1. Cell Cycle/DNA Damage
  2. CDK

1-NM-PP1 (Synonyms: PP1 Analog II; 1-NM-PP 1)

Cat. No.: HY-13942 Purity: 98.83%
Data Sheet SDS Handling Instructions

1-NM-PP1 inhibits Cdk7 recovered from the mutant, but not the wild-type cells with an IC50 of ~50 nM with either substrate.

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1-NM-PP1 Chemical Structure

1-NM-PP1 Chemical Structure

CAS No. : 221244-14-0

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  • Biological Activity

  • Protocol

  • Technical Information

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Description

1-NM-PP1 inhibits Cdk7 recovered from the mutant, but not the wild-type cells with an IC50 of ~50 nM with either substrate.

IC50 & Target

CDK7[1]

In Vitro

Cdk7 from Cdk7as/as or Cdk7+/+ cells is immunoprecipitated and tested its kinase activity towards both a Pol II CTD-containing fusion protein (GST-CTD) and human Cdk2. Cdk7 recovered from the mutant, but not the wild-type, cells is inhibited by 1-NM-PP1 (1-NMPP1), with an IC50 of ~50 nM with either substrate. Replacement of wild-type Cdk7 with Cdk7as/as also rendered growth of HCT116 cells sensitive to 1-NM-PP1. In the absence of 1-NM-PP1, the wild-type andCdk7as/as cells had population doubling times of ~17.9 and ~20.2 h, respectively, with similar cell-cycle distributions in asynchronous culture, indicating minimal impairment of Cdk7 function by the F91G mutation per se. The homozygous Cdk7as/as cells are sensitive to 1-NM-PP1, however, with an IC50 ~100 nM measured by cell viability (MTT) assays performed after 96 h of 1-NM-PP1 exposure. In contrast, wild-type HCT116 cells are resistant to 10 μM 1-NM-PP1. Addition of 10 μM 1-NM-PP1 retards G1/S progression by the mutant but not the wild-type cells. When added simultaneously with serum to the Cdk7as/as cells, 1-NM-PP1 prevents any progression into S phase in the next 15 h. After 24 h, there is evidence of progression into S-phase by a fraction of Cdk7as/as cells released from serum starvation directly into medium containing 1-NM-PP1, while a fraction remained in G1. The addition of 1-NM-PP1 3 h or 6 h after serum addition delays S-phase entry by ~7 h or by ~3 h, respectively[1].

References
Preparing Stock Solutions
Concentration Volume (DMSO) Mass 1 mg 5 mg 10 mg
1 mM 3.0174 mL 15.0871 mL 30.1741 mL
5 mM 0.6035 mL 3.0174 mL 6.0348 mL
10 mM 0.3017 mL 1.5087 mL 3.0174 mL
Kinase Assay
[1]

Immunoblotting and immunoprecipitation, and kinase assays of immune complexes, are carried out. To measure Cdk1/cyclin B assembly, extracts (200 μg total protein) from cells in mitosis or G2 are pre-incubated with 2 μM 1-NM-PP1 or DMSO, then added 500 ng purified cyclin B1, amino-terminally tagged with hexahistidine and the Myc epitope, and an ATP-regenerating system. Where indicated, incubations are supplemented with 400 ng purified Csk1 or 600 ng wild-type or analog-sensitive, T-loop-phosphorylated Cdk7/cyclin H/Mat1 complex. After 90 min at room temperature, Myc-cyclin B and associated proteins are immunoprecipitated with anti-Myc antibodies and immune complexes are subjected to immunoblotting, with anti-Myc and anti-Cdk1 antibodies, and tested for histone H1 kinase activity[1].

Cell Assay
[1]

1-NM-PP1 (1-NMPP1) is dissolved in DMSO and stored, and then diluted with appropriate media before use[1].

Wild-type or Cdk7as/as HCT116 cells are synchronized by incubation in serum-free medium for 48 h and released into medium containing 10% fetal calf serum. Synchronization with thymidine or nocodazole, and analysis of cell-cycle distribution by flow cytometry, are performed. Cell viability is measured by MTT assay[1].

References
M.Wt

331.41

Formula

C₂₀H₂₁N₅

CAS No.

221244-14-0

Storage
Powder -20°C 3 years
  4°C 2 years
In solvent -80°C 6 months
  -20°C 1 month
Shipping

Room temperature in continental US; may vary elsewhere

Solvent & Solubility

DMSO: 27.5 mg/mL

* "<1 mg/mL" means slightly soluble or insoluble. "≥" means soluble, but saturation unknown.

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1-NM-PP1
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