LMP - 400 by the cell checkpoint and Chk 1 - Chk 2 inhibitor , AZD 7762

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Novel topoisomerase I (Top1) inhibitors are in clinical development to circumvent the drawbacks of camptothecins. Here we report molecular investigations into LMP-400, an indenoisoquinoline Top1 inhibitor in Phase 1 clinical trial, by itself and in combination with the cell cycle checkpoint inhibitor, AZD7762. We examined drug effects on DNA replication and killing of cancer cells and found that LMP-400 showed synergistic antiproliferative activity when combined with AZD7762 in human colon carcinoma cells. Inhibition of S-phase progression and bromodeoxyuridine incorporation were similarly induced by LMP-400 and camptothecin (CPT) and were abrogated by AZD7762. Replication studied by single DNA molecule analyses and immunofluorescence microscopy (molecular combing) showed rapid inhibition of fork progression in response to LMP-400 treatment with subsequent recapitulation after AZD7762 addition. AZD7762 inhibited both the activation/autophosphosphorylation of Chk1 and Chk2 at nanomolar concentrations in LMP-400-treated cells. This potent dual inhibition of Chk1 and Chk2 by AZD7762 was below the drug concentrations required to abrogate cell cycle inhibition and produce synergism with LMP-400. Also, the synergism was independent of Chk2 both in Chk2-complemented cells and Chk2 knockout cells, suggesting additional mechanisms for cell cycle abrogation by AZD7762. Together, our findings demonstrate a rationale for combining cell cycle checkpoint inhibitors with the novel non-camptothecin indenoisoquinoline Top1 inhibitors. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.