Descoberta de novos compostos derivados de cloroquina como candidatos a antimaláricos
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Data
2024-03-21
Autores
Peres, Erica Paloma Maso Lopes [UNIFESP]
Orientadores
Aguiar, Anna Caroline Campos Aguiar [UNIFESP]
Tipo
Dissertação de mestrado
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Introdução: A malária é uma doença tropical grave, caracterizada por sua alta taxa de mortalidade e morbidade, representando um significativo problema de saúde pública com impacto socioeconômico. O controle da malária enfrenta desafios devido dificuldade no combate ao vetor e à rápida disseminação de linhagens de parasitos resistentes aos antimaláricos convencionais. O surgimento de resistência compromete a eficácia dos tratamentos atuais, tornando a luta contra a malária um desafio cada vez mais complexo sendo de extrema importância a busca por novos candidatos a antimaláricos potentes, de baixo custo e não tóxico ao hospedeiro vertebrado. Objetivo: Avaliar a atividade antiplasmodial e o alvo de ação contra estágios assexuados sanguíneos de 8 novos análogos de cloroquina, utilizando cepa de Plasmodium falciparum sensível e resistentes. Métodos: Realizamos ensaios in vitro para determinar o IC50 dos novos compostos contra as cepas de P. falciparum 3D7 (sensível), Dd2, K1 e SB1 (resistentes), utilizando o ensaio de SBYR Green I. Em seguida, prosseguimos com a avaliação citotóxica, a partir do método de rezarsurina, utilizando linhagens celulares de hepatoma humano (HepG2) e rim embrionário (HEK-293). Após essa fase, examinamos o mecanismo de resistência dos novos análogos de CQ na presença do verapamil, um inibidor de bombas de efluxo de P. falciparum e que está relacionada a resistência aos antimaláricos. Posteriormente, identificamos o vacúolo digestivo de P. falciparum como alvo intracelular dos novos compostos, utilizamos o marcador fluorescente Acridina Orange (AO). Em sequência, selecionamos os compostos 1, 2, 6 e 7 e realizamos ensaios ex vivo contra isolados de campo com P. falciparum e P. vivax circulantes na região amazônica brasileira. Resultados: Os compostos 6 e CEQ foram os mais potentes in vitro, apresentando IC50 de 7 nM e 15 nM, respectivamente, contra a cepa 3D7. Frente às cepas resistentes, os compostos mais potentes foram CEQ e 1 (cepa Dd2) com o IC50 de 30 nM e 75 nM, respectivamente, para a cepa K1, os compostos mais potentes foram 5, 7 e 8 com IC50 de 33 nM à 81 nM e para a cepa SB1 os compostos testados foram 1, 2 e 3 e todos se mostraram potentes com IC50 variando de 10 nM a 89 nM. A resistência cruzada dos compostos foi avaliada comparando seus valores de IC50 tanto para cepa sensível quanto resistentes à cloroquina. Os compostos 1, 2, 5, 7 e 8, não apresentaram índice de resistência cruzada (IRC) nos ensaios in vitro com valores de IRC <5. Os compostos CEQ, 3, 4 e 6 apresentaram IRC moderada a alta com valores de IRC >5. No ensaio com verapamil os compostos testados foram 1, 2, 3, 4 e 6 e foi observado uma redução do IC50, após incubação com verapamil, sugerindo resistência frente a cepa Dd2. Os compostos não se mostraram tóxicos para as células HepG2 e HEK-293. Para os ensaios de identificação do vacúolo digestivo (VD) com o marcador AO, pudemos identificar que ouve interação dos compostos com esta organela á partir da alteração da homeostase iônica, assim como observado para a CQ. Quando avaliamos a potência dos compostos frente aos isolados de campo circulantes em Porto Velho, foi possível observar que estes compostos foram mais potentes contra o P. vivax quando comparado com o P. falciparum, indicando uma baixa sensibilidade a essa espécie. Conclusão: Podemos concluir que esta nova classe de análogos de cloroquina apresentou resultados promissores in vitro, no entanto, não foram ativos contra os isolados circulantes na Amazônia brasileira de P. falciparum.
