VEGFR1 (IC50 = 4.3 nM); c-Fyn-as1 (IC50 = 3.2 nM); v-Src (IC50 = 28 μM); c-Fyn (IC50 = 1 μM); c-Abl (IC50 = 3.4 μM); CDK2 (IC50 = 29 μM); CAMKII (IC50 = 24 μM); cAbl-as2 (IC50 = 120 nM); CDK2-as1 (IC50 = 5 nM); CAMKII-as1 (IC50 = 8 nM)
Cdk7as/as [3]

Cdk7 from Cdk7^as/as or Cdk7^+/+ cells is immunoprecipitated, and its kinase activity is assessed against human Cdk2 and a Pol II CTD-containing fusion protein (GST-CTD). With an IC50 of ~50 nM for either substrate, 1-NM-PP1 (1-NMPP1) inhibits Cdk7 that is recovered from the mutant cells but not from the wild-type cells. The growth of HCT116 cells became sensitive to 1-NM-PP1 when Cdk7^as/as was substituted for wild-type Cdk7. The wild-type and Cdk7^as/as cells had population doubling times of approximately 17.9 and 20.2 hours, respectively, in the absence of 1-NM-PP1, with similar cell-cycle distributions in asynchronous culture, suggesting a minimal impairment of Cdk7 function per se by the F91G mutation. The homozygous Cdk7^as/as cells exhibit sensitivity to 1-NM-PP1, as demonstrated by cell viability (MTT) assays conducted 96 hours after 1-NM-PP1 exposure, revealing an IC50 of approximately 100 nM. Wild-type HCT116 cells, on the other hand, are resistant to 10 μM 1-NM-PP1. When 10 μM 1-NM-PP1 is added, the mutant cells' G1/S progression is retarded, but not that of the wild-type cells. When 1-NM-PP1 is added to the Cdk7^as/as cells concurrently with serum, it stops the cells from entering S phase for the next fifteen hours. A portion of Cdk7^as/as cells released from serum starvation and placed straight into medium containing 1-NM-PP1 showed evidence of progressing into S-phase after 24 hours, whereas a portion of the cells stayed in G1. 1-NM-PP1 is added either 3 or 6 hours after serum addition, which causes a ~7- or ~3-hour delay in S-phase entry[3].

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Cdk7 immunoprecipitated from Cdk7as/as cells can be inhibited by 1-NM-PP1, while Cdk7 immunoprecipitated from wild-type (Cdk7+/+) cells is not affected by it. In the experiment, 200 μM unlabeled ATP was used, and different concentrations of 1-NM-PP1 were added for GST-CTD or Cdk2 kinase assays [3]
In mitotic extracts from Cdk7as/as or Cdk7+/+ cells, after pre-incubation with 1-NM-PP1, purified recombinant Myc-tagged cyclin B, an ATP-regenerating system, and (or not) supplemented with Cdk7/cyclin H/Mat1 complex or Csk1 were added, followed by incubation at room temperature for 90 minutes. After immunoprecipitation with anti-Myc antibody, the binding of cyclin B and Cdk1 and histone H1 kinase activity were detected. The results showed that 1-NM-PP1 treatment would affect the assembly of Cdk1/cyclin B and related kinase activity [3]
In extracts from Cdk7as/as cells synchronized in G2 phase, Myc-tagged cyclin B was added, treated with 2 μM 1-NM-PP1 or DMSO, and supplemented with wild-type CAK (Cdk7wt) or analog-sensitive CAK (Cdk7as) at the same time. The detection found that 1-NM-PP1 treatment had an impact on Cdk1/cyclin B assembly and kinase activity [3]

