Furthermore, it was observed that binding occurs, neither to tubulin dimers nor to preformed microtubules. However, ligands are able to bind to tetramers. This, coupled with the fact that ligands compete for the binding site with model drugs, which bind in the interfacial region, indicates that the region of contact with the tubulin molecule is located in the vinca domain. Since interfacial ligands often require self-assembly of tubulin in order to bind, and since such self-assembly depends on the concentration of magnesium, it was determined the relation between ligand binding to target and magnesium concentration. The result showed a clear dependence between these two parameters, which means that binding and self-assembly are two coordinated processes in the studied system. Binding stoichiometry for all the studied compounds, measured in glycerol-buffer, fluctuates between 0.6:1 and 1.15:1, indicating the average oligomerization state induced by each ligand. This average oligomerization state tends to the tetrameric state, the more the value approaches to the relation 0.5:1, while it tends to big polymers, the more the value approaches to 1:1. Moreover, a method for determining equilibrium constants of ligands which bind to tubulin, by experiments of sedimentation velocity of analytical ultracentrifugation, has been optimized. This method is based on the assumption that the analyzed system follows the isodesmic condition. In such systems, each oligomerization step has the same binding constant as the previous step. The developed method allows to determine affinity constants for any ligand, as long as the studied ligand interacts with proteins, which assemble in a isodesmic manner, and changes the affinity of the protein self-assembly when it binds. Obtained results show that compounds coded as 1 (parental) and 2 are the most actives of the series. Finally, the structural characterization studies involved X-ray diffraction, electron microscopy, STD-NMR and bioinformatics. The tubulin tetramer structure, crystallized in the presence of the parental compound revealed the real conformation of the bound ligand, and it also allowed to determine its mechanism of action. When binding happens, the tetrameric curved apo-structure slightly straightens, what makes possible some degree of tubulin assembly. From the results obtained by electron microscopy, it is found that all the compounds induce the formation of helical polymers, while from the STDNMR analysis it was concluded that they contact with the vinca domain through the triazole ring, the trifluorobenzene ring and the substituent of the 3-position of the azabicyclo. Additionally, molecular modelling of the ligands showed that all of them bind to the tubulin in a very similar way, mainly through two aromatic stacks with two tubulin residues. It was also observed an inverse relationship between the polarity of the substituents at 3-position of the azabicyclo and the binding constant of the compounds. Finally, the effects induced by the studied ligands and the effects induced by vinblastine were compared. Vinblastine is a widely used antitumor drug which, as well as the studied compounds do, it binds to tubulin in the vinca domain. Comparison between data obtained in this work and those available in the literature about vinblastine, revealed several differences. While vinblastine, which is a drug with larger volume than the studied compounds, induces a wedge that promotes tubulin disassembly, these studied compounds access the vinca domain and straighten the tetramer structure they contact with. This fact suggests that the difference in the specific volume of the ligands of the vinca domain determines the induced effect on tubulin.

Nowadays, one of the major public health issues in developed countries is cancer and, for this reason, the study of new treatments against this disease receives today special attention. Among all existing therapies, chemotherapy is preferential choice in most cases because of their high success expectations. Chemotherapy involves the block of a specific cellular function by administering a chemical agent. The two cellular pathways, on which chemotherapy usually acts, are the genetic material replication and the cell division. Within the group of treatments that act on cell division, those using chemotherapy drugs that interact with the cytoskeleton, are at the forefront of the fight against cancer. Microtubules, the major components of cytoskeleton, are dynamic polymers, which look like hollow cylinders. They are made up of tubulin protofilaments, which establish lateral contacts with other protofilaments. Besides, such protofilaments are formed by the head-tail association of αβ-tubulin heterodimers. Balance between dimeric and polymeric state is regulated by a dynamics, which is GTP-hydrolysis dependent. GTP-heterodimers are more likely to polymerize, while GDP-heterodimers drive balance toward free species. There is a large collection of chemical compounds that are able to regulate this dynamics, either promoting tubulin assembly and/or stabilizing microtubules, or promoting disassembly and/or destabilizing polymers. In both cases, proper function of these structures is altered and thus, the whole functions they are responsible for, such as chromosome segregation or division of nuclear and cell envelopes during the replication process, are also altered. The work presented in this report, entitled “Evaluation of a new chemotype of tubulin modulators of the vinca domain”, shows the characterization of the binding process and the mechanism of action of a synthetic compound and its structural derivatives, from a biological, biochemical and structural point of view. Obtained information was further used in order to determine the implication of the different structural determinants in the activity of each compound. On one hand, performed tests on human cell lines demonstrated that the studied compounds have cytotoxic activity and they also demonstrated that the compounds interact with the microtubule network, being able to arrest cell cycle at G2/M phase. In addition, it was observed that none of the ligands is a substrate for the P-glycoprotein (PGP). This fact causes that these ligands are able to maintain, approximately, the same level of activity in cells that show the multidrug resistance phenotype (MDR), by overexpression of PGP. On the other hand, the study of the activity on the microtubule network of Aspergillus nidulans allowed to determine that both ligand binding and the consequent produced effect are reversible actions. Additionally, by in vitro biochemical experiments, it was analyzed the effect of compounds on tubulin. Its assembly capacity significantly increased as a result of the presence of studied compounds, so it was concluded that these compounds behave as tubulin polymerizers. Afterwards, by DOSY-NMR and analytical ultracentrifugation experiments, it was concluded that studied molecules induce the formation of oligomers of various sizes, without actually reaching microtubule generation.

173 p.-60 fig.-10 tab.-2 anexos.

Peer reviewed