Abstract
<jats:p>Human topoisomerase 1 (TOP1) resolves DNA supercoiling during replication and transcription and is a major target for anticancer therapy. TOP1 poisons exert cytotoxicity by stabilizing the TOP1-DNA cleavage complex (TOP1cc), thereby blocking DNA rejoining and generating lethal DNA damage. Several TOP1 poisons have been approved either as conventional therapeutics or as payloads in targeted delivery systems, and many additional candidates are under clinical development. Here, we resent cryo-EM structures of human TOP1cc bound to eight representative and clinically relevant TOP1 poisons: camptothecin (CPT), six CPT derivatives, and an indenoisoquinoline LMP-400. These cryo-EM structures reveal a TOP1cc conformation that differs substantially from canonical crystal structures. These structures also define how specific modifications on the CPT central scaffold and changing to an alternative non-CPT scaffold reshape drug intercalation geometry, molecular interaction networks, and TOP1cc protein architecture. Together with biochemical trapping data, these structural insights establish a foundation for designing next-generation TOP1 poisons with improved pharmacological properties and for their optimization as antibody-drug conjugate (ADC) payloads.</jats:p>