Lck kinase assays

To determine the inhibitory activity of WH-4-023 against Lck, kinase assays were conducted. The Lck HTRF kinase assay involved ATP-dependent phosphorylation of a biotinylated substrate peptide of gastrin with or without WH-4-023. The final concentration of ATP, gastrin and Lck were 0.5 μM, 1.2 μM and 250 pM, respectively. Buffer were made up as follows: 50 mM HEPES pH=7.5, 2 mM DTT, 5 mM MnCl2, 20 mM MgCl2, 50 mM NaCl, 0.05% BSA. The reaction was quenched and ended by adding 160 μL detection reagent. Detection reagent was prepared as follows: 50 mM Tris, pH=7.5, 3 mM EDTA, 100 mM NaCl, 0.05% BSA, 0.1% Tween-20. Before reading, streptavidin allophycocyanin and anti-phosphotyrosine Ab were added at a final concentration of 0.4 μg/mL and 0.025 nM, respectively. Assay plate was read with excitation at 320 nm and emission at 615 and 655 nm.

Cell lines

Naive human pluripotent cells

Preparation method

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

Reacting condition

1 μM

Applications

WH-4-023 is a potent and selective Src and Lck inhibitor with IC50 values of 6 and 2 nM, respectively[1].Naive human pluripotent cells were maintained in media containing WH-4-023. WH-4-023 improves the fraction of viable GFP+ cells. Withdrawal of the WH-4-023 results in a change in morphology and slight decrease of OCT4-DPE-GFP reporter activity[2].

WH-4-023 is a potent and selective Lck and Src inhibitor with IC50 values of 2 and 6 nM, respectively [1]. It is also an inhibitor of IC50 values of 10, 22 and 60 nM for SIK 1, 2 and 3, respectively [2].

Lck and Src are cytoplasmic tyrosine kinases of the Src family expressed in T cells and NK cells [1]. SIKs restrict the formation of regulatory macrophages and that their inhibition greatly stimulates the production of IL-10 and other anti-inflammatory molecules [2].

WH-4-023 inhibited a number of protein tyrosine kinases that possess a Thr residue at the gatekeeper site, such as FGF and Ephrin receptors, Src family members (Src, Lck, and Yes) and BTK. WH-4-023 also inhibited the SIKs and did not inhibit any other member of the AMPK-related kinase subfamily, which all possess a large hydrophobic residue (Met or Leu) at the gatekeeper site. WH-4-023 inhibited SIK 1, 2 and 3 with IC50 values of 10, 22 and 60 nM, respectively. Also, WH-4-023 increased LPS-stimulated IL-10 production and greatly suppressed proinflammatory cytokine secretion [2].

References:
[1].  Martin MW, Newcomb J, Nunes JJ, et al. Novel 2-aminopyrimidine carbamates as potent and orally active inhibitors of Lck: synthesis, SAR, and in vivo antiinflammatory activity. J Med Chem, 2006, 49(16): 4981-4991.
[2].  Clark K, MacKenzie KF, Petkevicius K, et al. Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages. Proc Natl Acad Sci U S A, 2012, 109(42): 16986-16991.