Cellular inhibition of checkpoint kinase 2 (Chk2) and potentiation of camptothecins and radiation by the novel Chk2 inhibitor PV1019 [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide].

Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide], which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiation-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.

Asparagine synthetase is a predictive biomarker of L-asparaginase activity in ovarian cancer cell lines.

We recently used RNA interference to show that a negative correlation of L-asparaginase (L-ASP) chemotherapeutic activity with asparagine synthetase (ASNS) expression in the ovarian subset of the NCI-60 cell line panel is causal. To determine whether that relationship would be sustained in a larger, more diverse set of ovarian cell lines, we have now measured ASNS mRNA expression using microarrays and a branched-DNA RNA assay, ASNS protein expression using an electrochemiluminescent immunoassay, and L-ASP activity using an MTS assay on 19 human ovarian cancer cell lines. Contrary to our previous findings, L-ASP activity was only weakly correlated with ASNS mRNA expression; Pearson's correlation coefficients were r = -0.21 for microarray data and r = -0.39 for the branched-DNA RNA assay, with just the latter being marginally statistically significant (P = 0.047, one-tailed). ASNS protein expression measured by liquid-phase immunoassay exhibited a much stronger correlation (r = -0.65; P = 0.0014, one-tailed). We conclude that ASNS protein expression measured by immunoassay is a strong univariate predictor of L-ASP activity in ovarian cancer cell lines. These findings provide rationale for evaluation of ASNS protein expression as a predictive biomarker of clinical L-ASP activity in ovarian cancer.