The naphthoquinone adduct 12,13-dihydro-N-methyl-6,11,13-trioxo-5H-benzo[4,5]cyclohepta[1,2-b] naphthalen-5,12-imine (hereafter called TU100) contains structural features of both the anthracycline and isoquinone chemotherapeutics. An initial characterization showed TU100 is cytotoxic to mammalian cells and can inhibit topoisomerase I and II. Analysis using topoisomerase I now reveals TU100 is a slow acting inhibitor targeting the enzyme in the absence of DNA. Diluting pre-incubated TU100 and topoisomerase I failed to alleviate inhibition, suggesting the enzyme is being covalently modified. Critical cysteine thiols were identified as the possible target based on the ability of reducing agents to reverse TU100 inhibition. Consistent with this idea, TU100 protected topoisomerase I from inactivation by the sulthydryl modifying agent N-ethylmaleimide (NEM). Unlike agents nonspecifically reacting with thiols, however, TU100 is specific for topoisomerase because it failed to inhibit a cysteine dependent protease. These results indicate TU100 is a novel naphthoquinone that inactivates free topoisomerase I via alkylation of cysteine residues. (C) 2011 Elsevier Inc. All rights reserved.