Identificação e caracterização de derivados furoxanos e seus mecanismos de ação para o tratamento oral da leishmaniose visceral e malária: uma contribuição para a agenda 2030-UNESP dentro dos objetivos de desenvolvimento sustentável da Organização das Nações Unidas
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2022-06-15
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Universidade Estadual Paulista (Unesp)
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As doenças negligenciadas representam um grande desafio para políticas em saúde pública, sendo as leishmanioses e a malária duas importantes doenças parasitárias, por acometerem um mesmo perfil populacional e necessitarem de novos agentes terapêuticos para somarem ao portfólio de quimioterápicos atualmente em uso. Considerando os objetivos de Desenvolvimento Sustentável proposto pela ONU, o qual estabelece uma agenda global que propicie o desenvolvimento sustentável como uma perspectiva de construção de um futuro comum, pautado nas Pessoas, Prosperidade, Planeta, Paz e Parcerias. Dentro deste contexto, nosso grupo de pesquisa vem estudando novas moléculas promissoras para o tratamento de doenças parasitárias, contribuindo para o tema Saúde e Bem-Estar que visa o desenvolvimento de pesquisas direcionadas ao entendimento e ações de prevenção, terapia e controle de doenças transmissíveis e não transmissíveis, desenvolvimento e avaliação de novos fármacos e medicamentos. Assim, ao longo do desenvolvimento desta pesquisa, furoxanos (4a-o) e benzofuroxanos (4p-x) foram caracterizados quanto ao potencial anti-amastigota para Leishmania infantum, identificando os furoxanos doadores de NO (4a-h, 4l-m) e o benzofuroxano 4r como os mais potentes (EC50 < 10 μM) e seletivos (SI > 10). Após clonagem do gene e expressão da correspondente enzima cisteíno protease de Leishmania mexicana LmCPB2.8ΔCTE (CPB 2.8) em Pichia pastoris, verificou-se que os furoxanos (4a-n), benzofuroxanos (4q-r e 4t-u) e N-acil-hidrazonas (14a-g) apresentam boa capacidade de inibir a protease parasitária (0,8 < IC50 < 14 μM). Como prova de conceito, destaca-se que a atividade leishmanicida dos compostos pode estar relacionada a um efeito dual provocado pela atividade microbicida do NO, associada à inibição da enzima, visto que (14a-g) inibem CPB2.8 (2 < IC50 < 12 μM), mas não apresentam atividade anti-amastigota na máxima concentração testada (10 μM). A análise in silico de parâmetros farmacocinéticos indicou 4f para avaliação quanto ao seu potencial terapêutico frente a camundongos Balb/c infectados com L. infantum. Verificou-se que a administração oral duas vezes ao dia com uma dose de 7.7 mg/kg/dia, foi capaz de reduzir em 90% a carga parasitária no fígado e baço, sem causar danos hepáticos ou renais, finalizando assim o trabalho desenvolvido para L. infantum. Todas as moléculas foram posteriormente avaliadas frente a formas intra-eritrocitárias de Plasmodium falciparum geneticamente modificados para expressar o indicador de Ca2+ GCaMP3, mediante estágio sanduíche na New Jersey Medical School, Rutgers, Newark – EUA. Dentre os furoxanos, 4m (EC50 2,8 μM) foi o mais promissor, enquanto 4r foi o benzofuroxano mais ativo, apresentando inibição em torno de 40% do crescimento parasitário. Estudos de mecanismos de ação mostraram que 4m não é capaz de alterar a morfologia do vacúolo digestivo, onde está localizada a cisteíno protease de plasmódio, como pode ser observado para o controle inibidor de proteases E64. Análise do extrato proteico de eritrócitos infectados e tratados por 10 horas com 4m ou E64 mostrou acúmulo de proteínas não degradadas apenas no tratamento com E64, indicando que a cisteíno protease de P. falciparum, potencialmente, não é o alvo de 4m. Foi analisada também a capacidade dos compostos interferir na homeostase do Ca2+ intracelular, sendo 4m capaz de alterar os níveis deste íon no parasita, não apresentando o mesmo efeito em hepatócitos murinos, após liberação do cátion a partir do retículo endoplasmático, sendo, portanto, este efeito seletivo para o parasita. A influência do NO liberado pela molécula 4m neste desbalanço foi descartada, visto que o composto doador de NO utilizado como controle não promoveu nenhuma alteração nos níveis de cálcio. Estes dados mostram que os furoxanos possuem grande potencial para o desenvolvimento de anti-leishmaniais e anti-maláricos.
