1-NM-PP1 (PP1 Analog II) is a cell-permeable PP1 analog that acts as a potent and selective inhibitor of mutant kinases over their wild-type progenitors.

PKD:0.398 μM

Cdk7蛋白从Cdk7as/as或Cdk7+/+细胞中免疫共沉淀后，其对含有Pol II CTD融合蛋白（GST-CTD）和人类Cdk2的激酶活性进行了检测。从突变体细胞中回收的 Cdk7会被1-NM-PP1（1-NMPP1）抑制，IC50约为50 nM。野生型Cdk7被Cdk7as/as替换后，也使HCT116细胞的生长对1-NM-PP1敏感。在没有1-NM-PP1的情况下，野生型和Cdk7as/as细胞的种群加倍时间分别约为17.9小时和20.2小时，异步培养下细胞周期分布类似，表明F91g突变本身对Cdk7功能的损害最小。然而，同源纯合的Cdk7as/as细胞对1-NM-PP1敏感，通过细胞活力（MTT）测定显示，经过96小时1-NM-PP1处理后，IC50约为100 nM。相比之下，野生型HCT116细胞对10 μM 1-NM-PP1具有抵抗力。10 μM 1-NM-PP1的添加通过突变体而非野生型细胞延缓了G1/S过渡。当1-NM-PP1与血清同时添加到Cdk7as/as细胞中时，在接下来的15小时内阻止了任何进入S期的进展。24小时后，有证据表明，一部分Cdk7as/as细胞从血清饥饿直接释放到含有1-NM-PP1的培养基中，进入S期，而另一部分仍停留在G1期。在血清添加3小时或6小时后添加1-NM-PP1，分别将S期入口延迟约7小时或约3小时。

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].

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].