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Opaganib (ABC294640) SPHK inhibitor

Cat.No.S7174

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

Chemical Structure

Molecular Weight: 380.91

Jump to Mechanism of Action Cell Culture & Working Concentration Solubility Chemical Information, Storage & Stability

Quality Control

Batch: Purity: 99.73%

99.73

Related Targets	Immunology & Inflammation related CD markers Interleukins Anti-infection Antioxidant COX Histamine Receptor TNF-alpha ROS Parasite PD-1/PD-L1 TLR Prostaglandin Receptor IFN CXCR Nrf2 NOS CCR NLRP3 eIF STING AhR IL Receptor Complement System IRAK β-lactamase IDO/TDO PGES FKBP NADPH-oxidase
Related Products	PF-543 hydrochloride SKI II SKI-V Defensamide (MHP) K6PC-5 SKI-178 SPHK1 Antibody [A2K18]

Cell Culture, Treatment & Working Concentration

Cell Lines	Assay Type	Incubation Time	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

Chemical Information, Storage & Stability

Molecular Weight	380.91	Formula	C₂₃H₂₅ClN₂O	Storage (From the date of receipt)	3 years-20°Cpowder
CAS No.	915385-81-8	Download SDF		Storage of Stock Solutions	1 year-80°Cin solvent 1 month-20°Cin solvent
Synonyms	N/A			Smiles	C1C2CC3(CC1CC(C2)(C3)C(=O)NCC4=CC=NC=C4)C5=CC=C(C=C5)Cl

Solubility

In vitro
Batch:

DMSO : 76 mg/mL (199.52 mM)
(Moisture-contaminated DMSO may reduce solubility. Use fresh, anhydrous DMSO.)

Ethanol : 28 mg/mL

Water : Insoluble

Molarity Calculator

Mass	Concentration	Volume	Molecular Weight
=	×	×

Dilution Calculator Molecular Weight Calculator

In vivo Batch:
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Clear solution	10%DMSO 1 40%PEG300 2 5%TWEEN80 3 45%ddH2O Validated by Selleck labs. Should you need adjustments to this formulation, contact our sales team for custom testing.	24.000mg/ml (63.01mM)	Taking the 1 mL working solution as an example, add 100 μL of 240 mg/ml clarified DMSO stock solution to 400 μL of PEG300, mix evenly to clarify it; add 50 μL of Tween80 to the above system, mix evenly to clarify; then continue to add 450 μL of ddH2O to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)

Dosage: mg/kg Average weight of animals: g Dosing volume per animal:μL Number of animals:

Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)

% DMSO + % + % Tween 80 + % ddH₂O

%DMSO+ %

Calculation results:

Working concentration: mg/ml;

Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such
as vortex, ultrasound or hot water bath can be used to aid dissolving.

Mechanism of Action

Targets/IC₅₀/Ki	SphK2 ^[1] (Cell-free assay) 60 μM
In vitro	Opaganib (ABC294640) markedly alters the ratio of ceramide/S1P consistent with inhibition of SK activity in MDA-MB-231 cells. It 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] This compound 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, it decreases E2-stimulated ERE-luciferase activity. ^[3]
Kinase Assay	Sphingosine Kinase Assays
Kinase Assay	The IC50 values for Opaganib (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 this compound, the ADP Quest assay system is used to measure kinase activity in the presence of varying concentrations of sphingosine and it. To determine the effects of Opaganib on cellular SK activity, near-confluent MDA-MB-231 cells are serum-starved overnight, and then treated with varying concentrations of it. 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.
In vivo	Opaganib (ABC294640) significantly reduces tumor growth in mice bearing mammary adenocarcinoma xenografts, associated with depletion of S1P levels. ^[1] In severe combined immunodeficient mice bearing A-498 xenografts, it delays tumor growth and elevates autophagy markers. ^[2] This compound also 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]
References	[1] https://pubmed.ncbi.nlm.nih.gov/20061445/ [2] https://pubmed.ncbi.nlm.nih.gov/20179157/ [3] https://pubmed.ncbi.nlm.nih.gov/20861237/ [4] https://pubmed.ncbi.nlm.nih.gov/22848628/

Applications

Methods	Biomarkers	Images	PMID
Western blot	c-Myc pRb / Rb H3K9ac / p21 p-STAT3 / STAT3		27517489
Growth inhibition assay	Cell proliferation		25122609

Clinical Trial Information

(data from https://clinicaltrials.gov, updated on 2024-05-22)

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

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