Búsqueda racional de agentes de asociación a tubulina con potencial acción anticancerígena
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2018
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es
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Universidad Andrés Bello
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Licencia CC
Resumen
Pelorusido A (PLA) y Laulimalida (LAU) son agentes estabilizantes de microtúbulos (MTs) que se unen en un sitio ubicado en la parte externa de β-tubulina, el cual ha sido caracterizado recientemente. El conocimiento estructural sobre el mecanismo de acción de estos compuestos ha sido materia de investigación los últimos años y representa un desafío para los avances relacionados al tratamiento de cáncer. En este trabajo, se realizaron simulaciones moleculares y cálculos de energía libre MM-GBSA/PBSA en complejos aJβ- tubulina con LAU y PLA, con el propósito de encontrar los parámetros y protocolos apropiados para el estudio de estos sistemas, ajustándose a lo reportado experimentalmente. Luego, se aplicaron estas metodologías en modelos reducidos de MT tetraméricos libres y asociados con LAU y PLA. Estos modelos permitieron emular las interacciones interdiméricas laterales y longitudinales existentes en la compactación de MTs, revelando que los compuestos confieren una fuerte estabilización en las interacciones longitudinales dímero-dímero. Además, los compuestos restringen notoriamente la estructura y movimientos de los tetrámeros, obteniéndose reconstrucciones de vuelta de MT congruentes con lo observado biológicamente (13-16 PFs por vuelta) y modelos de PFs más lineales acordes a estructuras de MT estable. Subsiguientemente, se realizó un screenirig virtual a partir una base de datos de 6 millones de compuestos utilizando AutodockVina con diferentes etapas de filtrado. Como resultado, se identificó un grupo de 7 compuestos que fue sometido a simulaciones de dinámica molecular y cálculos de energía libre MM GBSA en complejo con los modelos de dímero y tetrámeros. Los compuestos compartieron la mayoría de las características observadas en los ligandos activos, mostrando efectos energéticos similares a LAU/PLA y restringiendo la estructura de los tetrámeros. Ensayos de polimerización de tubulina mostraron que todos los compuestos tienen actividad promotora en concentraciones cercanas a los 100 tm. Finalmente, 3 compuestos expresaron actividad anticancerígena con un período de 48 horas de incubación, con reducciones de viabilidad celular superiores al 60%. Los resultados de esta tesis esperan contribuir con información valiosa para el estudio y diseño de agentes estabilizantes de MTs afines por el sitio LAU/PLA, utilizando como base los compuestos obtenidos en este trabajo.
Peloruside A (PLA) and Laulimalide (LAU) are microtubule-stabilizing agents that bind in a β-tubulin external pocket, which has been characterized recently. The knowledge about the mechanism of action of both compounds is still unclear and it has becomes a challenge for the development of new cancer treatments. In this work, it were performed molecular dynamics simulations and MM-GBSAIPBSA binding energy calculations in cíβ-tubulin complexed with LAU and PLA in order to define the protocols and their parameters for the proper study of these systems, according with experimental reports. Then, it were applied the aboye procedure to reduced tetrameric MT-models in free state and complexed with LAU and PLA. Tetrameric models incorporated lateral and longitudinal interdimeric interactions present in MT compaction, revealing that the association of compounds has a strong stabilization effect in the longitudinal contacts. Moreover, the compounds induce important restrictions on the structure and the interdimeric motions of tetramers, showing that computational reconstructions of one turn of the MT helix were consistent with biological observations (13-16 PFs) and more linear PFs according to a MT stable structure. Subsequently, it was performed a virtual screening with an initial database of 6 millions of compounds using AutodockVina combined with hierarchical filter stages. As result, it was proposed a set of 7 candidates that were subjected to molecular dynamics simulations and MM-GBSA binding energy calculations in complex with dimer and tetrameric models. The 7 compounds induce similar effects on MT as LAU and PLA, showing a strong stabilization of longitudinal interactions, important restrictions on interdimeric motions and consistency with 13-16 PFs per one turn of the MT helix by computational reconstructions. Tubulin polymerization assays show stabilization activity of 7 compounds at 200 and 100 tm of ligand concentration. Finally, 3 compounds have alsoactivity on HeLa cells in incubation period of 48 hours, reducing the cellular viability over 50%. These results are valuable to increase understanding about the molecular features involved in MT stabilization by compounds that bind to LAU/PLA pocket, and to design novel compounds using these ligands as reference.
Peloruside A (PLA) and Laulimalide (LAU) are microtubule-stabilizing agents that bind in a β-tubulin external pocket, which has been characterized recently. The knowledge about the mechanism of action of both compounds is still unclear and it has becomes a challenge for the development of new cancer treatments. In this work, it were performed molecular dynamics simulations and MM-GBSAIPBSA binding energy calculations in cíβ-tubulin complexed with LAU and PLA in order to define the protocols and their parameters for the proper study of these systems, according with experimental reports. Then, it were applied the aboye procedure to reduced tetrameric MT-models in free state and complexed with LAU and PLA. Tetrameric models incorporated lateral and longitudinal interdimeric interactions present in MT compaction, revealing that the association of compounds has a strong stabilization effect in the longitudinal contacts. Moreover, the compounds induce important restrictions on the structure and the interdimeric motions of tetramers, showing that computational reconstructions of one turn of the MT helix were consistent with biological observations (13-16 PFs) and more linear PFs according to a MT stable structure. Subsequently, it was performed a virtual screening with an initial database of 6 millions of compounds using AutodockVina combined with hierarchical filter stages. As result, it was proposed a set of 7 candidates that were subjected to molecular dynamics simulations and MM-GBSA binding energy calculations in complex with dimer and tetrameric models. The 7 compounds induce similar effects on MT as LAU and PLA, showing a strong stabilization of longitudinal interactions, important restrictions on interdimeric motions and consistency with 13-16 PFs per one turn of the MT helix by computational reconstructions. Tubulin polymerization assays show stabilization activity of 7 compounds at 200 and 100 tm of ligand concentration. Finally, 3 compounds have alsoactivity on HeLa cells in incubation period of 48 hours, reducing the cellular viability over 50%. These results are valuable to increase understanding about the molecular features involved in MT stabilization by compounds that bind to LAU/PLA pocket, and to design novel compounds using these ligands as reference.
Notas
Tesis Doctor en Fisicoquímica Molecular
Palabras clave
Cáncer, Tubulina