1-NM-PP1

1-NM-PP1, a cell-permeable PP1 analog, is a potent Src family kinases inhibitor with IC₅₀s of 4.3 nM and 3.2 nM for v-Src-as1 and c-Fyn-as1, respectively^[1][2]. IC50 & Target: IC50: 4.3 nM (v-Src-as1), 3.2 nM (c-Fyn-as1), 28 μM (v-Src), 1 μM (c-Fyn), 3.4 μM (c-Abl), 29 μM (CDK2), 24 μM (CAMKII), 120 nM (cAbl-as2), 5 nM (CDK2-as1), 8 nM (CAMKII-as1)^[2] In Vitro: Cdk7 from Cdk7^as/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 Cdk7^as/as also rendered growth of HCT116 cells sensitive to 1-NM-PP1. In the absence of 1-NM-PP1, the wild-type andCdk7^as/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 Cdk7^as/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 Cdk7^as/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 Cdk7^as/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^[3].