Kinase experiment:

Protein A-Sepharose beads (prepared as a 50% (w/v) suspension) are added to the antibody/lysate mixture at 250 μL/mL and allowed to incubate for 30 min at 4℃. The beads are then washed twice in 1 mL of lysis buffer and twice in 1 mL of kinase buffer (25 mM HEPES, 3 mM MnCl2, 5 mM MgCl2, and 100 μM sodium orthovanadate) and resuspended to 50% (w/v) in kinase buffer. Twenty-five microliters of the bead suspension is added to each well of the enolase-coated 96-well high protein binding plate together with an appropriate concentration of compound and [γ-32P]ATP (25 μL/well of a 200 μCi/mL solution in kinase buffer). After incubation for 20 min at 20℃, 60 μL of boiling 2× solubilization buffer containing 10 mM ATP is added to the assay wells to terminate the reactions. Thirty microliters of the samples is removed from the wells, boiled for 5 min, and run on a 7.5% SDS-polyacrylamide gel. The gels are subsequently dried and exposed to Kodak X-AR film. For quantitation, films are scanned using a Molecular Dynamics laser scanner, and the optical density of the major substrate band, enolase p46, is determined. Concentrations of compound that causes 50% inhibition of enolase phosphorylation (IC50) are determined from a plot of the density versus concentration of compound. In companion experiments for measuring the activity of compounds against Lck, the assay plate is washed with two wash cycles on a Skatron harvester using 50 mM EDTA, 1 mM ATP. Scintillation fluid (100 μL) is then added to the wells, and P incorporation is measured using a Pharmacia Biotech micro-β-counter. Concentrations of compound that causes 50% inhibition of enzyme activity (IC50) are determined from a plot of the percent inhibition of enzyme activity versus concentration of compound[1].

Cell experiment:

Inhibition of anti-CD3-stimulated tyrosine phosphorylation in purified human peripheral blood T cells is measured as follows. All incubations are carried out at 37℃ in an Eppendorf Thermomixer 5436 at a mixing setting of 11. Cells (1×106 in 100 μL of RPMI 1640 medium) are incubated for 15 min with drug prior to a 6-min incubation with 1 μg of anti-CD3/mL (anti-leu4, 100 μg/mL). The final volume of the reaction is 115 μL. Reactions are terminated by the addition of 57.5 μL of 3× solubilization buffer incubated at 100℃ prior to its addition. Samples are mixed, boiled for 5 min, and stored at -70℃. Western blots of these cell lysates, run on 10% SDS-polyacrylamide gels, are probed with a polyclonal anti-phosphotyrosine antibody, and immune complexes are detected with I-labeled protein A (ICN). For quantitation, films are scanned using a Molecular Dynamics laser scanner, and the optical densities of the major substrate band, p70, are quantitated in the presence of anti-CD3 (in the presence and absence of drug). Percent inhibition is calculated as follows: (1-(p70 optical density units in presence of drug/p70 units in absence of drug))×100. IC50 equals the concentration of compound at which 50% inhibition is measured[1].

IC50: A potent and selective inhibitor of Src-family tyrosine kinases, with an IC50 of 5 and 6 nM respectively for p56lck and p59fynT.

PP1 inhibits Src-family tyrosine kinases effectively and selectively. The non-receptor protein tyrosine kinase Src is a crucial enzyme in the signal transduction pathways involved in cell division, motility, adhesion, and survival. Src is regarded as a promising target for cancer therapy due to its importance in tumor progression, invasion, transition, angiogenesis, and the development of metastasis. [1]

In vitro: It was reported that PP1 specifically inhibited the expression and activity of Lyn, a Src family kinase, in RBL-2H3 cells. Based on the immune-complex kinase assays in vitro, PP1 suppressed the activity of Lyn at nanomolar levels without any effect on Syk kinase activity. In contrast, phosphorylation of both Syk and Lyn kinases were both blocked in RBL cells. FcεRI- and Thy-1-mediated early and late activation events were also interrupted by PP1 in a similar mannar. Moreover, PP1 was found to inhibited RET-derived oncoproteins with IC50 of 80 nM. RET/PTC3-transformed cells received PP1 treatment with a dose of 5 μM lost proliferative autonomy and showed morphological reversion. [2, 3]

In vivo: Under in vivo conditions PP1 was suggested to suppress tyrosine phosphorylation and proliferation in T cells stimulated with anti-CD3 and mitogen. Studies using mice tumor model also showed that PP1 upregulated the expression of the IL-2 gene rather than the granulocyte macrophage colony-stimulating factor or the IL-2 receptor genes. Based on these, PP1 could be adopted as a useful agent to investigate the role of Lck and Fyn T cell activation. [2]

Clinical trial: So far, no clinical trial has been conducted.

References:
[1]Sen B and Johnson FM.  Regulation of src family kinases in human cancers. J Signal Tr. 2010 Feb; doi:10.1155/2011/865819.
[2] Amoui M, Driber P and Driberovi L.  Src family-selective tyrosine kinase inhibitor, PP1, inhibits both FcERI- and Thy-1-mediated activation of rat basophilic leukemia cells. Eur. J. Immunol. 1997. 27: 1881-1886.
[3]Carlomagno F, Vitagliano D, Guida T, Napolitano M, Vecchio G, Fusco A, Gazit A, Levitzki A and Santoro M.  The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes. Cancer Res. 2002 Feb. 62: 1077–82