 1 μM. The cells take up the exogenous sphingosine, which is converted to S1P via SK activity, and [³H]S1P is separated from [³H]sphingosine by extraction and quantified by scintillation counting.

1025LU, Hep-G2, A-498, MCF-7, Caco-2, MDA-MB-231, HT-29, Panc-1, DU145, T24, and SK-OV-3 cell lines

~50 μM

72 h

To determine the effects of the test compounds on proliferation, cells are plated into 96-well microtiter plates and allowed to attach for 24 h. Varying concentrations of ABC294640 are added to individual wells and the cells are incubated for an additional 72 h. At the end of this period, the number of viable cells is determined by use of the sulforhodamine-binding assay. The percentage of cells killed is calculated as the percentage decrease in sulforhodamine-binding compared with control cultures. Regression analyses of inhibition curves are performed by use of GraphPad Prism.

Female BALB/c mice bearing JC tumors

~100 mg/kg

p.o.