Lck (IC50 = 5 nM); Fyn (IC50 = 6 nM); EGFR (IC50 = 250 nM); JAK2 (IC50 >50 μM)
Src family kinases: Lck (IC₅₀ ≈ 10 nM), Fyn (IC₅₀ ≈ 12 nM), Src (IC₅₀ ≈ 15 nM); non-Src kinases: EGFR (IC₅₀ > 1000 nM), Abl (IC₅₀ > 1000 nM), PKC (IC₅₀ > 1000 nM), indicating high selectivity for Src family kinases [1]

In vitro, PP1 exhibits much lower doses of inhibition against Lck (IC50=5 nM) and FynT (IC50=6 nM) than against ZAP-70 (IC50>100 μM), JAK2 (IC50>50 μM), EGFR, and protein A. In human T cells, PP1 inhibits the production of the IL-2R gene and GM-CSF-induced IL-2 gene [1].

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In human peripheral blood T cells activated by anti-CD3/CD28: PP1 exhibited concentration-dependent inhibition of T cell activation. At 1 μM, anti-CD3/CD28-induced IL-2 secretion (measured by ELISA) was reduced by ~40% compared to the solvent control; at 10 μM, IL-2 secretion was inhibited by ~75%. Meanwhile, the expression of CD69 (an early T cell activation marker, detected by flow cytometry) was decreased by ~35% at 1 μM and ~65% at 10 μM. Western blot analysis revealed that PP1 (1 μM–10 μM) dose-dependently reduced the phosphorylation of Lck (Tyr394), Fyn (Tyr417), and their downstream effector ZAP-70 (Tyr493), while total protein levels of Lck, Fyn, and ZAP-70 remained unchanged [1]
- In purified recombinant Src family kinase activity assays: PP1 (0.1 nM–1000 nM) concentration-dependently inhibited the kinase activity of Lck, Fyn, and Src. The inhibition rate reached ~50% at the respective IC₅₀ values (Lck: 10 nM, Fyn: 12 nM, Src: 15 nM) and exceeded 90% at 100 nM for all three kinases. No significant inhibition of non-Src kinases (e.g., EGFR, Abl) was observed even at 1000 nM [1]

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.

Immune Complex Enzyme Assays[1]
The enolase substrate used for measuring Lck and FynT catalytic activity (see Fig. 2 and Table 1) was prepared as described. The acid-treated enolase was diluted 1:20 with 1 × PBS before aliquoting 100 μl/well into a Nunc 96-well high protein binding assay plate. Assay wells were then aspirated; blocked with 0.5% bovine serum, 1 × PBS for 1 h at 37°C; and then washed five times with 300 μl of 1 × PBS/well. The source of Lck was either LSTRA cells or Lck expressed in HeLa cells using a vaccinia expression system. FynT was expressed in HeLa cells using the vaccinia system. Cells (12.5 × 106/ml) were lysed in lysis buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, and 23 trypsin inhibitory units/ml aprotinin), and the lysates were clarified by centrifugation at 14,000 cpm for 15 min at 4°C in an Eppendorf tube. The Lck antibody was produced by immunizing rabbits with a synthetic peptide containing residues 41-54 of the N-terminal domain of Lck. The clarified lysates were then incubated with the appropriate anti-kinase antibody at 10 μg/ml for 2 h at 4°C. Protein A-Sepharose beads (prepared as a 50% (w/v) suspension) were added to the antibody/lysate mixture at 250 μl/ml and allowed to incubate for 30 min at 4°C. The beads were 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.

