Caracterização funcional, estrutural e modificação racional da ASNaseM: Um novo fármaco para o tratamento da Leucemia Linfoide Aguda?
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Data
2019-04-10
Autores
Schultz, Leonardo [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
L-asparaginases (ASNases) bacterianas são importantes biofármacos utilizados no tratamento de leucemia linfoide aguda (LLA), uma vez que este tipo tumoral é dependente da disponibilidade de asparagina (Asn) extracelular. As ASNases bacterianas são capazes de hidrolisar eficientemente Asn em ácido aspártico (Asp) e amônia (NH3), diminuindo a disponibilidade de Asn para células tumorais e induzindo apoptose. Comercialmente, indústrias farmacêuticas internacionais produzem ASNases de Escherichia coli e Erwinia chrysanthemi, entretanto, ASNase de nenhuma origem é produzida pelas indústrias farmacêuticas brasileiras. Adicionalmente, o tratamento com estas enzimas produz efeitos colaterais, entre eles imunogênicos, que estão relacionados com a alta massa molecular da enzima (140kDa) e neurológicos, atribuídos a atividade secundária de glutaminase (GLNase). Neste contexto, fontes alternativas destas enzimas e também a auto-suficiência em suas produções são importantes para mitigar os efeitos colaterais e evitar falhas no tratamento devido a flutuações internacionais de sua fabricação. Neste trabalho, realizamos a caracterização de uma ASNase de levedura, denominada de ASNaseM que compartilha elevada homologia (maior que 30% de identidade e 40% de similaridade) com as enzimas bacterianas utilizadas no tratamento da LLA e que possui todos os aminoácidos envolvidos na catálise conservados, sugerindo uma fonte alternativa potencial para o tratamento da LLA. Experimentos de cromatografia de exclusão molecular revelaram uma enzima com tempo de retenção referente a um monômero (~45 kDa), e através de espectroscopia de dicroísmo circular, foi possível observar que ASNaseM é termorresistente, mantendo sua estrutura secundária em temperaturas maiores que 40ºC, apresentando Tm = 54,35 ± 0,37°C, o que pode ser resultado das 4 pontes de dissulfetos formados por 8 de 10 cisteínas, como observado pelo ensaio de quantificação de grupos sulfidrilas livres utilizando o reagente DTNB. Os parâmetros cinéticos foram determinados por ensaio espectrofotométrico acoplado a glutamato desidrogenase e a enzima apresentou comportamento alostérico com coeficiente de Hill = 2,125 e S0.5 ~ 0,35 mM e a eficiência catalítica foi na ordem de 101 M-1s-1 e atividade específica de aproximadamente 108 UI/mg. Adicionalmente, uma característica que está relacionada com diversos efeitos colaterais, observada nas enzimas comerciais, que é a atividade secundária de GLNase, não foi detectada para ASNaseM. Através de ensaios de citotoxicidade utilizando as linhagens ReH e Molt-4 (células neoplásicas), foi possível detectar atividade citotóxica. Também foram realizadas mutações sítio dirigidas visando conferir à ASNaseM a atividade de GLNase. Também buscamos o ganho de atividade de GLNase por meio de substituições pontuais de amnioacidos que conferem ou abolem em bactérias esta atividade, entretanto, nossos resultados revelaram que as enzimas mutadas não apresentaram ganho de atividade de GLNase, o que indica que apesar das semelhanças existentes com as enzimas bacterianas, o ganho de atividade de GLNase é um fenômeno complexo e necessita de investigações adicionais. Também realizamos análises in silico de sequências primárias de ASNases, em conjunto com a avaliação de estruturas tridimensionais, para ampliar o conhecimento de aspectos evolutivos destas enzimas e propor uma classificação criteriosa de ASNases. Os resultados da análise revelam que a ASNase de levedura, se posiciona evolutivamente distinta de outras enzimas e também auxiliaram na identificação de ASNases que podem possuir propriedades importantes como biofármacos alternativos apresentando alta atividade de ASNase ou ausência de atividade de GLNase.
