Kinase experiment:

In vitro kinase assays are performed on His(6)-tagged Lck (residues 62-509) and full-length c-Abl purified from Sf-9 cells, and commercial sources of Lyn, Src and PKC. Lck, Lyn, Src and Abl activities are measured with an ELISA-based assay. Flat bottom 96-well ELISA plates are incubated with a 200 μg/mL solution of Poly(Glu,Tyr) 4 : 1 substrate in phosphate buffered saline (PBS) for 1 h at 37℃ and then washed with PBS containing 0.1% Tween-20 (PBS-T). Inhibitor dilutions are added to the washed plates already containing the appropriate enzyme in kinase assay buffer (250 mM Mopso, pH 6.75, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTT, 0.02% BSA, 2 mM Na3VO4, 5% DMSO, 5 μM ATP). After incubation at room temperature for 20 min, plates are washed three times with PBS-T and plate-bound phosphotyrosine is detected with an anti-phosphotyrosine-HRP antibody conjugate and subsequent color development with K-Blue reagents. All assays are optimized to use the least amount of enzyme necessary for a reproducible signal-to-noise ratio[1].

Cell experiment:

K-562 cells are grown in RPMI 1640 supplemented with 10% fetal calf serum (FCS), and 50 g/mL Gentamycin. Meg-01 cells are cultured in Vitacell modified RPMI 1640 (ATCC), supplemented with 10% FCS and 50 μg/mL Gentamycin. The human GM-CSF-dependent myeloid leukemia cell line TF-1 is grown in RPMI 1640 supplemented with 10% FCS, 50 μg/mL Gentamycin, and 1 ng/mL of recombinant human GM-CSF. DAGM murine myeloid leukemia cells are cultured in RPMI 1640 supplemented with 10% FCS, 50 μg/mL Gentamycin, and 0.5 ng/mL recombinant IL-3. Concentrated stock solutions of PP2 (5 mM) and A-419259 (2 mM) are prepared in DMSO and stored at -20℃. Cellular proliferation is measured using the Live/Dead growth assay. This assay employs calcein-AM, a fluorogenic esterase substrate that is taken up by viable cells and hydrolyzed intracellularly, releasing a green fluorescent product. Briefly, 104 cells are plated per well in 96-well plates for each day of a 4-day time course. Various concentrations of PP2, A-419259 or vehicle control are added to the wells (five wells per concentration per day) and the plates are incubated at 37℃. At each time point, one plate is centrifuged at 1500 g for 10 min to pellet the cells. Cells are washed with phosphate buffered saline (PBS), and calcein-AM is added to each well to a final concentration of 1 μM. Plates are incubated in the dark at room temperature for 1 h. The plates are then read at 485/530 nm (excitation/emission) using a SpectraMax Gemini XS fluorescent plate reader and data are analysed with SoftMax Pro software[1].

A 419259 trihydrochloride is an orally available inhibitor of lck with IC50 value of 15 nM [1].

Lck is a src family tyrosine kinase. Inhibition of this enzyme results in a prevention of T-cell activation and provides an application for treating T-cell dependent processes such as autoimmune and inflammatory diseases. A 419259 trihydrochloride is screened out by its potent inhibitory activity against the recombinant lck (64–509) with IC50 value of 15nM. It is also a selective inhibitor. It shows inactive against a panel of other kinases including cMet, IGFR, PKC and PDGFR. Besides that, A 419259 trihydrochloride is found to inhibit the production of IL-2 with IC50 value of 25nM in Jurkat cells. Moreover, administration of A 419259 trihydrochloride reduces IL-2 production in mice with ED50 value of 2.5 mg/kg. A 419259 trihydrochloride also has efficacy of the antigen specific T-Cell immune response. Administration of this compound inhibits IFN-γ production during the in vivo priming phase [1].

Reference:
[1] Calderwood D J, Johnston D N, Munschauer R, et al.  Pyrrolo [2, 3-d] pyrimidines containing diverse N-7 substituents as potent inhibitors of Lck. Bioorganic & medicinal chemistry letters, 2002, 12(12): 1683-1686.