Characterization of novel topoisomerase IB inhibitors as a potential anticancer agent

Topoisomerase I is a target of active interest in cancer therapy and despite the fact that, up to now, only two camptothecin derivatives, topotecan and irinotecan, are in clinical use, there is a large effort by several groups in developing new compounds targeting this enzyme. With the aim to contribute in this field, we have investigated the effect of transition metal complexes and a natural organic compound on human DNA topoisomerase I. The work has been designed as follows: 1. Cloning, production and purification of human DNA topoisomerase IB. Checking of the purity and integrity of the protein by immunoblot assay. 2. Analysis of topoisomerase IB activity in vitro by plasmid relaxation assay. 3. Characterization of effects of metal complexes and natural compound on human DNA topoisomerase IB. Investigating the effects on each individual catalytic steps of the enzyme like equilibrium, cleavage and religation assays. 4. Analysis of the mechanism of action of topoisomerase poisons. 5. Prediction of putative binding site of the compound on topoisomerase IB, taking into account both geometrical and electrostatic match by molecular docking simulations. The results indicate that the natural compound thaspine can be considered a promising molecule and that its effect towards the enzyme can be improved by chemical modifications. As the same time, metal complexes are interesting since, the ability of the metals to induce different geometric conformations can be used to modulate their efficiency in the enzyme binding.