GS-9973 (Synonyms: GS-9973)

Kinase assays

Full-length baculovirus-expressed Syk kinase activity was measured in a Lance-based assay format in a final volume of 25 μL containing 25 mM Tris-HCl, pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, 0.5 μM Promega PTK biotinylated peptide substrate 1, 0.01% casein, 0.01% Triton X-100, 0.25% glycerol, and 40 mM ATP (Km for ATP) incubated at room temperature for 60 mins. Reactions were stopped with the addition of 30 mM EDTA containing 30 μL of SA-APC and 4 nM PT-66 antibody and the plates were measured on a Perkin-Elmer Envision. IC50 values for test compounds were determined using a four-parameter linear regression algorithm.

Cell lines

MV-4-11 cells

Preparation method

The solubility of this compound in DMSO is > 20.6 mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 °C for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 °C for several months.

Reacting condition

10 μM; 72 hrs

Applications

GS-9973 selectively inhibited Syk in cells, and potently suppressed BCR-mediated activation and proliferation of B-cells as well as immune-complex-induced cytokine production in monocytes.

Animal models

Rat collagen-induced arthritis (CIA) model

Dosage form

1 ~ 10 mg/kg; p.o.; b.i.d.

Applications

In a rat CIA model, GS-9973 (1 ~ 10 mg/kg; p.o.; b.i.d.) significantly reduced ankle inflammation. Moreover, GS-9973 also showed disease-modifying activity in several histological measurements, including inhibition of pannus formation, cartilage damage, bone resorption, as well as periosteal bone formation with the ED50 values ranging from 1.2 to 3.9 mg/kg.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Currie KS, Kropf JE, Lee T, et al. Discovery of GS-9973, a selective and orally efficacious inhibitor of spleen tyrosine kinase. J Med Chem, 2014, 57(9): 3856-3873.