Design, synthesis and screening of small libraries of potential topoisomerase I poisons

Topoisomerase I (topo I) is a ubiquitous enzyme that relaxes the supercoiling introduced into DNA during normal cellular processes such as replication and transcription. The topo I enzyme forms a covalent DNA adduct, called the cleavage complex, during the catalytic cycle. Compounds that trap the cleavage complex turn topo I into a cellular poison. Because cancer cells are replicating rapidly with frequently up-regulated topo I expression, they are more susceptible to topo I poisons than are normal cells. Thus, topo I poisons like the natural product camptothecin have potent anti-tumor activity. The successes of these drugs, along with their limitations, have made it desirable to develop new topo I poisons as potential chemotherapeutic agents. We hypothesized that a compound that simultaneously binds to the DNA minor groove and topo I would be such a poison. X-ray crystal structures of topo I bound to DNA were used to identify a hydrophobic pocket in the "nose-cone" region of the enzyme that faces the minor groove of the DNA. Potential enzyme-binding moieties were attached via linkers to Hoechst 33258, a minor groove binder and mild topo I poison. A gel-based assay was developed to evaluate increased concentrations of cleavage complexes as a function of drug. Small libraries of dipeptide-linker-Hoechst compounds were compared to Hoechst 33258 using this assay. Based on the results obtained with first- and second-generation libraries, a set of libraries with 49 di-peptides conjugated to Hoechst 33258 via a series of linkers was synthesized to optimize linker length. Deconvolution of the most active linker library led to the discovery of a single bivalent topo I poison with greater enhanced DNA cleavage than the monovalent parent compound Hoechst 33258 over concentrations from 2.56 nM to 300 nM. This result supports the utility of combining structure-based drug design with combinatorial chemistry as well as the potential gains in binding affinity and specificity obtained with bivalent inhibitors.