Thiadiazole-4β-sulfur-demethylepipodophyllotoxin traps human topoisomerase IIβ by distorting DNA and disrupting the tyrosyl-DNA linkage.

Type II topoisomerases (TOP2) are essential enzymes and established anticancer targets. However, clinical inhibitors like etoposide have been hindered by acquired resistance and secondary malignancies. The poisoning mechanism of human hTOP2β by Thiadiazole-4β-S-DMEP was elucidated through high-resolution structural studies, revealing active site disruption and DNA distortion. Crystallographic analysis of the hTOP2β-DNA complex provided a snapshot of the structural impact of FTh, showing a stabilized, religation-incompetent conformation with a disrupted active site, lacking the essential phosphotyrosyl linkage and exhibiting a distorted cleaved DNA terminus. Combined with biochemical assays and in-silico modeling, we propose that the unique 4'-S-thiadiazole moiety of FTh forms an additional anchoring interaction with the +4 DNA nucleotide downstream of the scissile phosphate. This interaction triggers an open conformational state of hTOP2β, exposes the active site to solvent, and accelerates hydrolytic cleavage of the phosphotyrosyl bond, leading to irreversible disabling of the religation machinery and permanent double-strand breaks. Unlike classical TOP2 poisons that merely trap the cleavage complex, FTh acts by actively dismantling the catalytic apparatus. Our findings establish a new paradigm for hTOP2 inhibition and validate the 4β-S-DMEP scaffold as a promising platform to develop next-generation anticancer agents capable of overcoming drug resistance.