Leishmanioses, derivados guanidínicos e cisteíno proteases: atividade biológica in vitro e in vivo para o desenvolvimento racional de fármacos
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Data
2022-10-04
Autores
Mela, Michele Ferreira da Silva
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
As leishmanioses são um conjunto de doenças causadas por parasitas do gênero Leishmania e acometem milhares de pessoas no mundo anualmente. Até o momento, há poucos fármacos disponíveis para o tratamento destas doenças, além de apresentarem elevada toxicidade, efeitos colaterais graves e baixa eficácia, o que torna urgente a busca por novas opções terapêuticas. Neste sentido, este trabalho teve como objetivo identificar compostos guanidínicos com atividade leishmanicida e potencial atividade inibitória da enzima cisteíno protease rCPB2.8, importante fator de virulência do parasita Leishmania. As moléculas foram avaliadas frente a Leishmania infantum e/ou Leishmania amazonensis, com destaque para os compostos G1, G2, G3, G7 e G30, os quais apresentaram potência frente à promastigotas de L. infantum, com destaque para G1, G6 e G7, as quais também mostraram-se promissoras frente a amastigotas intracelulares (IC50AMA < 10 µM), além de serem pouco citotóxicas quando avaliadas em macrófagos murinos, indicando índice de seletividade > 10. Em busca de potenciais alvos moleculares para entendimento do mecanismo de ação, estes derivados guanidínicos foram avaliados quanto à sua capacidade de inibição da enzima recombinante rCPB2.8, uma protease importante para sobrevivência e virulência de Leishmania spp, sugerindo que G6 tenha esta protease como potencial alvo de ação, (IC50CPB = 6 µM) , com análise do gráfico duplo-recíproco Lineweaver e Burk, indicando que esta molécula diminui a afinidade da enzima pelo substrato. Ensaios in vivo em modelo experimental Balb/c infectado com L. amazonensis, seguindo um protocolo de tratamento de administração intraperitoneal de 2 mg/Kg/dia, durante 15 dias, mostram que G6 e G7 levaram a diminuição das lesões cutâneas dos animais, com redução da carga parasitária de 79,4% e 66,5%, respectivamente, não indicando danos hepáticos e renais. Estes dados evidenciam o potencial desta classe de moléculas para as próximas etapas de desenvolvimento de fármacos antileishmaniais para o tratamento de leishmaniose cutânea e também para leishmaniose visceral.
Leishmaniasis is a set of diseases caused by parasites of the genus Leishmania and affects thousands of people worldwide annually. To date, there are few drugs available for the treatment of these diseases, in addition to presenting high toxicity, severe side effects and low efficacy, which makes the search for new therapeutic options urgent. In this sense, this work aimed to identify guanidinic compounds with leishmanicidal activity and potential inhibitory activity of the cysteine protease enzyme rCPB2.8, an important virulence factor of the Leishmania parasite. The molecules were evaluated against Leishmania infantum and/or Leishmania amazonensis, with emphasis on compounds G1, G2, G3, G7 and G30, which showed potency against L. infantum promastigotes, especially G1, G6 and G7, which also showed promise against intracellular amastigotes (IC50AMA < 10 µM), in addition to being little cytotoxic when evaluated in murine macrophages, indicating a selectivity index > 10. In search of potential molecular targets to understand the mechanism of action, these guanidinium derivatives were evaluated for their ability to inhibit the recombinant enzyme rCPB2.8, an important protease for the survival and virulence of Leishmania spp, suggesting that G6 has this protease as a potential target of action (IC50CPB = 6 µM), with analysis of the Lineweaver and Burk double-reciprocal plot, indicating that this molecule decreases the enzyme's affinity for the substrate. In vivo assays in an experimental model Balb/c infected with L. amazonensis, following a treatment protocol of intraperitoneal administration of 2 mg/kg/day for 15 days, show that G6 and G7 led to a decrease in the skin lesions of the animals, with reduction in parasite load of 79.4% and 66.5%, respectively, not indicating liver or kidney damage. These data show the potential of this class of molecules for the next steps in the development of antileishmanial drugs for the treatment of cutaneous leishmaniasis and also for visceral leishmaniasis.
Leishmaniasis is a set of diseases caused by parasites of the genus Leishmania and affects thousands of people worldwide annually. To date, there are few drugs available for the treatment of these diseases, in addition to presenting high toxicity, severe side effects and low efficacy, which makes the search for new therapeutic options urgent. In this sense, this work aimed to identify guanidinic compounds with leishmanicidal activity and potential inhibitory activity of the cysteine protease enzyme rCPB2.8, an important virulence factor of the Leishmania parasite. The molecules were evaluated against Leishmania infantum and/or Leishmania amazonensis, with emphasis on compounds G1, G2, G3, G7 and G30, which showed potency against L. infantum promastigotes, especially G1, G6 and G7, which also showed promise against intracellular amastigotes (IC50AMA < 10 µM), in addition to being little cytotoxic when evaluated in murine macrophages, indicating a selectivity index > 10. In search of potential molecular targets to understand the mechanism of action, these guanidinium derivatives were evaluated for their ability to inhibit the recombinant enzyme rCPB2.8, an important protease for the survival and virulence of Leishmania spp, suggesting that G6 has this protease as a potential target of action (IC50CPB = 6 µM), with analysis of the Lineweaver and Burk double-reciprocal plot, indicating that this molecule decreases the enzyme's affinity for the substrate. In vivo assays in an experimental model Balb/c infected with L. amazonensis, following a treatment protocol of intraperitoneal administration of 2 mg/kg/day for 15 days, show that G6 and G7 led to a decrease in the skin lesions of the animals, with reduction in parasite load of 79.4% and 66.5%, respectively, not indicating liver or kidney damage. These data show the potential of this class of molecules for the next steps in the development of antileishmanial drugs for the treatment of cutaneous leishmaniasis and also for visceral leishmaniasis.
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Palavras-chave
Leishmania, Antiparasitários, Inibidores de proteases, Guanidina, Fármacos