Avaliação do pH como efeito modulador da interação entre a proteína Grb2 monomérica e a molécula de cumarina (1,2-benzopirona)
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Data
2022-11-21
Autores
Moreira, Giovana Casteluci
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Universidade Estadual Paulista (Unesp)
Resumo
O câncer é uma das principais causas de morte na atualidade e se manifesta através da proliferação acelerada e descontrolada de células. Uma das principais vias de sinalização ativadas para o desenvolvimento da doença é a via da MAPK/ERK, responsável pelos processos de proliferação e diferenciação celular. O reconhecimento de fatores de crescimento por proteínas de membrana, como a FGFR2, recruta a proteína Grb2, que atua como uma ponte entre a proteína quinase e outras proteínas que ativam a proteína ERK, responsável por ativar fatores de transcrição no núcleo. Apesar de ser classificada como proteína adaptadora, a Grb2 regula a via da MAPK/ERK através de um equilíbrio monômero-dímero, em que o monômero permite a transdução de sinal e o dímero inibe esse processo. Além disso, a Grb2 é capaz de inibir a FGFR2 – que está relacionada com diversos tipos de cânceres, como o câncer de estômago, pele e mama. Desta maneira, a Grb2 é um alvo promissor para o desenvolvimento de terapias antitumorais. Um dos principais desafios da medicina moderna é o desenvolvimento de medicamentos e estratégias que sejam cada vez menos adversas aos pacientes e, nesse contexto, as moléculas de origem natural têm ganhado destaque no meio científico, devido sua grande biodisponibilidade e inúmeras propriedades benéficas para o ser humano. Devido à pouca literatura disponível a respeito de mecanismos moleculares de interação entre Grb2 e moléculas de origem natural, neste trabalho nós realizamos a caracterização dos mecanismos de interação entre o monômero de Grb2 e a molécula de cumarina (1,2-benzopirona), que possui atividade anticarcinogênica e inibe a proliferação celular. Os estudos biofísicos foram conduzidos em duas condições de pH (7,0 e 8,0) por se aproximarem do pH intracelular de células saudáveis e cancerosas, respectivamente. Os resultados da caracterização estrutural indicam que o monômero é mais flexível e estável em pH 8,0 do que em pH 7,0. A interação entre a proteína e a molécula foi classificada como favorável e hidrofóbica (ΔG < 0; ΔH e ΔS > 0), com constantes de afinidade (Ka) moderadas e da ordem de 104 M-1 para o pH 7,0 e 105 M-1 para o pH 8,0. A combinação dos resultados de STD-RMN e análises computacionais mostraram que a molécula de cumarina interage com o sítio de interação por meio de encaixes distintos, comprovando que mudanças conformacionais induzidas pelo aumento de pH modulam o mecanismo de interação de Grb2 com cumarina. Além disso, foi verificado que a estabilidade estrutural do monômero se mostrou dependente da concentração de cumarina, sendo fortemente perturbada na proporção de [2:1] de cumarina:Grb2. A partir desses resultados, concluímos que a conformação estrutural da Grb2 é sensível às mudanças de pH e isso pode influenciar na interação desta proteína com outras moléculas e proteínas parceiras. Estes resultados, se levados em consideração, podem contribuir para o desenvolvimento de medicamentos e tratamentos, contra o câncer, e baseados em Grb2, mais eficazes.