Introduction: Malaria is a severe tropical disease characterized by its high mortality and morbidity rates, posing a significant public health problem with socioeconomic impact. Malaria control faces challenges due to difficulties in combating the vector and the rapid spread of parasite strains resistant to conventional antimalarials. The emergence of resistance compromises the effectiveness of current treatments, making the fight against malaria an increasingly complex challenge. It is of utmost importance to search for new candidates for potent, lowcost, and nontoxic antimalarials to the vertebrate host. Objective: To assess the antiplasmodial activity and target action against the bloodstage asexual forms of 8 new CQ analogues, using sensitive and resistant strains of Plasmodium falciparum. Methods: In vitro assays were conducted to determine the IC50 of the new compounds against the strains of P. falciparum 3D7 (sensitive), Dd2, K1, and SB1 (resistant), using the SBYR Green I assay. Subsequently, cytotoxic evaluation was carried out using the resazurin method, employing human hepatoma (HepG2) and embryonic kidney (HEK293) cell lines. After this phase, we examined the resistance mechanism of the new CQ analogues in the presence of verapamil, an inhibitor of P. falciparum efflux pumps related to antimalarial resistance. Subsequently, we identified the digestive vacuole of P. falciparum as the intracellular target of the new compounds, using the fluorescent marker Acridine Orange (AO). Following this, we selected compounds 1, 2, 6, and 7 and evaluated ex vivo assays were conducted against them against field isolates of circulating P. falciparum and P. vivax in the Brazilian Amazon region. Results: Compounds 6 and CEQ were the most potent in vitro, with IC50 values of 7 nM and 15 nM, respectively, against the 3D7 strain. Faced with resistant strains, the most potent compounds were CEQ and 1 (Dd2 strain) with IC50 of 30 nM and 75 nM, respectively; for the K1 strain, the most potent compounds were 5, 7, and 8 with IC50 ranging from 33 nM to 81 nM, and for the SB1 strain, compounds 1, 2, and 3 were tested and all showed potency with IC50 ranging from 10 nM to 89 nM. For crossresistance assays, compounds 1, 2, 5, 7, and 8 showed no crossresistance in vitro with crossresistance index (CRI) values <5. Compounds CEQ, 3, 4, and 6 showed moderate to high CRI with values of CRI >5. In the verapamil assay, the tested compounds were 1, 2, 3, 4, and 6, and a reduction in IC50 was observed after incubation with verapamil, suggesting resistance against the Dd2 strain. The compounds showed no toxicity to HepG2 and HEK293 cells. In the digestive vacuole identification assays with the AO marker, we could identify interaction of the compounds with this organelle through alteration of ionic homeostasis, similar to what was observed for CQ. When evaluating the potency of the compounds against circulating field isolates in Porto Velho, it was observed that these compounds were more potent against P. vivax compared to P. falciparum, indicating low sensitivity to this species. Conclusion: We can conclude that this new class of chloroquine analogs showed promising in vitro results; however, they were not active against circulating isolates in the Brazilian Amazon of P. falciparum.
Introduction: Malaria is a severe tropical disease characterized by its high mortality and morbidity rates, posing a significant public health problem with socioeconomic impact. Malaria control faces challenges due to difficulties in combating the vector and the rapid spread of parasite strains resistant to conventional antimalarials. The emergence of resistance compromises the effectiveness of current treatments, making the fight against malaria an increasingly complex challenge. It is of utmost importance to search for new candidates for potent, lowcost, and nontoxic antimalarials to the vertebrate host. Objective: To assess the antiplasmodial activity and target action against the bloodstage asexual forms of 8 new CQ analogues, using sensitive and resistant strains of Plasmodium falciparum. Methods: In vitro assays were conducted to determine the IC50 of the new compounds against the strains of P. falciparum 3D7 (sensitive), Dd2, K1, and SB1 (resistant), using the SBYR Green I assay. Subsequently, cytotoxic evaluation was carried out using the resazurin method, employing human hepatoma (HepG2) and embryonic kidney (HEK293) cell lines. After this phase, we examined the resistance mechanism of the new CQ analogues in the presence of verapamil, an inhibitor of P. falciparum efflux pumps related to antimalarial resistance. Subsequently, we identified the digestive vacuole of P. falciparum as the intracellular target of the new compounds, using the fluorescent marker Acridine Orange (AO). Following this, we selected compounds 1, 2, 6, and 7 and evaluated ex vivo assays were conducted against them against field isolates of circulating P. falciparum and P. vivax in the Brazilian Amazon region. Results: Compounds 6 and CEQ were the most potent in vitro, with IC50 values of 7 nM and 15 nM, respectively, against the 3D7 strain. Faced with resistant strains, the most potent compounds were CEQ and 1 (Dd2 strain) with IC50 of 30 nM and 75 nM, respectively; for the K1 strain, the most potent compounds were 5, 7, and 8 with IC50 ranging from 33 nM to 81 nM, and for the SB1 strain, compounds 1, 2, and 3 were tested and all showed potency with IC50 ranging from 10 nM to 89 nM. For crossresistance assays, compounds 1, 2, 5, 7, and 8 showed no crossresistance in vitro with crossresistance index (CRI) values <5. Compounds CEQ, 3, 4, and 6 showed moderate to high CRI with values of CRI >5. In the verapamil assay, the tested compounds were 1, 2, 3, 4, and 6, and a reduction in IC50 was observed after incubation with verapamil, suggesting resistance against the Dd2 strain. The compounds showed no toxicity to HepG2 and HEK293 cells. In the digestive vacuole identification assays with the AO marker, we could identify interaction of the compounds with this organelle through alteration of ionic homeostasis, similar to what was observed for CQ. When evaluating the potency of the compounds against circulating field isolates in Porto Velho, it was observed that these compounds were more potent against P. vivax compared to P. falciparum, indicating low sensitivity to this species. Conclusion: We can conclude that this new class of chloroquine analogs showed promising in vitro results; however, they were not active against circulating isolates in the Brazilian Amazon of P. falciparum.
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Citação
PERES, Erica Paloma Maso Lopes. Descoberta de novos compostos derivados de cloroquina como candidatos a antimaláricos. 2024. 91 f. Dissertação (Mestrado em Bioprodutos e Bioprocessos) - Universidade Federal de São Paulo, Instituto de Saúde e Sociedade, Santos, 2024.