After wild-type (Cdk7+/+) and Cdk7as/as cells were treated with different concentrations of 1-NM-PP1 for 48 hours and 96 hours, cell viability was detected by MTT assay. The results showed that Cdk7as/as cells had dose-dependent sensitivity to 1-NM-PP1, while wild-type cells were not sensitive [3]
Asynchronously growing Cdk7+/+ and Cdk7as/as HCT116 cells were treated with different concentrations of 1-NM-PP1 for 14 hours, then cell extracts were prepared for Western blot analysis to detect the total protein levels of Cdk1 and Cdk2, their T-loop phosphorylation levels (P-T161 site of Cdk1, P-T160 site of Cdk2), as well as the total protein level of Pol II subunit Rpb1 and the phosphorylation level of Ser5 site in its CTD region. The results showed that 1-NM-PP1 treatment could dose-dependently affect the phosphorylation status of Cdk1, Cdk2 and Pol II in Cdk7as/as cells, but had no obvious effect on wild-type cells [3]
Cells were arrested by serum starvation and then released into serum-containing medium. DMSO or 10 μM 1-NM-PP1 was added at different time points. DNA content was monitored by flow cytometry. The results showed that 1-NM-PP1 treatment could prevent Cdk2 activation and delay G1/S transition [3]
After Cdk7as/as cells were released from serum starvation, DMSO or 1-NM-PP1 was added at 6 hours. Cells were harvested at different time points to prepare extracts. The isoforms of Cdk2 in total extracts were detected by Western blot, and Cdk2 was immunoprecipitated to detect its recovery and histone H1 kinase activity. The results showed that 1-NM-PP1 treatment could terminate Cdk2 activation [3]
Serum-starved cells were treated with DMSO or 10 μM 1-NM-PP1 in serum-free medium for 9 hours, then extracts were prepared. Western blot detection showed that 1-NM-PP1 treatment could reduce the T-loop phosphorylation level of Cdk2 [3]
Cdk7as/as cells were arrested at the G1/S boundary by double-thymidine block, then released into fresh medium without or with 10 μM 1-NM-PP1 (added at different time points). DNA content was monitored by flow cytometry to track cell cycle progression. The results showed that adding 1-NM-PP1 in S/G2 phase could prevent cells from entering mitosis [3]
Wild-type and Cdk7as/as cells were released into nocodazole-containing medium, 1-NM-PP1 was added at 4 hours, and observed by phase-contrast microscopy at 15 hours. The results showed that Cdk7as/as cells could not enter mitosis after 1-NM-PP1 treatment [3]
Cells were released from G1/S (double-thymidine) block into nocodazole-containing medium, mock-treated or treated with 1-NM-PP1 at 6 hours or 4 hours respectively. Cdk1-associated histone H1 kinase activity was detected. The results showed that 1-NM-PP1 treatment could inhibit the kinase activity of Cdk1 [3]
Cdk7as/as cells were released from double-thymidine block into nocodazole-containing medium, mock-treated or treated with 1-NM-PP1 at different time points. The total cyclin B level was detected, and the results showed that 1-NM-PP1 treatment had no obvious effect on the total level of cyclin B [3]
After Cdk7as/as cells were treated as described above, immunoprecipitation was performed with anti-Cdk1 antibody or anti-cyclin B antibody, followed by Western blot to detect the binding of cyclin B and Cdk1. The results showed that adding 1-NM-PP1 at 4 hours or 6 hours would disrupt the assembly of Cdk1/cyclin B complex [3]
Cdk7as/as cells were released from thymidine block into nocodazole-containing medium, and 10 μM 1-NM-PP1 or 10 μg/ml actinomycin D was added at different time points. Cells were harvested at 15 hours, and the binding of Cdk1/cyclin B was detected. The results showed that 1-NM-PP1 treatment could inhibit the assembly of Cdk1/cyclin B, while actinomycin D treatment had no such effect [3]

Assays for kinase of immune complexes, immunoblotting, and immunoprecipitation are performed. A system for regenerating ATP is added, along with 500 ng of purified cyclin B1, which is amino-terminally tagged with hexahistidine and the Myc epitope, to extracts (200 μg total protein) from cells in mitosis or G2. This pre-incubation is done with 2 μM 1-NM-PP1 or DMSO. When indicated, 400 ng of purified Csk1 or 600 ng of the wild-type or analog-sensitive, T-loop-phosphorylated Cdk7/cyclin H/Mat1 complex are added to the incubations. The process involves letting Myc-cyclin B and related proteins sit at room temperature for 90 minutes. Then, immune complexes are immunoprecipitated using anti-Myc and anti-Cdk1 antibodies, and their histone H1 kinase activity is assessed[3].

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Protocol 1: Extract total proteins from Cdk7as/as and Cdk7+/+ cells, and obtain Cdk7 protein by immunoprecipitation; prepare reaction system containing 200 μM unlabeled ATP, add GST-CTD or Cdk2 as substrates, and add different concentrations of 1-NM-PP1 to perform kinase reaction to detect the effect of 1-NM-PP1 on Cdk7 kinase activity [3]
Protocol 2: Prepare mitotic extracts from Cdk7as/as or Cdk7+/+ cells, and pre-incubate the extracts with (or without) 1-NM-PP1; add purified recombinant Myc-tagged cyclin B, ATP-regenerating system, and (or not) add Cdk7/cyclin H/Mat1 complex or Csk1 to the reaction system; after incubation at room temperature for 90 minutes, add anti-Myc antibody for immunoprecipitation; detect the presence of cyclin B and Cdk1 in the precipitate by Western blot, and perform histone H1 kinase activity assay to evaluate the effect of 1-NM-PP1 on Cdk1/cyclin B assembly and related kinase activity [3]
Protocol 3: Obtain extracts from Cdk7as/as cells synchronized in G2 phase, and add Myc-tagged cyclin B to the extracts; divide the reaction system into two groups, add 2 μM 1-NM-PP1 or DMSO respectively; at the same time, supplement wild-type CAK (Cdk7wt) or analog-sensitive CAK (Cdk7as) to each group; after incubation, detect the binding of Cdk1/cyclin B by immunoprecipitation and Western blot, and perform kinase activity assay [3]

After 48 hours of incubation in serum-free medium, wild-type or Cdk7^as/as HCT116 cells are synchronized and released into medium containing 10% fetal calf serum. Cell-cycle distribution is analyzed using flow cytometry, and thymidine or nocodazole synchronization is carried out. The MTT assay is used to determine cell viability[3].

