Discovery of potent bisindole-based pyrazolopyridine derivatives as topoisomerase inhibitors: DNA damage induction and synergistic antileukemic activity.

[INTRODUCTION] The development of novel anticancer agents targeting DNA replication and repair mechanisms remains a priority in leukemia therapy. In this study, newly synthesized derivatives incorporating bis-indole and pyrazolo[3,4-]pyridine scaffolds were evaluated for their antiproliferative potential against leukemia cell lines.

[METHODS] The antiproliferative activity of the synthesized compounds was assessed in four cancer cell lines, including acute myeloid leukemia (MV4-11) and chronic myeloid leukemia (K562). Growth inhibition (GI) values were determined. DNA relaxation assays were performed to evaluate inhibition of topoisomerase I and IIα activities. Cell cycle distribution, apoptosis induction, and DNA damage response markers were analyzed using cellular and molecular assays. Combination studies were conducted using CHK1, ATR, and PARP-1 inhibitors.

[RESULTS] Compounds , , and demonstrated the most potent antiproliferative activity, with GI values below 2.5 μM in leukemic cell lines. Compound exhibited notable cytotoxicity, with GI values of 1.1 μM (MV4-11) and 2.7 μM (K562). Compounds and significantly inhibited topoisomerase I activity and effectively suppressed topoisomerase IIα-mediated DNA relaxation. Cellular studies revealed S-phase cell cycle arrest, activation of apoptotic pathways (caspase cleavage and PARP-1 degradation), and induction of DNA damage response markers (γH2AX, p-CHK1, p53). In MV4-11 cells, combination treatment with CHK1 or ATR inhibitors resulted in pronounced synergistic cytotoxicity, whereas co-treatment with a PARP-1 inhibitor produced minimal synergy.

[DISCUSSION] These findings identify bis-indole and pyrazolo[3,4-]pyridine derivatives, particularly compound , as potent dual topoisomerase inhibitors with significant antileukemic activity. Their ability to induce DNA damage and enhance cytotoxicity in combination with DNA damage response inhibitors highlights their potential therapeutic value, especially in combination strategies targeting replication stress pathways in leukemia.