3‐substituted‐1H‐indazoles as Catalytic Inhibitors of the Human DNA Topoisomerase IIα

Catalytic inhibitors represent an attractive possibility of revisiting the established anticancer target human DNA topoisomerase IIα, taking advantage of the many different inhibition steps in its catalytic cycle. In this study we present our efforts to expand the chemical space of inhibitors of the human DNA topoisomerase IIα with a combination of in silico and in vitro methods. Using our previously reported compounds as a base, we constructed a ligand-based pharmacophore and conducted a virtual screening campaign. This led to the discovery of 1H-indazole inhibitors for which the SAR data was subsequently expanded. Additionally, several in vitro methods; cleavage assay, competitive cleavage assay, ATPase assay and microscale thermophoresis (MST) binding studies were employed to study the inhibitory mechanism, and it was confirmed that our compounds are catalytic inhibitors that bind to the ATPase domain. IC50 values were in the lower micromolar range with multiple compounds showing stronger inhibitory activity than the established topoisomerase poison etoposide. Using in silico methods we proposed a binding mode and evaluated it using molecular dynamics simulation. 1H-indazoles represent a new versatile scaffold that could be utilized for further development of catalytic inhibitors of the human DNA topoisomerase IIα targetting the ATPase domain where ATP binding site is located.