Estudo da interação da piperina com potenciais alvos moleculares de interesse farmacológico
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Data
2021-06-17
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Universidade Estadual Paulista (Unesp)
Resumo
O estudo de produtos naturais com propriedades farmacológicas é uma área de interesse para a comunidade científica. Estudos recentes mostraram que a piperina, uma molécula extraída de diversas espécies de pimenta, é um agente anti-inflamatório que atua inibindo a cascata de ativação da proteína NF-κB em várias etapas desta via, ocasionando o bloqueio da transcrição de genes de citocinas e outras proteínas pró-inflamatórias. Devido a este fato, o objetivo deste trabalho foi a caracterização biofísica a nível molecular da piperina e seus derivados com proteínas envolvidas neste processo, nunca antes reportados na literatura. A caracterização dos complexos piperina-proteína, foi feita por meio da técnica de espectroscopia de fluorescência de modo a determinar o número de sítios de ligação, a descrição dos parâmetros termodinâmicos de interação, bem como, as constantes e os modos de ligação dos complexos. Além disto, o dicroísmo circular foi empregado de modo a entender as mudanças conformacionais que as proteínas sofreram devido às interações com as moléculas. As técnicas computacionais de docagem e dinâmica molecular foram utilizadas para encontrar os prováveis sítios de interação e para descrever o microambiente de interação dos ligantes. A primeira etapa deste trabalho foi dedicada ao estudo da interação da piperina com as proteínas interleucina-1β (IL-1β), domínio NBD da proteína de choque térmico 70 (Hsp70) e albumina sérica de camundongo (RSA). Os resultados experimentais mostraram que a piperina se liga as proteínas espontaneamente e que o processo é entropicamente dirigido para as proteínas IL-1β e NBD e entalpicamente dirigido para a RSA. Além disto, os dados experimentais revelaram a constante de afinidade das interações, o número de sítios e o modo de interação (cooperativo ou não). Com as ferramentas de docagem molecular e dinâmica verificaram-se os sítios de interação, as interações envolvidas e a energia livre de ligação teórica. A segunda etapa deste trabalho foi dedicada à síntese e à caracterização de moléculas inspiradas na estrutura da piperina e ao estudo computacional dos efeitos das modificações estruturais na interação com as proteínas envolvidas nos processos inflamatórios IL-1β e NF-κB. O conjunto de resultados adquirido permitiu uma avaliação espectroscópica auxiliada por modelagem computacional que elucidou em detalhes as interações entre a piperina e derivados com as proteínas IL-1β, NBD e RSA que servirá como base para futuros estudos na linha de descoberta de fármacos.
The search of natural products with pharmacological properties is of utmost interest to the scientific community. Recent studies have shown that piperine, a natural metabolite extracted from several pepper species, presents anti-inflammatory properties, playing inhibitory effect on the activation of the NF-κB protein at several stages of the pathway, which causes the blocking of the transcription of cytokine genes and other pro-inflammatory proteins. From this perspective, the objective of this work was the biophysical characterization at the molecular level of piperine and its analogues with proteins involved in inflammatory process. The characterization of the protein-piperine complex was carried out applying fluorescence spectroscopy technique in order to determine the number of binding sites, the description of the thermodynamic parameters of interaction, as well as the binding constants of the complexes. In addition, circular dichroism was employed in order to understand the conformational changes that proteins experienced due to interactions with the small molecules. The computational techniques of molecular docking and dynamics were used to find the sites of interaction and to describe the microenvironment of interaction of the ligands. The first stage of this work was dedicated to the study of the interaction of piperine with the proteins interleukin-1β (IL-1β), NBD domain of heat shock protein 70 (Hsp70) and rat serum albumin (RSA). The experimental results revealed that piperine binds to proteins spontaneously and that the process was entropically driven in its interaction with IL-1β and NBD proteins and enthalpically driven in its interaction with RSA. In addition, the experimental data revealed the constant affinity of the interactions, the number of sites and the mode of interaction (either cooperative or non-cooperative). With molecular docking and dynamics analyses, the microenvironment of the interaction was disclosed and the theoretical free energy of binding was calculated. The second stage of this work was dedicated to the synthesis and characterization of piperine-inspired molecules and to the computational study of the effects of structural changes in the interaction with the proteins involved in the inflammatory processes: IL-1β and NF-κB. The set of results presented in this work elucidated in detail the interactions between piperine and pipeirne-inspired with the IL-1β, NBD and RSA proteins and it can shed light on future studies in drug discovery.
The search of natural products with pharmacological properties is of utmost interest to the scientific community. Recent studies have shown that piperine, a natural metabolite extracted from several pepper species, presents anti-inflammatory properties, playing inhibitory effect on the activation of the NF-κB protein at several stages of the pathway, which causes the blocking of the transcription of cytokine genes and other pro-inflammatory proteins. From this perspective, the objective of this work was the biophysical characterization at the molecular level of piperine and its analogues with proteins involved in inflammatory process. The characterization of the protein-piperine complex was carried out applying fluorescence spectroscopy technique in order to determine the number of binding sites, the description of the thermodynamic parameters of interaction, as well as the binding constants of the complexes. In addition, circular dichroism was employed in order to understand the conformational changes that proteins experienced due to interactions with the small molecules. The computational techniques of molecular docking and dynamics were used to find the sites of interaction and to describe the microenvironment of interaction of the ligands. The first stage of this work was dedicated to the study of the interaction of piperine with the proteins interleukin-1β (IL-1β), NBD domain of heat shock protein 70 (Hsp70) and rat serum albumin (RSA). The experimental results revealed that piperine binds to proteins spontaneously and that the process was entropically driven in its interaction with IL-1β and NBD proteins and enthalpically driven in its interaction with RSA. In addition, the experimental data revealed the constant affinity of the interactions, the number of sites and the mode of interaction (either cooperative or non-cooperative). With molecular docking and dynamics analyses, the microenvironment of the interaction was disclosed and the theoretical free energy of binding was calculated. The second stage of this work was dedicated to the synthesis and characterization of piperine-inspired molecules and to the computational study of the effects of structural changes in the interaction with the proteins involved in the inflammatory processes: IL-1β and NF-κB. The set of results presented in this work elucidated in detail the interactions between piperine and pipeirne-inspired with the IL-1β, NBD and RSA proteins and it can shed light on future studies in drug discovery.
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RSA, IL1β, HSP70/NBD, NF-κB, Piperina, Espectroscopia de fluorescência, Dicroísmo circular, Docagem molecular, Dinâmica molecular, Piperine, Fluorescence spectroscopy, Circular dichroism, Molecular docking, Molecular dynamics