Design, synthesis, and evaluation of new fluorescent antimitotic compounds

Abstract

[EN]Tubulin is a key protein in eukaryotic cells, participating in numerous processes as the fundamental units of microtubules, and its proper functioning is linked to the cell's fate. The search for new agents that can inhibit the activity of this protein in tumor cells or harmful organisms is essential. There are many drug-binding sites in the protein that alter its polymerization into microtubules, and some clinically used drugs target these sites, such as taxol© and colchicine, the latter lending its name to the binding site targeted by our compounds. In this thesis, we have designed, synthesized, isolated, characterized, and evaluated more than 230 tubulin inhibitors with potential antitumor and/or antiparasitic activity by binding to the colchicine site oftubulin. The compounds were designed based on known structures, such as CA4 (a well-known ligand of the colchicine site), or antiparasitic compounds discovered by the research group, and through the use of computational technologies aimed at identifying more potent compounds that interact better with the target protein. This approach combines both rational drug design and experimental validation to identify new tubulin inhibitors with enhanced therapeutic potential. The compounds have been classified into two major families. The first consists of CA-4 analogs, which are further divided into two groups depending on the bridge connecting both rings: sulfonamides and tetrazoles. The structure of CA-4 consists of two rings connected by an olefinic bridge. Modifications have been introduced in these three elements, allowing us to gain extensive knowledge of the structure-activity relationship of these derivatives. The compounds in this family were designed to have intrinsic fluorescence, aiding the study of the compounds themselves without the need for adding external fluorophores. The second family comprises amide compounds, focused on their use as antiparasitics, designed based on computer models and comparisons between parasite and mammalian sequences, with the aim of exploiting differences between them to achieve selectivity. The synthesized compounds were evaluated in vitro against a broad panel of tumor cell lines to assess their antitumor activity and against four parasites: Strongyloides spp., Trichinella spp., Schistosoma spp., and Leishmania spp. Studies on their mechanism of action have been conducted to understand how they interact with their biological target. These studies included cell cycle analysis, apoptosis assays, confocal microscopy, studies on cell migration, and inhibition of tubulin polymerization. The studies on tumor cells showed that the CA-4 analog compounds exhibited antiproliferative effects, in the best cases at a near-nanomolar range, causing apoptosis in the cells and disrupting the structure of their microtubules. Since the emergence of resistance is also one of the causes of therapeutic failure, it was studied and confirmed that these compounds are not substrates of MDR pumps, which are associated with resistance processes. Finally, it was demonstrated in vitro that these compounds were capable of inhibiting tubulin polymerization. On the other hand, molecules with interesting antiparasitic effects, especially against Strongyloides venezuelensis and Trichinella spiralis, have been reported, with some even tested in in vivo models of parasitosis. Since parasite tubulin is much less known, during this thesis work, experi ments were conducted to obtain the functional protein from the four studied parasites for further study and characterization.