pp1

Names

[ Name ]:
pp1

[Synonym ]:
1-(tert-Butyl)-3-(p-tolyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine
3-(4-Methylphenyl)-1-(2-methyl-2-propanyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine
1-tert-butyl-3-(4-methylphenyl)pyrazolo[3,4-d]pyrimidin-4-amine
1H-Pyrazolo[3,4-d]pyrimidin-4-amine, 1-(1,1-dimethylethyl)-3-(4-methylphenyl)-
4-Amino-5-(methylphenyl)-7-(t-butyl)pyrazolo-(3,4-d)pyrimidine
1H-Pyrazolo[3,4-d]pyrimidin-4-amine (1-(1,1-dimethylethyl)-3-(4-methylphenyl)
1-tert-butyl-3-(4-methylphenyl)-1h-pyrazolo[3,4-d]pyrimidin-4-amine
pp1

Biological Activity

[Description]:

PP1 is a potent, and Src family-selective tyrosine kinase inhibitor with IC50 of 5 and 6 nM for Lck and Fyn, respectively.

[Related Catalog]:

Signaling Pathways >> Protein Tyrosine Kinase/RTK >> Src

Research Areas >> Cancer

[Target]

IC50: 5 nM (Lck), 6 nM (Fyn), 250 nM (EGFR), >50 μM (JAK2)[1]

[In Vitro]

PP1 inhibits Lck (IC50=5 nM) and FynT (IC50=6 nM) in vitro at concentrations significantly lower than those required to inhibit ZAP-70 (IC50>100 μM), JAK2 (IC50>50 μM), the EGFR kinase, and protein kinase A. PP1 inhibits whole cell tyrosine phosphorylation and proliferation in T cells stimulated with anti-CD3 and mitogens. PP1 selectively inhibits IL-2 gene expression over GM-CSF and IL-2R gene induction in human T cells[1].

[Kinase Assay]

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].

[Cell Assay]

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].

[References]

[1]. Hanke JH, et al. 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.

[Related Small Molecules]

Chemical & Physical Properties

[ Density]:
1.2±0.1 g/cm3

[ Boiling Point ]:
478.8±40.0 °C at 760 mmHg

[ Melting Point ]:
205-207ºC

[ Molecular Formula ]:
C₁₆H₁₉N₅

[ Molecular Weight ]:
281.356

[ Flash Point ]:
243.4±27.3 °C

[ Exact Mass ]:
281.164032

[ PSA ]:
69.62000

[ LogP ]:
3.11

[ Vapour Pressure ]:
0.0±1.2 mmHg at 25°C

[ Index of Refraction ]:
1.652

[ Storage condition ]:
Desiccate at +4°C

[ Water Solubility ]:
DMSO: >20mg/mL

Safety Information

[ Symbol ]:

GHS06

[ Signal Word ]:
Danger

[ Hazard Statements ]:
H301

[ Precautionary Statements ]:
P301 + P310

[ Hazard Codes ]:
Xn

[ Risk Phrases ]:
22

[ RIDADR ]:
UN 2811 6.1 / PGIII

[ HS Code ]:
2933990090

Customs

[ HS Code ]: 2933990090

[ Summary ]:
2933990090. heterocyclic compounds with nitrogen hetero-atom(s) only. VAT:17.0%. Tax rebate rate:13.0%. . MFN tariff:6.5%. General tariff:20.0%

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pp1