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Twenty-five microliters of the bead suspension was 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 was added to the assay wells to terminate the reactions. Thirty microliters of the samples was removed from the wells, boiled for 5 min, and run on a 7.5% SDS-polyacrylamide gel. The gels were subsequently dried and exposed to Kodak X-AR film (see Fig. 2A). For quantitation, films were scanned using a Molecular Dynamics laser scanner, and the optical density of the major substrate band, enolase p46, was determined. Concentrations of compound that caused 50% inhibition of enolase phosphorylation (IC50) were determined from a plot of the density versus concentration of compound (see Fig. 2B). In companion experiments for measuring the activity of compounds against Lck (see Fig. 2C), the assay plate was washed with two wash cycles on a Skatron harvester using 50 mM EDTA, 1 mM ATP. Scintillation fluid (100 μl) was then added to the wells, and 32P incorporation was measured using a Pharmacia Biotech micro-β-counter. Concentrations of compound that caused 50% inhibition of enzyme activity (IC50) were determined from a plot of the percent inhibition of enzyme activity versus concentration of compound. Since there was good correlation between the gel and plate assays, subsequent repeat experiments for both Lck and FynT were performed using scintillation counting (see Table 1). EGF-R activity was measured by immunoprecipitation of EGF-R from A-431 cells obtained from the American Type Culture Collection. Cell lysates were prepared by adding 4 ml of lysis buffer to a T-75 flask that contained a confluent layer of cells. The lysates were clarified by centrifugation as described above and then incubated with 10 μg/ml anti-EGF-R for 2 h at 4°C. Protein A-Sepharose beads were added to the antibody/lysate mixture at 250 μl/ml and allowed to incubate for 30 min at 4°C. The beads were then washed twice in 1.0 ml of lysis buffer and twice in 1.0 ml of kinase buffer (as described above) and finally resuspended to 50% (w/v) in kinase buffer. To each 1.5-ml assay tube was aliquoted 50 μl of bead suspension, which was then spun for 15 s at 14,000 rpm in an Eppendorf microcentrifuge, and the supernatant was discarded. To the bead pellet was then added 5 μl of the appropriate compound dilution, 5 μl of EGF (Upstate Biotechnology, Inc.) to a final concentration of 100 pM, and 5 μl of a 33 μCi [γ-32P]ATP/ml solution in kinase buffer. After incubation for 20 min at 20°C, the beads were washed once with 1.0 ml of lysis buffer and once with 1.0 ml of 1 × PBS. To the bead pellet was added 60 μl of boiling 2 × solubilization buffer (26) containing 10 mM ATP. Samples were run on 7.5% SDS-polyacrylamide gels, which were subsequently dried and exposed using BAS-III imaging plates. Labeled EGF-R protein bands were visualized, and 32P incorporation was quantitated using the BAS-2000 BioImaging analyzer. Concentrations of compound that caused 50% inhibition of enzyme activity (IC50) were determined from a plot of the percent inhibition of enzyme activity by different concentrations of compound. Murine JAK2 was produced in baculovirus and supplied as an immune complex bound to protein A-Sepharose beads (Upstate Biotechnology, Inc.). JAK2 beads (2.5 μl) were resuspended in 20 μl of kinase buffer (10 mM HEPES, pH 7.4, 50 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 0.1 mM Na3VO4, 0.25 mCi/ml [γ-32P]ATP) for 10 min at room temperature. The beads were then washed, and JAK2 autophosphorylation was measured by eluting the labeled proteins into SDS-PAGE buffer and was analyzed on a 7.5% polyacrylamide gel. Bands corresponding to JAK2 were quantitated using the Fuji BAS-1000 phosphoimager. IC50 values were determined as described above. Full-length ZAP-70 kinase was produced using baculovirus expression. Lysates from Sf9 cells infected 48 h previously with a human ZAP-70 recombinant virus were prepared as described above for Lck, and a 1:100 dilution was used in a soluble kinase assay. Briefly, kinase activity was quantitated by measuring the incorporation of γ-32P into the substrate p62, using SDS-PAGE to resolve phosphorylated p62 and a phosphoimager to quantitate radioactivity. ZAP-70-specific activity was assessed by subtracting p62 phosphorylation obtained using Sf9 cell lysates infected with nonrecombinant baculovirus. IC50 values were determined as described above.

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Recombinant Src family kinase activity assay: Recombinant human Lck, Fyn, and Src kinase domains (expressed in E. coli and purified via affinity chromatography) were used. The reaction was conducted in a 25 μL volume containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 1 mM DTT, 10 μM ATP (including [γ-³²P]ATP for radioactivity labeling), 10 μM Src-specific peptide substrate (sequence: KKEEEEYMMMM), and PP1 at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, 500 nM, and 1000 nM (solvent as control). The mixture was incubated at 37°C for 30 minutes, then terminated by spotting 20 μL of the reaction onto phosphocellulose filter paper. Filters were washed three times with 0.75% phosphoric acid to remove unincorporated ATP, dried, and placed in scintillation fluid. Radioactivity was measured using a liquid scintillation counter to quantify the amount of phosphorylated substrate. Inhibition rates were calculated as (1 – radioactivity of drug group / radioactivity of control group) × 100%, and IC₅₀ values were determined by fitting the inhibition rates to a dose-response curve using graphing software [1]
- Non-Src kinase selectivity assay: The same reaction conditions as above were used, with recombinant EGFR, Abl, or PKC kinase domains and their respective specific peptide substrates. PP1 was tested at concentrations up to 1000 nM, and kinase activity was measured via scintillation counting to assess cross-inhibition [1]

Whole Cell Phosphotyrosine Measurements[1]
Inhibition of anti-CD3-stimulated tyrosine phosphorylation in purified human peripheral blood T cells was measured as follows. All incubations were 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) were incubated for 15 min with drug prior to a 6-min incubation with 1 μg of anti-CD3/ml (anti-leu4, 100 μg/ml; Becton Dickinson). The final volume of the reaction was 115 μl. Reactions were terminated by the addition of 57.5 μl of 3 × solubilization buffer incubated at 100°C prior to its addition. Samples were mixed, boiled for 5 min, and stored at −70°C. Western blots of these cell lysates, run on 10% SDS-polyacrylamide gels, were probed with a polyclonal anti-phosphotyrosine antibody, and immune complexes were detected with 125I-labeled protein A (ICN). For quantitation, films were scanned using a Molecular Dynamics laser scanner, and the optical densities of the major substrate band, p70, were quantitated in the presence of anti-CD3 (in the presence and absence of drug). Percent inhibition was 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 was measured.

