Home Immunology & Inflammation SPHK inhibitor Opaganib (ABC294640) For research use only.

Opaganib (ABC294640)

Opaganib (ABC294640) is an orally bioavailable and selective sphingosine kinase-2 (SphK2) inhibitor with IC50 of approximately 60 μM. Phase 1/2.

Opaganib (ABC294640) Chemical Structure

Opaganib (ABC294640) Chemical Structure

CAS No. 915385-81-8

Purity & Quality Control

Opaganib (ABC294640) Related Products

Cell Data

Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
Sf9 Function assay Inhibition of human recombinant SphK2 expressed in Sf9 cells assessed as radiolabeled products using 10 uM sphingosine and 10 uM gamma[32P]ATP by liquid scintillation counting, Ki=9.8μM 25643074
Sf9 Function assay 2 hrs Inhibition of recombinant human full-length N-terminal His-tagged SK2 expressed in baculovirus infected Sf9 insect cells using NBD-Sph as substrate measured after 2 hrs by fluorescence based HPLC analysis, Ki=9.8μM 30889352
Click to View More Cell Line Experimental Data

Biological Activity

Description Opaganib (ABC294640) is an orally bioavailable and selective sphingosine kinase-2 (SphK2) inhibitor with IC50 of approximately 60 μM. Phase 1/2.
Targets
SphK2 [1]
(Cell-free assay)
60 μM
In vitro
In vitro ABC294640 markedly alters the ratio of ceramide/S1P consistent with inhibition of SK activity in MDA-MB-231 cells. ABC294640 inhibits tumor cell proliferation with IC50 values ranging from approximately 6 to 48 μM, and impairs tumor cell migration concomitant with loss of microfilaments. [1] ABC294640 induces nonapoptotic cell death, morphological changes in lysosomes, formation of autophagosomes, and increases in acidic vesicles in A-498, PC-3, and MDA-MB-231 cells. [2] In both MCF-7 and ER-transfected HEK293 cells, ABC294640 decreases E2-stimulated ERE-luciferase activity. [3]
Kinase Assay Sphingosine Kinase Assays
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.
Cell Research Cell lines 1025LU, Hep-G2, A-498, MCF-7, Caco-2, MDA-MB-231, HT-29, Panc-1, DU145, T24, and SK-OV-3 cell lines
Concentrations ~50 μM
Incubation Time 72 h
Method 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.
Experimental Result Images Methods Biomarkers Images PMID
Western blot c-Myc pRb / Rb H3K9ac / p21 p-STAT3 / STAT3 27517489
Growth inhibition assay Cell proliferation 25122609
In Vivo
In vivo In mice bearing mammary adenocarcinoma xenografts, ABC294640 (100 mg/kg, p.o.) significantly reduce tumor growth, associated with depletion of S1P levels. [1] In severe combined immunodeficient mice bearing A-498 xenografts, ABC294640 delays tumor growth and elevates autophagy markers. [2] ABC294640 protects against liver transplantation-induced inflammation and cross-talk between innate and adaptive immunities, major events precipitating and exacerbating graft injury, and improves liver function and survival. [4]
Animal Research Animal Models Female BALB/c mice bearing JC tumors
Dosages ~100 mg/kg
Administration p.o.
NCT Number Recruitment Conditions Sponsor/Collaborators Start Date Phases
NCT02939807 Withdrawn
Carcinoma Hepatocellular
Medical University of South Carolina|National Cancer Institute (NCI)|Apogee Biotechnology Corporation
 September 30 2019 Phase 2
NCT03377179 Completed
Cholangiocarcinoma|Cholangiocarcinoma Non-resectable|Cholangiocarcinoma Perihilar|Cholangiocarcinoma Extrahepatic|Cholangiocarcinoma Intrahepatic
RedHill Biopharma Limited
 March 7 2018 Phase 2
NCT02757326 Terminated
Multiple Myeloma
RedHill Biopharma Limited|Apogee Biotechnology Corporation|Duke University|National Cancer Institute (NCI)
 December 13 2016 Phase 1|Phase 2
NCT02229981 Withdrawn
Diffuse Large B Cell Lymphoma|Kaposi Sarcoma
RedHill Biopharma Limited|Louisiana State University Health Sciences Center in New Orleans|Apogee Biotechnology Corporation
 July 2014 Phase 1|Phase 2
NCT01488513 Completed
Pancreatic Cancer|Unspecified Adult Solid Tumor Protocol Specific
RedHill Biopharma Limited|Apogee Biotechnology Corporation|Medical University of South Carolina|FDA Office of Orphan Products Development|National Cancer Institute (NCI)
 August 2011 Phase 1

Chemical Information & Solubility

Molecular Weight 380.91 Formula

C23H25ClN2O
CAS No. 915385-81-8 SDF Download Opaganib (ABC294640) SDF
Smiles C1C2CC3(CC1CC(C2)(C3)C(=O)NCC4=CC=NC=C4)C5=CC=C(C=C5)Cl
Storage (From the date of receipt)

In vitro
Batch:

DMSO : 76 mg/mL ( (199.52 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Ethanol : 28 mg/mL

Water : Insoluble


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