Evaluation of Rhamnetin as an Inhibitor of the Pharmacological Effect of Secretory Phospholipase A2

Rhamnetin (Rhm), 3-O-methylquercetin (3MQ), and Rhamnazin (Rhz) are methylated derivatives of quercetin commonly found in fruits and vegetables that possess antioxidant and anti-inflammatory properties. Phospholipase A2 (PLA2) displays several important roles during acute inflammationtherefore, this study aimed at investigating new compounds able to inhibit this enzyme, besides evaluating creatine kinase (CK) levels and citotoxicity. Methylated quercetins were compared with quercetin (Q) and were incubated with secretory PLA2 (sPLA2) from Bothrops jararacussu to determine their inhibitory activity. Cytotoxic studies were performed by using the J774 cell lineage incubated with quercertins. In vivo tests were performed with Swiss female mice to evaluate decreasing paw edema potential and compounds' CK levels. Structural modifications on sPLA2 were made with circular dichroism (CD). Despite Q and Rhz showing greater enzymatic inhibitory potential, high CK was observed. Rhm exhibited sPLA2 inhibitory potential, no toxicity and, remarkably, it decreased CK levels. The presence of 3OH on the C-ring of Rhm may contribute to both its anti-inflammatory and enzymatic inhibition of sPLA2, and the methylation of ring A may provide the increase in cell viability and low CK level induced by sPLA2. These results showed that Rhm can be a candidate as a natural compound for the development of new anti-inflammatory drugs.Universidade Estadual Paulista (UNESP)Universidade Federal de São Paulo (UNIFESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Fed São Paulo, Postgrad Program Food Nutr & Hlth, BR-11015020 São Paulo, BrazilUniv Estadual Paulista, Biosci Inst, BR-11330900 São Paulo, BrazilBrazil Univ, Prorector Res, BR-08230030 São Paulo, BrazilUniv São Paulo, Pathol Lab Infect Dis LIM50, Dept Pathol, Sch Med, BR-01246903 São Paulo, BrazilUniv Fed São Paulo, Postgrad Program Food Nutr & Hlth, BR-11015020 São Paulo, BrazilWeb of Scienc

Avaliação Do Inibidor De Peroxirredoxina 1 E Derivados De Quercetina Sobre As Atividades Farmacológicas Induzidas Pela Spla2 De Bothrops Jararacussu

The present work evaluated the relationship between the structure and function of four quercetins (3-O-Methylquercetin, Rhamnetin, Rhamnazin and Quercetin) in relation to the inhibitory activity of sPLA2 from Bothrops jararacussu. Among the flavonoids evaluated, Rhamnetin revealed inhibitory activity in vitro, in vivo, in addition to inhibiting creatine kinase (CK) and not showing cytotoxicity against J774 macrophages. The presence of methylation at the C-7 position of ring A seems to be essential for cell protection activity. In addition, the molecule has all the essential characteristics to maintenance of anti-inflammatory activity, such as the presence of catechol group, the double bond in ring C (C-2 and C-3), and the presence of hydroxyl in C-3 of the C-ring. In order to correlate inflammatory process with oxidative stress, as well as the relevance of peroxiredoxins in this process, Adenantin inhibitor was administered 60 minutes prior to application of sPLA2. Results showed that the inhibition of Prx 1 and 2 extended edematogenic process, besides leads an increase in serum CK levels, which shows an increase in muscle damage. Thus, knowing the correlation between inflammation and oxidative stress, this study reveals the importance of new anti- inflammatory compounds’ discovery as new therapeutic targets or prevention of inflammatory and oxidative processes. In addition, it emphasizes the role of the Prx in the protection of oxidative stress caused by acute inflammatory process.Este trabalho avaliou a relação entre a estrutura e função de quatro quercetinas (3-O- Metilquercetina, Ramnetina, Ramnazina e Quercetina) em relação a atividade inibitória da sPLA2 de Bothrops jararacussu. Dentre os flavonoides avaliados, a Ramnetina revelou atividade inibitória in vitro, in vivo, além de inibir a creatina quinase (CK) e não mostrar citotoxicidade contra macrófagos J774. A presença da metilação na posição C-7 do anel A, mostrou-se fundamental para a atividade de proteção celular. Além disso, a molécula apresenta todas as características essenciais para a manutenção da atividade anti-inflamatória, como a presença do grupo catecol, a dupla ligação no anel C (C-2 e C-3), e a presença da hidroxila no C-3 do anel C. A fim de correlacionar o processo inflamatório com o estresse oxidativo, bem como a importância das peroxirredoxinas neste processo, o inibidor Adenantina foi administrado 60 minutos antes da aplicação da sPLA2. Os resultados mostraram que a inibição das Prx 1 e 2, prolongou o processo edematogênico, além de causar um aumento nos níveis de Ck séricos, o que demonstra aumento da lesão muscular. Deste modo, sabendo a correlação entre a inflamação e o estresse oxidativo, este estudo revela a importância da descoberta de novos compostos anti-inflamatórios, como novos alvos terapêuticos ou prevenção de processos inflamatórios e oxidativos. Além disso, enfatiza a importância das Prx na proteção ao estresse oxidativo provocado pelo processo inflamatório agudo.Dados abertos - Sucupira - Teses e dissertações (2017