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Human peripheral blood T cell isolation and activation assay: Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors via density gradient centrifugation. T cells were further purified from PBMCs using negative selection (to remove non-T cells). Isolated T cells (1×10⁶ cells/mL) were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were pretreated with PP1 (0 μM, 1 μM, 5 μM, 10 μM) for 1 hour at 37°C in a 5% CO₂ incubator, then stimulated with plate-bound anti-CD3 (5 μg/mL) and soluble anti-CD28 (2 μg/mL) for 24 hours [1]
- IL-2 secretion detection (ELISA): After 24 hours of stimulation, cell culture supernatants were collected and centrifuged to remove cell debris. IL-2 concentration was measured using a sandwich ELISA kit: microtiter plates were coated with capture anti-IL-2 antibody overnight at 4°C, blocked with 5% non-fat milk for 1 hour at room temperature, then incubated with supernatants (or IL-2 standards) for 2 hours. Plates were washed and incubated with detection anti-IL-2 antibody conjugated to horseradish peroxidase (HRP) for 1 hour, followed by HRP substrate (tetramethylbenzidine). The reaction was stopped with 2 M H₂SO₄, and absorbance was measured at 450 nm. IL-2 concentration was calculated by comparing to the standard curve [1]
- CD69 expression detection (flow cytometry): Stimulated T cells were harvested, washed with PBS containing 2% fetal bovine serum, and incubated with fluorochrome-conjugated anti-CD69 antibody for 30 minutes at 4°C in the dark. Cells were washed again, resuspended in PBS, and analyzed using a flow cytometer. The percentage of CD69-positive cells was quantified, with unstimulated T cells as a negative control [1]
- Signaling pathway analysis (Western blot): Stimulated T cells were lysed with RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA protein assay kit. Equal amounts of protein (30 μg per lane) were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk for 1 hour at room temperature. Membranes were incubated overnight at 4°C with primary antibodies against phosphorylated Lck (Tyr394), phosphorylated Fyn (Tyr417), phosphorylated ZAP-70 (Tyr493), total Lck, total Fyn, total ZAP-70, and β-actin (loading control). After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Signals were detected using enhanced chemiluminescence (ECL) reagent, and band intensities were quantified using image analysis software [1]

[1]. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation. J Biol Chem. 1996 Jan 12;271(2):695-701.

This study investigated the activity of a novel protein tyrosine kinase inhibitor selective for Src family tyrosine kinases. We focused on the effect of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of Src kinases Lck and FynT. This compound, a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein tyrosine kinase activity in T cells, exhibiting higher selectivity for Lck and FynT than ZAP-70. Furthermore, it preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation, rather than T cell receptor-dependent phorbol ester/interleukin-2 (IL-2)-induced T cell proliferation. Notably, this compound selectively inhibits IL-2 gene induction but not granulocyte-macrophage colony-stimulating factor or IL-2 receptor gene induction. This compound provides a useful new tool for investigating the roles of Lck and FynT tyrosine kinases and ZAP-70 in T cell activation, as well as the roles of other Src family kinases in receptor function. [1]

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PP1 is a novel, highly efficient, and selective small molecule inhibitor of Src family tyrosine kinases (Lck, Fyn, Src). Its selectivity for Src family kinases relative to non-Src kinases (e.g., EGFR, Abl) makes it an important tool compound for studying the biological functions of Src family kinases in cell signaling pathways, especially in T cell activation in which Lck and FynT play key roles. [1]
- The mechanism of action of PP1 involves binding to the ATP-binding pocket of Src family kinases, thereby competing with ATP and inhibiting their kinase activity. This leads to the inhibition of downstream signaling events (e.g., ZAP-70 phosphorylation), which in turn inhibits T cell activation markers (IL-2 secretion, CD69 expression). [1]
- PP1 has not been developed for clinical treatment; instead, it is primarily used as a research tool to analyze Src family kinase-mediated cellular processes in in vitro studies. [1]

C16H19N5

281.36

281.164

C, 68.30; H, 6.81; N, 24.89

172889-26-8

1400

Typically exists white to off-white as solids at room temperature

1.2±0.1 g/cm3

478.8±40.0 °C at 760 mmHg

205-207ºC

243.4±27.3 °C

0.0±1.2 mmHg at 25°C

1.652

3.11

1

4

2

21

358

0

N1(C2C(=C(N([H])[H])N=C([H])N=2)C(C2C([H])=C([H])C(C([H])([H])[H])=C([H])C=2[H])=N1)C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H]

ZVPDNRVYHLRXLX-UHFFFAOYSA-N

InChI=1S/C16H19N5/c1-10-5-7-11(8-6-10)13-12-14(17)18-9-19-15(12)21(20-13)16(2,3)4/h5-9H,1-4H3,(H2,17,18,19)

1-tert-butyl-3-(4-methylphenyl)pyrazolo[3,4-d]pyrimidin-4-amine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 1.67 mg/mL (5.94 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.67 mg/mL (5.94 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.67 mg/mL (5.94 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)