PP1 (AGL 1872), a specific and effective Src inhibitor, is with IC50 for Lck/Fyn is 5 nM/ 6 nM, respectively.

Lck:5 nM, Fyn:6 nM

PP1是一种纳摩级别的Lck和FynT抑制剂，能够抑制T细胞中抗CD3诱导的蛋白酪氨酸激酶活性（IC50为0.5 μM），对Lck和FynT具有选择性，优先抑制T细胞受体依赖的抗CD3诱导T细胞增殖（IC50为0.5 μM），而对非T细胞受体依赖的phorbol 12-myristate 13-acetate/白细胞介素-2（IL-2）诱导的T细胞增殖的抑制作用较弱。PP1（1 μM）选择性抑制IL-2基因的诱导，但不影响粒细胞-巨噬细胞集落刺激因子或IL-2受体基因。此外，PP1还能抑制Src（IC50为170 nM）和Hck（IC50为20 nM）。其抑制A-431表皮生长因子受体自磷酸化的活性较低，为0.25 μM的IC50的50-100倍。PP1还能以IC50约75 nM和1 μM的浓度抑制Kit和Bcr-Abl酪氨酸激酶。PP1能完全阻止M07e细胞对SCF的增殖反应，IC50为0.5-1 μM。在完整细胞中，PP1（1 μM）抑制SCF诱导的c-Kit自磷酸化，并阻断了丝裂原激活蛋白激酶和Akt的激活。PP1抑制在肥大细胞疾病中发现的c-Kit的突变活性形式（D814V和D814Y），并在表达突变c-Kit的大鼠嗜碱性白血病细胞系RBL-2H3中诱导凋亡。PP1降低FDCP1细胞中Bcr-Abl的构成性激活，并触发凋亡，这些FDCP1细胞表达信号转导和转录激活因子5以及丝裂原活化蛋白激酶。

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°C. 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°C, 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].

PP1 is dissolved in DMSO and stored, and then diluted with appropriate medium before use[2]. 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°C 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°C prior to its addition. Samples are mixed, boiled for 5 min, and stored at -70°C. 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].