Bacterial L-asparaginases (ASNases) are important biopharmaceuticals used in the treatment of acute lymphoid leukemia (ALL), since this tumor type is dependent on the availability of extracellular asparagine (Asn). Bacterial ASNases are able to efficiently hydrolyze Asn in aspartic acid (Asp) and ammonia (NH3), decreasing the availability of Asn to tumor cells and inducing apoptosis. Commercially, international pharmaceutical industries produce ASNases from Escherichia coli and Erwinia chrysanthemi, however none ASNase is produced by the Brazilian pharmaceutical companies. Additionally, the treatment with these enzymes can produce side effects, among them immunogenic ones, that are related to the high molecular weight of the enzyme (140kDa) and neurological, attributed to the glutaminase secondary activity (GLNase), being glutamine (Gln) the most abundant amino acid in the bloodstream. In this context, alternative sources of these enzymes as also the self-sufficiency of the production are important to mitigate side effects and avoid treatment failures due to international fluctuations in their manufacture. In this work, we performed the characterization of a yeast ASNase, called ASNaseM, which shares high homology (higher than 30% identity and 40% similarity) with the bacterial enzymes used in the treatment of ALL, and which has all the amino acids involved in the catalysis conserved, suggesting a potential alternative source for the treatment of ALL. Molecular exclusion chromatography experiments revealed an enzyme with retention time for a monomer (~ 45 kDa), and through circular dichroism spectroscopy, it was possible to observe that ASNaseM is heat-resistant, maintaining its secondary structure at temperatures higher than 40ºC, presenting Tm = 54.35 ± 0.37 °C, which may be the result of 4 disulfide bonds formed by 8 of 10 cysteines, as indicated by the free sulfhydryl group quantification assay using the DTNB reagent. The kinetic parameters were determined by spectrophotometric assay coupled to glutamate dehydrogenase, the enzyme presented allosteric behavior with Hill coefficient of 2.125 and S0.5 of ~ 0.35 mM and the catalytic efficiency was in the order of 101 M-1S-1 and activity specific of approximately 108 UI/mg. Additionally, a characteristic that is related to several side effects of the bacterial ASNases, observed in the commercial enzymes, the secondary GLNase activity was not detected to ASNaseM. Through cytotoxicity assays using ReH and Molt-4 (neoplastic cells) strains, it was possible to observe cytotoxic activity to neoplastic cells. Site-directed mutations were also performed to confer GLNase activity to ASNaseM based in known substitutions that confers or abolish this kind of activity to the bacterial enzymes. Our results revealed that the mutated enzymes showed no gain in GLNase activity, indicating that despite the similarities to the bacterial enzymes, gain of GLNase activity is a complex phenomenon and requires further investigation. In addition, we performed "in silico" analyzes of ASNase primary sequences, together with the evaluation of three-dimensional structures, to increase the knowledge of evolutionary aspects of these enzymes and propose a careful classification of ASNases. The results of the analysis reveal that yeast ASNase is evolutionarily distinct from other enzymes and also aided in the identification of ASNases that may possess important properties as alternative biopharmaceuticals having high ASNase activity or absence of GLNase activity.
Bacterial L-asparaginases (ASNases) are important biopharmaceuticals used in the treatment of acute lymphoid leukemia (ALL), since this tumor type is dependent on the availability of extracellular asparagine (Asn). Bacterial ASNases are able to efficiently hydrolyze Asn in aspartic acid (Asp) and ammonia (NH3), decreasing the availability of Asn to tumor cells and inducing apoptosis. Commercially, international pharmaceutical industries produce ASNases from Escherichia coli and Erwinia chrysanthemi, however none ASNase is produced by the Brazilian pharmaceutical companies. Additionally, the treatment with these enzymes can produce side effects, among them immunogenic ones, that are related to the high molecular weight of the enzyme (140kDa) and neurological, attributed to the glutaminase secondary activity (GLNase), being glutamine (Gln) the most abundant amino acid in the bloodstream. In this context, alternative sources of these enzymes as also the self-sufficiency of the production are important to mitigate side effects and avoid treatment failures due to international fluctuations in their manufacture. In this work, we performed the characterization of a yeast ASNase, called ASNaseM, which shares high homology (higher than 30% identity and 40% similarity) with the bacterial enzymes used in the treatment of ALL, and which has all the amino acids involved in the catalysis conserved, suggesting a potential alternative source for the treatment of ALL. Molecular exclusion chromatography experiments revealed an enzyme with retention time for a monomer (~ 45 kDa), and through circular dichroism spectroscopy, it was possible to observe that ASNaseM is heat-resistant, maintaining its secondary structure at temperatures higher than 40ºC, presenting Tm = 54.35 ± 0.37 °C, which may be the result of 4 disulfide bonds formed by 8 of 10 cysteines, as indicated by the free sulfhydryl group quantification assay using the DTNB reagent. The kinetic parameters were determined by spectrophotometric assay coupled to glutamate dehydrogenase, the enzyme presented allosteric behavior with Hill coefficient of 2.125 and S0.5 of ~ 0.35 mM and the catalytic efficiency was in the order of 101 M-1S-1 and activity specific of approximately 108 UI/mg. Additionally, a characteristic that is related to several side effects of the bacterial ASNases, observed in the commercial enzymes, the secondary GLNase activity was not detected to ASNaseM. Through cytotoxicity assays using ReH and Molt-4 (neoplastic cells) strains, it was possible to observe cytotoxic activity to neoplastic cells. Site-directed mutations were also performed to confer GLNase activity to ASNaseM based in known substitutions that confers or abolish this kind of activity to the bacterial enzymes. Our results revealed that the mutated enzymes showed no gain in GLNase activity, indicating that despite the similarities to the bacterial enzymes, gain of GLNase activity is a complex phenomenon and requires further investigation. In addition, we performed "in silico" analyzes of ASNase primary sequences, together with the evaluation of three-dimensional structures, to increase the knowledge of evolutionary aspects of these enzymes and propose a careful classification of ASNases. The results of the analysis reveal that yeast ASNase is evolutionarily distinct from other enzymes and also aided in the identification of ASNases that may possess important properties as alternative biopharmaceuticals having high ASNase activity or absence of GLNase activity.
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Palavras-chave
Leucemia linfoblástica aguda, Asparaginase, Biofarmacêutico, Acute Lymphoblastic Leukemia, Asparaginase, Biopharmaceutical