Investigation of new human DNA topoisomerase IB inhibitors

DNA topoisomerases perform a key role in the relief of torsional strain, which occurs in DNA during many cellular processes, including transcription, replication, chromosome condensation, also DNA repair (Coleman, 2002; Stewart et al., 1998). They can be classified into two types and each one of them subdivide in A or B subfamily (Wang, 1996). In particular, the human topoisomerase IB is a monomeric enzyme composed of four major domains: N- terminal, core, linker and C-terminal domain, which contains the active site tyrosine 723. It catalyses the relaxation of negative and positive DNA supercoils, by introducing a transient singlestrand break and a covalent link with the 3’-phosphotyrosil end of the broken DNA strand, leaving a free 5’-OH strand (Champoux, 1981). This enzyme is of great interest to medical applications because, it can be target by natural and also non-natural compounds, classified as poisons if they act like camptothecin (CPT) by blocking the religation step, or catalytic inhibitors if they inhibit binding or cleavage of the DNA (Bailly, 2000). In the first part of this thesis, the characterization of the inhibition effect of berberine chloride and three derivatives belonging to the 13diphenylalkyl, has been tested as hTopo IB inhibitors. Berberine is a natural alkaloid and it has been used for many medical applications especially in China. Therefore, additional knowledge regarding its antitumor activity seems to be of great interest. The results obtained demonstrated that all the compounds display an efficient inhibition in a dose dependent manner. This inhibition is mostly like due to an interaction between enzyme-compound, since it clearly increases upon pre-incubation. Although, this interaction is reversible, similarly to the camptothecin behavior. In presence of the derivatives the cleavage step is inhibited but not for the religation of the hTopo IB enzyme catalytic cycle. The pre-incubation of enzyme and compound, increases the inhibition of the cleavage, however it just slightly increases for religation. Docking simulation indicates that the compounds bind in proximity of the catalytic pentad, still permitting the binding but not the cleavage of the DNA. So, all berberine derivatives act as catalytic inhibitors. The second part consists in an investigation of transition-metal based complexes. Since its discovery, cisplatin has been largely studied and nowadays is used in chemotherapy. However due to its side effects and subsequently resistance, the design and test of new drugs have been an essential key to improve cancer treatments. Ruthenium complexes have been proposed to overcome the problems related to cisplatin, being more effective to kill cancer cell, with less toxicity effects and variable structures. Thereby, three ruthenium compounds have been tested. Their structure consists in a common metal-precursor linked to different ligands forming the compounds: M-IMI, IMIDAZOL and MTZ. By testing the three compounds as hTopo IB inhibitors, the results demonstrated that all of them inhibit the relaxation of supercoiled DNA, and the effect of this inhibition is increased with a preincubation of compound-enzyme before the addition of supercoiled DNA. The ligands alone do not affect the enzyme activity, demonstrating that the precursor containing the transition metal plays an essential role in the inhibition effect. Only one compound, IMIDAZOL, presents a strong interaction with the supercoiled DNA. The cleavage step is inhibited and this effect is increased upon pre-incubation. Furthermore, regarding the religation step, two among them inhibit this step, suggesting that the interactions between the compounds, enzyme and DNA strongly depends upon the structure of the compound and the different ligands. Therefore, compounds M-IMI and IMIDAZOL, acts as mix inhibitors, showing behavior from the two classes of inhibitors; on the other side, MTZ acts like a catalytic inhibitor. To sum up, both, berberine derivatives and ruthenium compounds can then be considered as possible topoisomerase inhibitors. Moreover, understanding the how the different ligand or introduction of distinct side chains can affect the interactions of the compounds and the enzyme, contributes for the development of new antitumor drugs.