Cancer is one of the leading causes of death today and is manifested through accelerated and uncontrolled cell proliferation. One of the main signaling pathways involved in the development of the disease is the MAPK/ERK pathway, which is responsible for cell proliferation and differentiation processes. Recognition of growth factors by membrane proteins such as FGFR2 recruits the Grb2 protein, which acts as a bridge between the protein kinase and other proteins that activate ERK, responsible for activating transcription factors in the nucleus. Despite being classified as an adapter protein, Grb2 regulates the MAPK/ERK pathway through a monomer-dimer equilibrium, in which the monomer activates the signaling pathway and the dimer inhibits FGFR2 – which is related to several cancers, such as stomach, skin, and breast cancer. Thus, Grb2 is a promising target for the development of antitumor therapies due to its role in regulating cell signaling pathways related to cancer development. One of the main challenges of modern medicine is the development of drugs and strategies that are less and less adverse to patients. In this context, molecules of natural origin have gained prominence in the scientific community due to their excellent bioavailability and numerous beneficial properties for humans. Due to the limited literature available about the molecular mechanisms of interaction between Grb2 and molecules of natural origin, we carried out in this work the characterization of the mechanisms of interaction between the Grb2 monomer and the coumarin molecule (1,2-benzopyrone), which has anticarcinogenic activity and inhibits cell proliferation. The biophysical studies were conducted under two pH conditions (7.0 and 8.0) as they approximate the intracellular pH of healthy and cancer cells, respectively. Structural characterization results indicate that the Grb2 monomer is more flexible and stable at pH 8.0 when compared to pH 7.0. The interaction between the protein and the molecule was classified as favorable and hydrophobic (ΔG < 0; ΔH and ΔS > 0), with moderate binding constants (Ka) of the order of 104 M-1 for pH 7.0 and 105 M-1 for pH 8.0. The combination of STD-NMR results and computational analyses identified two fits of the coumarin molecule within the interaction site, proving conformational changes induced by increasing the pH modulate the interaction mechanism of Grb2 with coumarin. It was also found that the structural stability of the monomer was dependent on the concentration of coumarin, being strongly disturbed in the proportion of [2:1] coumarin:Grb2. From these results, we conclude that the structural conformation of Grb2 is sensitive to changes in pH, which may influence the interaction of this protein with other molecules and partner proteins. If considered, these results may contribute to developing more effective Grb2-based drugs and treatments against cancer.
Cancer is one of the leading causes of death today and is manifested through accelerated and uncontrolled cell proliferation. One of the main signaling pathways involved in the development of the disease is the MAPK/ERK pathway, which is responsible for cell proliferation and differentiation processes. Recognition of growth factors by membrane proteins such as FGFR2 recruits the Grb2 protein, which acts as a bridge between the protein kinase and other proteins that activate ERK, responsible for activating transcription factors in the nucleus. Despite being classified as an adapter protein, Grb2 regulates the MAPK/ERK pathway through a monomer-dimer equilibrium, in which the monomer activates the signaling pathway and the dimer inhibits FGFR2 – which is related to several cancers, such as stomach, skin, and breast cancer. Thus, Grb2 is a promising target for the development of antitumor therapies due to its role in regulating cell signaling pathways related to cancer development. One of the main challenges of modern medicine is the development of drugs and strategies that are less and less adverse to patients. In this context, molecules of natural origin have gained prominence in the scientific community due to their excellent bioavailability and numerous beneficial properties for humans. Due to the limited literature available about the molecular mechanisms of interaction between Grb2 and molecules of natural origin, we carried out in this work the characterization of the mechanisms of interaction between the Grb2 monomer and the coumarin molecule (1,2-benzopyrone), which has anticarcinogenic activity and inhibits cell proliferation. The biophysical studies were conducted under two pH conditions (7.0 and 8.0) as they approximate the intracellular pH of healthy and cancer cells, respectively. Structural characterization results indicate that the Grb2 monomer is more flexible and stable at pH 8.0 when compared to pH 7.0. The interaction between the protein and the molecule was classified as favorable and hydrophobic (ΔG < 0; ΔH and ΔS > 0), with moderate binding constants (Ka) of the order of 104 M-1 for pH 7.0 and 105 M-1 for pH 8.0. The combination of STD-NMR results and computational analyses identified two fits of the coumarin molecule within the interaction site, proving conformational changes induced by increasing the pH modulate the interaction mechanism of Grb2 with coumarin. It was also found that the structural stability of the monomer was dependent on the concentration of coumarin, being strongly disturbed in the proportion of [2:1] coumarin:Grb2. From these results, we conclude that the structural conformation of Grb2 is sensitive to changes in pH, which may influence the interaction of this protein with other molecules and partner proteins. If considered, these results may contribute to developing more effective Grb2-based drugs and treatments against cancer.
Descrição
Palavras-chave
Grb2, Cumarina, Interação proteína-ligante, Modulação estrutural por pH, Coumarin, Structural characterization, Protein-ligand interaction, pH-induced structural modulation