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Atividade anti-inflamatória e anti-viral de bromoditerpenos isolados da alga Sphaerococcus coronopifolius
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Abstract(s)
A inflamação é uma resposta inata para manter a homeostasia do organismo, atuando como mecanismo de defesa contra diversos fatores, incluindo patógenos, células danificadas e compostos tóxicos. Contudo, quando essa reação é demasiado prolongada, pode causar problemas ao nível dos tecidos e órgãos, inclusive conduzir à morte. A COVID-19 é uma doença infeciosa causada pelo vírus SARS-CoV-2, do grupo dos coronavírus, que, frequentemente, se manifesta com sintomas de infeção respiratória aguda, com grande impacto na saúde e nas economias globais. O desenvolvimento de medicamentos neste contexto é complexo e vai desde a compreensão do mecanismo de ação até à avaliação da sua eficácia e toxicidade. Como tal, o desenvolvimento de agentes terapêuticos contra o vírus SARS-CoV-2, que mitiguem alguns efeitos da doença como a inflamação severa, é uma necessidade, já que a terapia convencional tem limitações, incluindo a possível redução da eficácia das vacinas contra as variantes com mutações em genes-chave. Neste contexto, os organismos marinhos constituem fontes excecionais de novas moléculas com potencial terapêutico, como os bromoditerpenos presentes na alga vermelha Sphaerococcus coronopifolius, os quais já demonstraram atividades anticancerígenas, antifouling e antimicrobianas. Contudo a sua capacidade anti-inflamatória ainda permanece desconhecida. Utilizando técnicas de cromatografia em coluna preparativa (CC) e semipreparativa
(HPLC) foi possível isolar quatro compostos desta alga. Através de análise por ressonância magnética nuclear de protão (1H RMN) foi possível identificar o sesquiterpeno Aloaromadendreno, e três bromoditerpenos, Sphaerococcenol A, Bromosphaerol e 12Rhidroxi-bromosphaerol. Para avaliar o potencial anti-inflamatório destes compostos, foi previamente realizado um teste de citotoxicidade em macrófagos RAW 264.7 (0,01 – 10 μM; 24 h) para definição das concentrações sub-tóxicas. Nenhum dos compostos estimulou a produção de
óxido nítrico (NO). Nos macrófagos RAW 264.7 tratados com lipopolissacarídeos (LPS), o Aloaromadendreno (1 μM) reduziu significativamente a produção de NO de 171,5 ± 8,9% para 111,0 ± 4,8%. Por sua vez, o Bromosphaerol (10 μM) foi o composto que exibiu a maior redução da concentração do fator de necrose tumoral (TNF-α) (637,5 ± 192 pg/mL) comparado com o tratamento com LPS (1209,0 ± 84,6 pg/mL). No que diz respeito, à interleucina-6 (IL-6), o Sphaerococcenol A (0,1 μM) reduziu significativamente os seus níveis (129,3 ± 31,3 pg/mL), quando comparado com o tratamento com LPS (862,7±39,9 pg/mL). Finalmente, o composto 12R-hidroxi-bromosphaerol (1μM) foi o único que demonstrou estimular significativamente a produção da citocina anti-inflamatória interleucina-10 (1301.0 ± 381,1 pg/mL). No que diz respeito à atividade antiviral, foi realizado um ensaio dinâmico in-silico, com recurso à ferramenta informática Dockthor, utilizando as estruturas (PDB:6LU7) da Mpro, proteína essencial no processo de replicação do SARS-COV-2 , (PDB: 6M0J) da Spike, e (PDB:1O9K), proteína essencial na entrada do vírus na células hospedeira, e da pRb, proteína essencial na eliminação da propagação de células cancerígenas O composto 12R-hidroxi-bromosphaerol demonstrou a maior afinidade, assim como estabilidade nas ligações com a proteína Mpro, com uma energia de afinidade de -8.1 (kcal/mol) e uma interação hidrofóbica com Cys145 (distância 5,35 Å). Por sua vez, o Bromosphaerol exibiu interação com a proteína Spike, apresentando uma energia de afinidade de -7.9 (kcal/mol). Relativamente à proteína pRb, o composto 12R-hidroxi-bromosphaerol foi o que exibiu uma
maior interação com uma energia de -8.4 (kcal/mol). No entanto o Sphaerococcenol A foi o único composto que interagiu com o sítio ativo da pRb, mediante uma ponte de hidrogénio com Arg467 (distância 2,58 Å).