Edema Induced by a Crotalus durissus terrificus Venom Serine Protease (Cdtsp 2) Involves the PAR Pathway and PKC and PLC Activation

Snake venom serine proteases (SVSPs) represent an essential group of enzymatic toxins involved in several pathophysiological effects on blood homeostasis. Some findings suggest the involvement of this class of enzymatic toxins in inflammation. In this paper, we purified and isolated a new gyroxin isoform from the Crotalus durissus terrificus (Cdt) venom, designated as Cdtsp 2, which showed significant proinflammatory effects in a murine model. In addition, we performed several studies to elucidate the main pathway underlying the edematogenic effect induced by Cdtsp 2. Enzymatic assays and structural analysis (primary structure analysis and three-dimensional modeling) were closely performed with pharmacological assays. The determination of edematogenic activity was performed using Cdtsp 2 isolated from snake venom, and was applied to mice treated with protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, dexamethasone (Dexa), antagonists for protease-activated receptors (PARs), or saline (negative control). Additionally, we measured the levels of cyclooxygenase 2 (COX-2), malondialdehyde (MDA), and prostaglandin E2 (PGE2). Cdtsp 2 is characterized by an approximate molecular mass of 27 kDa, an isoelectric point (pI) of 4.5, and significant fibrinolytic activity, as well as the ability to hydrolyze Nα-benzoyl-l-arginine 4-nitroanilide (BAPNA). Its primary and three-dimensional structures revealed Cdtsp 2 as a typical snake venom serine protease that induces significant edema via the metabolism of arachidonic acid (AA), involving PARs, PKC, PLC, and COX-2 receptors, as well as inducing a significant increase in MDA levels. Our results showed that Cdtsp 2 is a serine protease with significant enzymatic activity, and it may be involved in the degradation of PAR1 and PAR2, which activate PLC and PKC to mobilize AA, while increasing oxidative stress. In this article, we provide a new perspective for the role of SVSPs beyond their effects on blood homeostasis

“Biotecnological war” uma ferramenta de avaliação conceitual e de percepção para o ensino de biotecnologia e química de proteínas para os alunos de graduação em ciências biológicas

Biochemistry in general is practically unanimous as a discipline with a high degree of difficulty, complex and "boring". So, practical and creative play games as teaching methodology has been disseminated in several disciplines of biological sciences. “Biotecnological war” board game is a proposal that was initially conceived as an alternative complementary tool for biochemistry teaching of proteins and peptides, challenging students, aiming to review concepts transmitted in classroom, stimulating student’s abilities, such as their creativity, competitiveness, resource management and making possible to correlate biochemistry importance of proteins and peptides as new products. This game proved to be an excellent tool for complementary evaluation of students, which besides stimulating teamwork, also stimulated "a strong competitive spirit" within the classroom, which allowed to analyze students' perception in relation to the theme and mainly articulated group work.A bioquímica no geral é taxada como uma disciplina com alto grau de dificuldade, complexa e “chata”. Por outro lado, a aplicação de jogos lúdicos práticos e criativos como metodologia de ensino vem se disseminando em várias disciplinas em ciências biológicas. O jogo de tabuleiro “Biotecnological war” é uma proposta pensada como uma ferramenta complementar para o ensino de bioquímica de proteínas e peptídeos, desafiando os discentes, visando rever conceitos transmitidos em sala de aula, e estimulando aptidões dos estudantes, como sua criatividade, competitividade e gestão de recursos. Possibilitando ainda correlacionar a importância da bioquímica de proteínas e peptídeos com o desenvolvimento de produtos. Este jogo se mostrou uma excelente ferramenta de avaliação complementar dos alunos, e além de estimular o trabalho em equipe, também estimulou “um forte espírito competitivo”, o que permitiu analisar a percepção dos alunos em relação ao tema e principalmente o trabalho em grupo

