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The different steps of the topoisomerase I catalytic cycle have been analyzed in the presence of the plant alkaloid thaspine (1- (2-(Dimethylamino)ethyl)-3,8-dimethoxychromeno[5,4,3-cde]chromene-5,10-dione), known to induce apoptosis in colon carcinoma cells. The experiments indicate that thaspine inhibits both the cleavage and the religation steps of the enzyme reaction. The inhibition is reversible and the effect is enhanced upon pre-incubation. Molecular docking simulations of thaspine over topoisomerase I, in the presence or absence of the DNA substrate, show that thaspine, when interacting with the enzyme alone in the closed or in the open state, can bind in proximity of the active residues preventing the cleavage reaction, whilst when docked with the enzyme-DNA cleavable complex intercalates between the DNA bases in a way similar to that found for camptothecin, explaining its religation inhibition. These results unequivocally demonstrate that thaspine targets human topoisomerase I .

Topoisomerases I are ubiquitous enzymes that control DNA topology within the cell. They are the unique target of the antitumor drug camptothecin that selectively recognizes the DNA-topoisomerase covalent complex and reversibly stabilizes it. The biochemical and structural-dynamical properties of the Asp677Gly-Val703Ile double mutant with enhanced CPT sensitivity have been investigated. The mutant displays a lower religation rate of the DNA substrate when compared to the wild-type protein. Analyses of the structural dynamical properties by molecular dynamics simulation show that the mutant has reduced flexibility and an active site partially destructured at the level of the Lys532 residue. These results demonstrate long-range communication mechanism where reduction of the linker flexibility alters the active site geometry with the consequent lowering of the religation rate and increase in drug sensitivity.