Em suma, os compostos da alga vermelha Sphaerococcus coronopifolius, demonstram capacidade de mediar efeitos em diferentes biomarcadores relacionados com o processo inflamatório, assim como interagir com diferentes proteínas relevantes no processo de infeção do SARS-CoV2. Como tal, estes compostos poderão contribuir para inspirar o desenvolvimento de novos agentes terapêuticos para tratamento de doenças virais, com SARS-CoV2.
Inflammation is an innate response to maintain the body's homeostasis, acting as a defense mechanism against various factors, including pathogens, damaged cells, and toxic compounds. However, when this reaction is too prolonged, it can cause problems at the level of tissues and organs, including leading to death. COVID-19 is an infectious disease caused by the SARS-CoV-2 virus, from the coronavirus group, which often manifests itself with symptoms of acute respiratory infection, with a major impact on health and global economies. The development of medicines in this context is complex and ranges from understanding the mechanism of action to evaluating their efficacy and toxicity. Therefore, the development of therapeutic agents against the SARS-CoV-2 virus, which mitigate some effects of the disease such as severe inflammation, is mandatory since conventional therapy has limitations, including the possible reduction in the effectiveness of vaccines against the SARS-CoV-2 virus variants with mutations in key genes. In this context, marine organisms constitute exceptional sources of new molecules with therapeutic potential, such as the bromoditerpenes present in the red alga Sphaerococcus coronopifolius, which have already demonstrated anticancer, antifouling, and antimicrobial activities. However, its antiinflammatory capacity remains unknown. Using preparative (CC) and semi-preparative (HPLC) column chromatography techniques, it was possible to isolate four compounds from this alga. Through analysis by proton nuclear magnetic resonance (1H NMR) it was possible to identify the sesquiterpene Alloaromadendrene, and three bromoditerpenes, Sphaerococcenol A, Bromosphaerol and 12Rhydroxy-bromosphaerol. To evaluate the anti-inflammatory potential of these compounds, a cytotoxicity test was previously carried out on RAW 264.7 macrophages (0.01 – 10 μM; 24 h) to define sub-toxic concentrations. None of the compounds stimulated the production of nitric oxide (NO). In RAW 264.7 macrophages treated with lipopolysaccharides (LPS), Alloaromadendrene (1 μM) significantly reduced NO production from 171.5 ± 8.9% to 111.0 ± 4.8%. In turn, Bromosphaerol (10 μM) was the compound that exhibited the greatest reduction in the concentration of tumor necrosis factor (TNF-α) (637.5 ± 192 pg/mL) compared to treatment with LPS (1209.0 ± 84.6 pg/mL). Regarding interleukin-6 (IL-6), Sphaerococcenol A (0.1 μM) significantly reduced its levels (129.3 ± 31.3 pg/mL) when compared to treatment with LPS (862.7±39.9 pg/mL). Finally, the compound 12R-hydroxy-bromosphaerol (1μM) was the only one that was shown to significantly stimulate the production of the anti-inflammatory cytokine interleukin -10 (1301.0 ± 381.1 pg/mL). Concerning the antiviral activity, an in-silico dynamic assay was carried out through the Dockthor computer tool, using the structures (PDB:6LU7) of Mpro, an essential protein in the SARS-COV-2 replication process (PDB : 6M0J) of Spike, and (PDB:1O9K), an essential protein in the entry of the virus into host cells, and of pRb, an essential protein in eliminating the spread of cancer cells. The compound 12R-hydroxy-bromosphaerol demonstrated the highest affinity, as well as stability in bonds with the Mpro protein, with an affinity energy of - 8.1 (kcal/mol) and a hydrophobic interaction with Cys145 (distance 5.35 Å). In turn, Bromosphaerol exhibited interaction with the Spike protein, presenting an affinity energy of - 7.9 (kcal/mol). Regarding the pRb protein, the compound 12R-hydroxy-bromosphaerol exhibited the greatest interaction with an energy of -8.4 (kcal/mol). However, Sphaerococcenol A was the only compound that interacted with the active site of pRb, through a hydrogen bond with Arg467 (distance 2.58 Å). In short, compounds from the red alga Sphaerococcus coronopifolius demonstrate the ability to mediate effects on different biomarkers related to the inflammatory process, as well as to interact with different proteins relevant to the SARS-CoV2 infection process. Therefore, these compounds could help to inspire the development of new therapeutic agents for the treatment of viral diseases, such as SARS-CoV2.
