Novel Azapodophyllotoxin Induces DNA Cleavage via Groove Binding: An Advancement in Exploration of Anticancer Activity of a Known Inhibitor of Tubulin Polymerization and Topoisomerase

Abstract

The present study embodies exploration of new potential targets for bioactive azapodophyllotoxins (AZP) that have been mainly considered as inhibitor of tubulin polymerization and topoisomerases. The interaction of a novel AZP, HTDQ, with potential target DNA (calf thymus DNA) has been investigated alongwith its mechanism of action by using combined biophysical spectroscopic techniques and in silico approach. The steady state absorption and fluorescence studies highlights binding interaction of HTDQ with the nucleic acid and the value of Stern Volmer quenching constant (8.07×103 L mol−1) hints towards minor groove binding interaction. The KI quenching studies, helix melting and viscosity measurements further supports our speculation and is substantiated by fluorescence displacement assay using standard minor groove binder, Hoechst 33258. The conformational alterations due to HTDQ‐ctDNA binding is evident from circular dichroism studies and have been further validated from the in silico molecular docking and molecular dynamics simulation studies. Additionally, with a view to get thorough understanding of the mechanism, the DNA cleavage studies have been performed using gel electrophoresis that proved instrumental in determining the ability of HTDQ to damage the structural homogeneity of the nucleic acid.