Design, synthesis, and evaluation of new fluorescent antimitotic compounds

• Autores: Miguel Marín Folgado
• Directores de la Tesis: Rafael Peláez Lamamie de Clairac (dir. tes.), Laura Gallego Yerga (codir. tes.)
• Lectura: En la Universidad de Salamanca ( España ) en 2024
• Idioma: inglés
• Títulos paralelos:
  • Diseño, síntesis y evaluación de nuevos compuestos antimitóticos fluorescentes
• Tribunal Calificador de la Tesis: Alba Cortés Carbonell (presid.), Raquel Álvarez Lozano (secret.), Bárbara de Filippis (voc.)
• Programa de doctorado: Programa de Doctorado en Farmacia y Salud por la Universidad de Salamanca
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• Resumen
  • español

    La tubulina es una proteína clave en las células eucariotas, participando en numerosos procesos como unidades fundamentales de los microtúbulos, los cuales intervienen en una amplia variedad de procesos celulares, como el transporte de vesículas y orgánulos, funciones estructurales o la formación del huso mitótico para la división celular. Existen múltiples sitios de unión de fármacos en la proteína que alteran su polimerización en microtúbulos, clasificándose en agentes estabilizantes, como el taxol, o agentes desestabilizantes, como la colchicina. Los compuestos que se unen a este sitio han demostrado su potencial para el tratamiento de enfermedades como el cáncer o las parasitosis.

    En esta tesis hemos diseñado, sintetizado, aislado, caracterizado y evaluado más de 230 compuestos derivados de amidas o sulfonamidas aromáticas con el objetivo de que sean capaces de unirse al sitio de la colchicina de tubulina de mamíferos y parásitos y actúen como antimitóticos. Los compuestos fueron diseñados basándose en estructuras de ligandos de tubulina de mamífero conocidos, como CA-4, o en compuestos antiparasitarios descubiertos por el grupo de investigación, y mediante el uso de tecnologías computacionales, como estudios de docking o de dinámica molecular destinadas a identificar compuestos más potentes que interactúen mejor con la proteína objetivo. De esta manera se plantea un trabajo basado en ciclos de diseño-síntesis-evaluación y rediseño, que combina el diseño racional de fármacos y la validación experimental para identificar nuevos inhibidores de la tubulina con mayor potencial terapéutico.

    Los compuestos sintetizados se evaluaron in vitro frente a un amplio panel de líneas celulares tumorales para evaluar su actividad antitumoral y frente a cuatro parásitos: Strongyloides spp., Trichinella spp., Schistosoma spp. y Leishmania spp.

    Se seleccionaron los compuestos con menores IC50 en los experimentos en células tumorales o parásitos para estudiar su mecanismo de acción.

  • English

    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 astaxol© 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, experiments were conducted to obtain the functional protein from the four studied parasites for further study and characterization.