In Silico Evaluation of Quercetin Methylated Derivatives on the Interaction with Secretory Phospholipases A2 from <i>Crotalus durissus terrificus</i> and <i>Bothrops jararacussu</i>

Quercetin derivatives have already shown their anti-inflammatory potential, inhibiting essential enzymes involved in this process. Among diverse pro-inflammatory toxins from snake venoms, phospholipase A2 is one of the most abundant in some species, such as Crotalus durissus terrificus and Bothrops jararacussu from the Viperidae family. These enzymes can induce the inflammatory process through hydrolysis at the sn-2 position of glycerophospholipids. Hence, elucidating the main residues involved in the biological effects of these macromolecules can help to identify potential compounds with inhibitory activity. In silico tools were used in this study to evaluate the potential of quercetin methylated derivatives in the inhibition of bothropstoxin I (BthTX-I) and II (BthTX-II) from Bothrops jararacussu and phospholipase A2 from Crotalus durissus terrificus. The use of a transitional analogous and two classical inhibitors of phospholipase A2 guided this work to find the role of residues involved in the phospholipid anchoring and the subsequent development of the inflammatory process. First, main cavities were studied, revealing the best regions to be inhibited by a compound. Focusing on these regions, molecular docking assays were made to show main interactions between each compound. Results reveal that analogue and inhibitors, Varespladib (Var) and p-bromophenacyl bromide (BPB), guided quercetins derivatives analysis, revealing that Leu2, Phe5, Tyr28, glycine in the calcium-binding loop, His48, Asp49 of BthTX-II and Cdtspla2 were the main residues to be inhibited. 3MQ exhibited great interaction with the active site, similar to Var results, while Q anchored better in the BthTX-II active site. However, strong interactions in the C-terminal region, highlighting His120, seem to be crucial to decreasing contacts with phospholipid and BthTX-II. Hence, quercetin derivatives anchor differently with each toxin and further in vitro and in vivo studies are essential to elucidate these data

Gallic Acid as a Non-Selective Inhibitor of &alpha;/&beta;-Hydrolase Fold Enzymes Involved in the Inflammatory Process: The Two Sides of the Same Coin

(1) Background: Gallic acid (GA) has been characterized as an effective anti-inflammatory, antivenom, and promising drug for therapeutic use. (2/3) Methods and Results: GA was identified from ethanolic extract of fresh pitanga (Eugenia uniflora) leaves, which was identified using commercial GA. Commercial GA neutralized the enzymatic activity of secretory PLA2 (sPLA2) by inhibiting the active site and inducing changes in the secondary structure of the enzyme. Pharmacological edema assays showed that GA strongly decreased edema when the compound was previously incubated with sPLA2. However, prior treatment of GA (30 min before) significantly increased the edema and myotoxicity induced by sPLA2. The molecular docking results of GA with platelet-acetylhydrolase (PAF-AH) and acetylcholinesterase reveal that this compound was able to interact with the active site of both molecules, inhibiting the hydrolysis of platelet-activating factor (PAF) and acetylcholine (ACh). (4) Conclusion: GA has a great potential application; however, our results show that this compound can also induce adverse effects in previously treated animals. Additionally, the increased edema and myotoxicity observed experimentally in GA-treated animals may be due to the inhibition of PAF-AH and Acetylcholinesterase