Development of a screening assay for surrogate markers of CHK1 inhibitor-induced cell cycle release.
Chk1 is a key regulator of the S and G2/M checkpoints and is activated following DNA damage by agents such as the topoisomerase I inhibitor camptothecin (CPT). It has been proposed that Chk1 inhibitors used in combination with such a DNA damaging agent to treat tumors would potentiate cytotoxicity and increase the therapeutic index, particularly in tumors lacking functional p53. The aim of this study was to determine whether gene expression analysis could be used to inform lead optimization of a novel series of Chk1 inhibitors. The candidate small-molecule Chk1 inhibitors were used in combination with CPT to identify potential markers of functional Chk1 inhibition, as well as resulting cell cycle progression, using cDNA-based microarrays. Differential expression of several of these putative marker genes was further validated by RT-PCR for use as a medium-throughput assay. In the presence of DNA damage, Chk1 inhibitors altered CPT-dependent effects on the expression of cell cycle and DNA repair genes in a manner consistent with a Chk1-specific mechanism of action. Furthermore, differential expression of selected marker genes, cyclin E2, EGR1, and DDIT3, was dose dependent for Chk1 inhibition. RT-PCR results for these genes following treatment with a panel of Chk1 inhibitors showed a strong correlation between marker gene response and the ability of each compound to abrogate cell cycle arrest in situ following CPT-induced DNA damage. These results demonstrate the utility of global expression analysis to identify surrogate markers, providing an alternative method for rapid compound characterization to support advancement decisions in early drug discovery.
Fanton
Christie P
CP
Biopharma Research and Development, Chiron Corporation, Emeryville, CA, USA. christie.fanton@chiron.com
Rowe
Michael W
MW
Moler
Edward J
EJ
Ison-Dugenny
Marjorie
M
De Long
Susan K
SK
Rendahl
Katherine
K
Shao
Yi
Y
Slabiak
Trina
T
Gesner
Thomas G
TG
MacKichan
Mary Lee
ML
D015415:Biomarkers / Q000032:analysis; D002166:Camptothecin / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects* / Q000235:genetics; D000071877:Checkpoint Kinase 1; D004249:DNA Damage / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects*
D015415:Biomarkers; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D002166:Camptothecin