DNA Topoisomerase I Inhibitors: Chemistry, Biology, and Interfacial Inhibition

DNA topoisomerases I and II (Top1 and Top2) are established molecular targets of anticancer drugs.1–5 Mammalian somatic cells express six topoisomerase genes: two TOP1 (TOP1 and TOP1mt), two TOP2 (TOP2α and β), two topoisomerase III (TOP3α and β)6,7 (Figure 1A). The most recently discovered eukaryotic topoisomerase is mitochondrial Top1 (Top1mt), which we reported in 2001.8,9 Figure 1 Schematic architecture of the topoisomerase cleavage complexes A common feature of topoisomerases is their catalytic mechanism, which in all cases consists in a nucleophilic attack of a DNA phosphodiester bond by a catalytic tyrosyl residue from the topoisomerase. The resulting covalent attachment of the tyrosine to the DNA phosphate is either at the 3′-end of the broken DNA in the case of Top1 enzymes (Top1 and Top1mt) or at the 5′-end of the broken DNA for the other topoisomerases (Figure 1). Thus, Top1 enzymes are the only topoisomerases that form a covalent link with the 3′-end of the broken DNA while generating a 5′-hydroxyl end at the other end of the break. In that respect, the eukaryotic Top1 enzymes belong to the broader family of site-specific tyrosine recombinases of prokaryotes and yeast (e.g., XerCD of Escherichia coli, bacteriophage λ integrase and Cre recombinase, and Flp of Saccharomyces cerevisiae). Another unique feature of the Top1 enzymes is their DNA relaxation mechanism by “controlled rotation” rather than by “strand passage”.10–12 In other words, Top1 enzymes relax DNA by letting the 5′-hydroxyl end swivel around the intact strand. This processive reaction does not require ATP or divalent metal binding, which is different from Top2 enzymes, which require both ATP hydrolysis and Mg2+.5,13 Top3 enzymes, which, like other type IA topoisomerases require Mg2+ (but no ATP) for catalysis14 are not very active in relaxing DNA supercoiling. They can relax DNA when it is very negatively supercoiled (single-stranded) one turn at a time.15 Moreover, both Top2 and Top3 enzymes change DNA topology by a strand passage distributive mechanism rather than by the processive controlled rotation of the Top1 enzymes. In the case of the Top2 enzymes, a full DNA duplex [referred to as the T (transported) strand] goes through the double-strand break made by an enzyme homodimer5,16,17 (Figure 1A). In the case of the Top3 enzymes, a single strand goes through the single-stranded break,14 typically at double-Holliday junction crossovers.18 The remarkable efficiency of the nicking-closing activity of Top1 enables the enzyme to relax both negatively and positively supercoiled DNA (even at 0°C)19 with similar efficiency.12 This is in contrast with Top2α, which relaxes more efficiently positive supercoiling.20 Of note, Top2β, like Top1 relaxes both positive and negative supercoils similarly.20 Removing positive supercoils is required for replication and transcription progression. Otherwise their accumulation in advance of replication and transcription complexes hinders the melting of the DNA duplex (by helicases) and consequently polymerase translocation along the DNA template. The normal nicking-closing activity of Top1 can however be uncoupled when the 5′-hydroxyl end generated by the nicking reaction becomes misaligned; for instance at preexisting base lesions or DNA nicks.21,22 In such cases, the Top1 cleavage complex (Top1cc) remains without effective legitimate religation partner. Those Top1-DNA covalent complexes are commonly referred to as “suicide complexes”. Under such conditions, Top1 can nevertheless religate an illegitimate (“foreign”) 5-hydroxyl-DNA end and act as a recombinase.23 This property is routinely used for molecular cloning (TOPO® Cloning, Invitrogen) using vaccinia Top1.24