Neglected diseases represent a major challenge for public health policies. Among these diseases, we highlight leishmaniasis and malaria: two important parasitic diseases that affect poor people with no political voice and heavily affected by the etiologic agents Leishmania spp. and Plasmodium spp. There are few available therapeutic drugs, which present high toxicity and low efficacy; thus, new therapeutic agents are urgently needed. Considering the Sustainable Development goals proposed by The United Nations, which establishes a global agenda to promote, among other goals, Health and wellness for the human population until 2030, our research group has been involved contributing to this theme in order to develop new therapy to control leishmaniasis and more recently, malaria on the context of this research project. Therefore, furoxan (4a-o) and benzofuroxan (4p-x) compounds were characterized regarding the anti-Leishmania infantum effect, which identified the furoxan NO donors (4a-h, 4l-m) and benzofuroxan 4r as the most potent and selective compounds (EC50 < 10 μM; SI > 10). Due to the presence of a N-acyl-hydrazone group that seems to target proteases, we cloned and expressed the corresponding Leishmania mexicana cysteine protease enzyme LMCPB2.8∆CTE (CPB2.8) in Pichia pastoris in order to evaluate the inhibitory effect of these compounds against the purified enzyme. The furoxans (4a-n), benzofuroxan (4q-r and 4t-u) and N-acyl- hydrazone compounds (14a-g) show good ability to inhibit the parasite protease (0.8 μM < IC50 < 14 μM). It is also important to highlight that the leishmanicidal activity of these compounds might be related to a dual effect caused by the microbicidal activity of NO and associated to the enzyme inhibition, since 14a-g, which are not able to release NO, thus not present any anti-amastigote activity at the maximum tested concentration (10 μM), although causing protease inhibition (2 μM < IC50 < 12 μM). In silico, pharmacokinetic analysis shows the therapeutic potential of 4f a L.infantum- infected Balb/c mice model. After oral administration, twice daily at a 7.7 mg/kg/day dose, 4f reduced the parasite burden by 90% in the liver and spleen, with no observed toxicity to the liver or kidney, finishing the studies involving L. infantum. All molecules were subsequently evaluated against intra-erythrocyte forms of Plasmodium falciparum genetically modified to express the GCaMP3 Ca2+ indicator through a sandwich stage at Rutgers New Jersey Medical School in Newark – USA. Among the furoxan, 4m (EC50 2.8 μM) while 4r was the most active benzofuroxan, showing inhibition around 40% of parasite growth inhibition. Studies regarding the mechanism of action suggests that 4m probably is not targeting the P. falciparum proteases falcipains through the analyses of morphological modifications of the parasite digestive vacuole, or hemoglobin degradation. In the meantime, the ability of the compounds to interfere in intracellular calcium homeostasis was also analyzed. The compound 4m is selectively capable to preferentially alter the levels of this ion in the parasite rather than in murine hepatocytes through the release of calcium from the endoplasmic reticulum not related to an indirect effect due to NO release. This data shows that the furoxan compounds have potential to develop antileishmanial and antimalarial drugs.
Neglected diseases represent a major challenge for public health policies. Among these diseases, we highlight leishmaniasis and malaria: two important parasitic diseases that affect poor people with no political voice and heavily affected by the etiologic agents Leishmania spp. and Plasmodium spp. There are few available therapeutic drugs, which present high toxicity and low efficacy; thus, new therapeutic agents are urgently needed. Considering the Sustainable Development goals proposed by The United Nations, which establishes a global agenda to promote, among other goals, Health and wellness for the human population until 2030, our research group has been involved contributing to this theme in order to develop new therapy to control leishmaniasis and more recently, malaria on the context of this research project. Therefore, furoxan (4a-o) and benzofuroxan (4p-x) compounds were characterized regarding the anti-Leishmania infantum effect, which identified the furoxan NO donors (4a-h, 4l-m) and benzofuroxan 4r as the most potent and selective compounds (EC50 < 10 μM; SI > 10). Due to the presence of a N-acyl-hydrazone group that seems to target proteases, we cloned and expressed the corresponding Leishmania mexicana cysteine protease enzyme LMCPB2.8∆CTE (CPB2.8) in Pichia pastoris in order to evaluate the inhibitory effect of these compounds against the purified enzyme. The furoxans (4a-n), benzofuroxan (4q-r and 4t-u) and N-acyl- hydrazone compounds (14a-g) show good ability to inhibit the parasite protease (0.8 μM < IC50 < 14 μM). It is also important to highlight that the leishmanicidal activity of these compounds might be related to a dual effect caused by the microbicidal activity of NO and associated to the enzyme inhibition, since 14a-g, which are not able to release NO, thus not present any anti-amastigote activity at the maximum tested concentration (10 μM), although causing protease inhibition (2 μM < IC50 < 12 μM). In silico, pharmacokinetic analysis shows the therapeutic potential of 4f a L.infantum- infected Balb/c mice model. After oral administration, twice daily at a 7.7 mg/kg/day dose, 4f reduced the parasite burden by 90% in the liver and spleen, with no observed toxicity to the liver or kidney, finishing the studies involving L. infantum. All molecules were subsequently evaluated against intra-erythrocyte forms of Plasmodium falciparum genetically modified to express the GCaMP3 Ca2+ indicator through a sandwich stage at Rutgers New Jersey Medical School in Newark – USA. Among the furoxan, 4m (EC50 2.8 μM) while 4r was the most active benzofuroxan, showing inhibition around 40% of parasite growth inhibition. Studies regarding the mechanism of action suggests that 4m probably is not targeting the P. falciparum proteases falcipains through the analyses of morphological modifications of the parasite digestive vacuole, or hemoglobin degradation. In the meantime, the ability of the compounds to interfere in intracellular calcium homeostasis was also analyzed. The compound 4m is selectively capable to preferentially alter the levels of this ion in the parasite rather than in murine hepatocytes through the release of calcium from the endoplasmic reticulum not related to an indirect effect due to NO release. This data shows that the furoxan compounds have potential to develop antileishmanial and antimalarial drugs.
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Doenças negligenciadas, Óxido nítrico, Mecanismo de ação (Bioquímica), Malária, Cálcio - Metabolismo