## 2 Pharmacological Activities
### Antitumour Properties
#### 2.6.2 Mechanism of Action of Dimeric Alkaloids  
Microtubules (MTs) are components of the cytoskeleton and play very important role in a great numbers of cellular processes. They are involved in chromosome separation during mitosis and meiosis, and are the major constituents of mitotic spindles, besides they are involved in maintaining cell structure, transport and many others cell functions. MTs are synthesized from \(\alpha\),\(\beta\)-tubulin heterodimers which polymerize end-to-end into linear protofilaments. These structures are, in a dynamic polymerization and depolymerization, at their ends. The assembly and disassembly of the MTpolymers are regulated by the binding of tubulin and guanosine 5-triphosphate. Any interference with this MT dynamics can provoke a cell cycle arrest and lead to programmed cell death or apoptosis [42]. MT dynamics, and therefore cell division, can also be perturbed by small molecules, which are usually divided into two groups: (i) MT-stabilizing agents that prevent then depolymerization; and (ii) MT-depolymerizing agents that inhibit their formation. Vinca alkaloids fall inside the second group, as they arrest tumour cells during mitosis by binding at the surface between two tubulin heterodimers next to the exchangeable guanosine 5-triphosphate-binding site [43] and depolymerizing the MTs. This leads to cell cycle arrest in mitosis [44,45]. At high concentration Vinca alkaloids lead to the formation of large tubulin polymers called \"Paracristal\" and as a result, the tumoral cells are stopped in mitosis and immediately they die [46]. However, when the levels of Vinca alkaloids are low, the cells are arrested in mitosis and the cells die after a long time of incubation. Since the binding of natural Vinca alkaloids, vinblastine and vincristine, as well as their semi-synthetic analogues, vinorelbine and vinflunine, is linked to tubulin self-association, their affinities can be determined and their binding are entropically driven so that the overall affinities decrease in the following order: vincristine \(>\) vinblastine \(>\) vinorelbine \(>\) vinflunine (Figure 1). The affinity of the four Vinca alkaloids for tubulin heterodimers appears to be almost identical however [47], the major differences amongst them are due to the distinct affinities of the resulting ligand heterodimers for polymerized spirals. All of Vinca alkaloids, in their respective complexes with \(a\),\(\beta\)-tubulin, exhibit overall similar van der Waals forces and electrostatic interaction energy terms thereby defining a common binding site. However, the vinodine domain is used to bind tubulin  
Figure 1: Chemical structures of the four _Vinca_ alkaloids vincristine (**A**), vinblastine (**B**), vinorelbine (**C**) and vinflunine (**D**). The arrow indicates the principal difference between vincristine (A) and vinblastine (B). The arrow heads indicate the principal difference between vinorelbine (C) and vinflunine (D). heterodimers, while the catharanthine domain provides a cytotoxic effect [48]. Their binding site to MTs is lined by the side chains of a number of mostly non-polar amino acids from both tubulin monomers. The formyl group on the indole ring of vincristine that replaces the methyl in vinblastine is relevant for different spectra of pharmacological activity and dose-related toxicities, as well as differential cellular uptake and retention characteristics. On the other hand, the saturated double bond and the two fluorine atoms that differentiate vinflunine from vinorelbine provide only a marginally improved electrostatic interaction [49]. Even though their interaction with tubulin has been characterized, additional studies have revealed new mechanisms of action, such as their interplay with MT associated protein, inhibition of amino acid metabolism and interaction with calmodulin.