Publication: RMN y reconocimiento molecular:relación estructura-actividad de péptidos implicados en el dolor e interacciones proteína-carbohidrato
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2017-05-11
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Universidad Complutense de Madrid
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Abstract
El reconocimiento molecular es el punto de partida de numerosos procesos biológicos. La comprensión del reconocimiento entre los ligandos y sus dianas es clave en la optimización y diseño de complejos de mayor afinidad. La combinación de datos de RMN con cálculos teóricos facilita desarrollar una aproximación a la comprensión de las bases moleculares de estos procesos de reconocimiento ligando-receptor. En esta tesis hemos estudiado cuatro sistemas ligando-receptor de interés biológico empleando esta estrategia combinando de RMN y modelización molecular (MM, MD y ab initio). - Estudio estructural de análogos del péptido opiorfina. Opiorfina (QRFSR) es un péptido que impide la degradación de las encefalinas mediante la inhibición de las proteasas NEP y APN. Esta acción sobre el catabolismo de las encefalinas determina un efecto analgésico, demostrado en modelos animales. En este capítulo se han esudiado distintos análogos de la opiorfina cuya actividad ha sido correlacionada con los cambios estructurales observados en RMN. El barrido de alanina en la secuencia de la opiorfina reveló que la sustitución del residuo Phe3 produce una perdida drástica de la actividad frente a NEP y APN. Seguidamente, distintas modificaciones en esta posición mostraron la posibilidad de afectar la selectividad de la capacidad inhibitoria de este péptido. La sustitución de L-Phe por D-Phe mejoró la potencia de la opiorfina frente a APN, perdiendo su actividad frente a NEP. Esta modulación de su actividad se correlaciona con cambios en los espectros de RMN de los péptidos que contienen el enantiómero D-Phe. La diferencia experimental más notable es el apantallamiento de los protones de la Arg2 en los péptidos que contienen D-Phe. Los cálculos de MM mostraron la proximidad espacial entre el anillo aromático y la Arg2, confirmándose el apantallamiento en los espectros RMN. Este estudio muestra una posible interacción catión/ entre la D-Phe3 y la Arg2 que favorece una restricción conformacional que determina una interacción selectiva con APN, mientras que la flexibilidad del péptido natural permite la inhibición dual, pero de menor afinidad frente a APN...
Molecular recognition is involved in numerous life processes. Understanding the variables which are involved in the interactions between biological targets and their ligands in these life processes has the potential to rationalize binding and selectivity features. A combined approach of NMR and theoretical MM and MD could clarify the system dynamics and provide deeper comprehension of the molecular basis of ligand-receptor recognition. In this thesis the analysis of four different ligand-receptor systems of biological relevance has been accomplished by this combined approach. - Structural analysis of opiorphin analogs by NMR Opiorphin (QRFSR) slows down enkephalin degradation by inhibiting both NEP and APN. This peptide produces antinociceptive effects as it was demonstrated in rodent models. In this thesis different opiorphin analogs were characterized by their activity against NEP and APN, and the most remarkable activity findings were analyzed in structural context by a combined approach NMR and MM. The activity of these analogs was measured versus NEP and APN by fluorimetric assays. Alanine scanning showed Phe3 should play a relevant role in the recognition. Subsequently, distinct modifications at Phe3 modulate its dual inhibition. D-Phe3-containing peptides were one order of magnitude more potent than the parent peptide in APN assays, but inactive in NEP assays. NMR corroborates the different tendencies between L-Phe3-containing and D-Phe3-containing peptides. The most remarkable experimental difference is severe shielding of Arg2 H2 and H3 protons to higher field in D-Phe3-containing peptides. MM calculations confirmed the spatial proximity between the aromatic ring and the H protons of Arg2, explaining the shielding at higher field observed by NMR. In contrast, flexible native opiorphin cannot be explained with a single conformation. In this study a higher-affinity selective APN inhibitor has been identified, whose potency enhancement depends on its conformational rearrangement in solution...
Molecular recognition is involved in numerous life processes. Understanding the variables which are involved in the interactions between biological targets and their ligands in these life processes has the potential to rationalize binding and selectivity features. A combined approach of NMR and theoretical MM and MD could clarify the system dynamics and provide deeper comprehension of the molecular basis of ligand-receptor recognition. In this thesis the analysis of four different ligand-receptor systems of biological relevance has been accomplished by this combined approach. - Structural analysis of opiorphin analogs by NMR Opiorphin (QRFSR) slows down enkephalin degradation by inhibiting both NEP and APN. This peptide produces antinociceptive effects as it was demonstrated in rodent models. In this thesis different opiorphin analogs were characterized by their activity against NEP and APN, and the most remarkable activity findings were analyzed in structural context by a combined approach NMR and MM. The activity of these analogs was measured versus NEP and APN by fluorimetric assays. Alanine scanning showed Phe3 should play a relevant role in the recognition. Subsequently, distinct modifications at Phe3 modulate its dual inhibition. D-Phe3-containing peptides were one order of magnitude more potent than the parent peptide in APN assays, but inactive in NEP assays. NMR corroborates the different tendencies between L-Phe3-containing and D-Phe3-containing peptides. The most remarkable experimental difference is severe shielding of Arg2 H2 and H3 protons to higher field in D-Phe3-containing peptides. MM calculations confirmed the spatial proximity between the aromatic ring and the H protons of Arg2, explaining the shielding at higher field observed by NMR. In contrast, flexible native opiorphin cannot be explained with a single conformation. In this study a higher-affinity selective APN inhibitor has been identified, whose potency enhancement depends on its conformational rearrangement in solution...
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Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Bioquímica y Biología Molecular I, leída el 18-12-2015