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Protocol 1 (Cell Viability Detection): Culture Cdk7+/+ and Cdk7as/as cells, and seed the cells into culture plates; add different concentrations of 1-NM-PP1 and incubate for 48 hours and 96 hours respectively; add MTT reagent, measure the absorbance value after incubation, calculate cell viability, and analyze the sensitivity of cells to 1-NM-PP1 [3]
Protocol 2 (Western Blot Detection of Phosphorylation Level): Culture asynchronously growing Cdk7+/+ and Cdk7as/as HCT116 cells; treat the cells with different concentrations of 1-NM-PP1 for 14 hours; collect the cells, extract total proteins and perform protein quantification; separate proteins by SDS-PAGE electrophoresis and transfer to membranes; perform Western blot detection with antibodies against Cdk1, phosphorylated Cdk1 (P-T161), Cdk2, phosphorylated Cdk2 (P-T160), total Rpb1 protein and phosphorylated Rpb1 (CTD P-Ser-5); analyze the effect of 1-NM-PP1 on the phosphorylation level of related proteins [3]
Protocol 3 (Cell Cycle Progression Monitoring): Arrest Cdk7as/as cells by serum starvation; release the cells into serum-containing medium, and add DMSO or 10 μM 1-NM-PP1 at different time points; collect cells at different time points, fix and stain DNA; detect DNA content by flow cytometry to analyze cell cycle progression [3]
Protocol 4 (G1/S Boundary Arrest and Release Experiment): Arrest Cdk7as/as cells at the G1/S boundary by double-thymidine method; release the cells into fresh medium without or with 10 μM 1-NM-PP1 (added at different time points); collect cells at different time points, fix and stain DNA; detect DNA content by flow cytometry to monitor cell cycle progression [3]
Protocol 5 (Mitotic Entry Detection): Release wild-type and Cdk7as/as cells into nocodazole-containing medium; add 1-NM-PP1 at 4 hours; after culturing for 15 hours, observe cell morphology by phase-contrast microscopy to determine whether they enter mitosis [3]
Protocol 6 (Cdk2 Activation Detection): Subject Cdk7as/as cells to serum starvation and then release them; add DMSO or 1-NM-PP1 at 6 hours; collect cells at different time points and extract total proteins; detect the isoforms of Cdk2 in total extracts by Western blot; perform immunoprecipitation with anti-Cdk2 antibody; detect the recovery of immunoprecipitated Cdk2 and perform histone H1 kinase activity assay to analyze the activation state of Cdk2 [3]

[1]. Generation of Monospecific Nanomolar Tyrosine Kinase Inhibitors via a Chemical Genetic Approach. Journal of the American Chemical Society, 1999, 121(4):627-631.

[2]. A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature. 2000 Sep 21;407(6802):395-401.

[3]. Requirements for Cdk7 in the assembly of Cdk1/cyclin B and activation of Cdk2 revealed by chemical genetics in human cells. Mol Cell. 2007 Mar 23;25(6):839-50.

1-NM-PP1 is a pyrazolopyrimidine compound that can act as a tyrosine kinase inhibitor. 1-NM-PP1 is a cell-permeable small molecule compound used in chemogenetic studies to specifically target analog-sensitive mutant kinases (e.g., Cdk7as) without affecting the function of wild-type kinases. This helps to elucidate the role of specific kinases in biological processes such as cell cycle progression [3].

C20H21N5

331.4142

331.179

C, 72.48; H, 6.39; N, 21.13

221244-14-0

221244-14-0

5154691

white solid powder

1.3±0.1 g/cm3

545.7±45.0 °C at 760 mmHg

175-176ºC

283.8±28.7 °C

0.0±1.5 mmHg at 25°C

1.675

3.96

1

4

3

25

462

0

N1(C2C(=C(N([H])[H])N=C([H])N=2)C(C([H])([H])C2=C([H])C([H])=C([H])C3=C([H])C([H])=C([H])C([H])=C23)=N1)C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H]

GDQXJQSQYMMKRA-UHFFFAOYSA-N

InChI=1S/C20H21N5/c1-20(2,3)25-19-17(18(21)22-12-23-19)16(24-25)11-14-9-6-8-13-7-4-5-10-15(13)14/h4-10,12H,11H2,1-3H3,(H2,21,22,23)

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

1NM PP1; 1NM-PP1; 1NMPP1l PP1 analog II

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)

DMSO: ~27.5 mg/mL (~82.9 mM）

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.54 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 25.0 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: ≥ 2.5 mg/mL (7.54 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 25.0 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.)