Inflammation is an innate response to maintain the body's homeostasis, acting as a defense mechanism against various factors, including pathogens, damaged cells, and toxic compounds. However, when this reaction is too prolonged, it can cause problems at the level of tissues and organs, including leading to death. COVID-19 is an infectious disease caused by the SARS-CoV-2 virus, from the coronavirus group, which often manifests itself with symptoms of acute respiratory infection, with a major impact on health and global economies. The development of medicines in this context is complex and ranges from understanding the mechanism of action to evaluating their efficacy and toxicity. Therefore, the development of therapeutic agents against the SARS-CoV-2 virus, which mitigate some effects of the disease such as severe inflammation, is mandatory since conventional therapy has limitations, including the possible reduction in the effectiveness of vaccines against the SARS-CoV-2 virus variants with mutations in key genes. In this context, marine organisms constitute exceptional sources of new molecules with therapeutic potential, such as the bromoditerpenes present in the red alga Sphaerococcus coronopifolius, which have already demonstrated anticancer, antifouling, and antimicrobial activities. However, its antiinflammatory capacity remains unknown. Using preparative (CC) and semi-preparative (HPLC) column chromatography techniques, it was possible to isolate four compounds from this alga. Through analysis by proton nuclear magnetic resonance (1H NMR) it was possible to identify the sesquiterpene Alloaromadendrene, and three bromoditerpenes, Sphaerococcenol A, Bromosphaerol and 12Rhydroxy-bromosphaerol. To evaluate the anti-inflammatory potential of these compounds, a cytotoxicity test was previously carried out on RAW 264.7 macrophages (0.01 – 10 μM; 24 h) to define sub-toxic concentrations. None of the compounds stimulated the production of nitric oxide (NO). In RAW 264.7 macrophages treated with lipopolysaccharides (LPS), Alloaromadendrene (1 μM) significantly reduced NO production from 171.5 ± 8.9% to 111.0 ± 4.8%. In turn, Bromosphaerol (10 μM) was the compound that exhibited the greatest reduction in the concentration of tumor necrosis factor (TNF-α) (637.5 ± 192 pg/mL) compared to treatment with LPS (1209.0 ± 84.6 pg/mL). Regarding interleukin-6 (IL-6), Sphaerococcenol A (0.1 μM) significantly reduced its levels (129.3 ± 31.3 pg/mL) when compared to treatment with LPS (862.7±39.9 pg/mL). Finally, the compound 12R-hydroxy-bromosphaerol (1μM) was the only one that was shown to significantly stimulate the production of the anti-inflammatory cytokine interleukin -10 (1301.0 ± 381.1 pg/mL). Concerning the antiviral activity, an in-silico dynamic assay was carried out through the Dockthor computer tool, using the structures (PDB:6LU7) of Mpro, an essential protein in the SARS-COV-2 replication process (PDB : 6M0J) of Spike, and (PDB:1O9K), an essential protein in the entry of the virus into host cells, and of pRb, an essential protein in eliminating the spread of cancer cells. The compound 12R-hydroxy-bromosphaerol demonstrated the highest affinity, as well as stability in bonds with the Mpro protein, with an affinity energy of - 8.1 (kcal/mol) and a hydrophobic interaction with Cys145 (distance 5.35 Å). In turn, Bromosphaerol exhibited interaction with the Spike protein, presenting an affinity energy of - 7.9 (kcal/mol). Regarding the pRb protein, the compound 12R-hydroxy-bromosphaerol exhibited the greatest interaction with an energy of -8.4 (kcal/mol). However, Sphaerococcenol A was the only compound that interacted with the active site of pRb, through a hydrogen bond with Arg467 (distance 2.58 Å). In short, compounds from the red alga Sphaerococcus coronopifolius demonstrate the ability to mediate effects on different biomarkers related to the inflammatory process, as well as to interact with different proteins relevant to the SARS-CoV2 infection process. Therefore, these compounds could help to inspire the development of new therapeutic agents for the treatment of viral diseases, such as SARS-CoV2.
Description
À Fundação para a Ciência e a Tecnologia (FCT) pelo financiamento através dos projetos estratégicos concedidos ao MARE – Centro de Ciências do Mar e do Ambiente (UIDP/04292/2020 e UIDB/04292/2020), Laboratório ssociado ARNET (LA/P/0069/2020), e projeto de investigação “NEURONS4 – New edge in the therapeutics of Parkinson’s disease from Seaweeds” (2022.09196.PTDC; https://doi.org/10.54499/2022.09196.PTDC). Ao projeto “BEAP – MAR – Blue Bioeconomy in the atlantic Area: New Products from Marine Organims (EAPA_0032/2022)” financiado pelo programa Europeu INTERREG Espaço Atlântico.
Keywords
Anti-inflamatório Anti-viral Sphaerococcus coronopifolius Bromoditerpenos SARS-CoV2