Oportunidades de controle das leishmanioses: planejamento, síntese, caracterização e bioconjugação de peptídeos a agentes leishmanicidas
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2022-10-18
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Universidade Estadual Paulista (Unesp)
Resumo
As leishmanioses correspondem a um grupo de doenças causada por protozoários do gênero Leishmania. O controle das leishmanioses depende de intervenção quimioterápica, com destaque para a anfotericina B, objeto de estudo deste trabalho. A anfotericina B é tóxica e causa severos efeitos colaterais nos pacientes. Dentro deste cenário, parte de nossos objetivos visa a modificação da anfotericina B via acoplamento de peptídeos antimicrobianos, em um processo chamado bioconjugação terapêutica. Neste estudo, peptídeos leishmanicidas das classes temporinas e histatinas foram exploradas para desenvolvimento de novos bioconjugados. Para isso os peptídeos TSHa (IC₅₀ama=5µmol L -1 e IS>200), TSHd (IC₅₀ama=7,2µmol L -1 e IS=14) e Hist 5 (IC₅₀ama=8 µmol L -1 e IS>125) foram funcionalizados à anfotericina B (IC₅₀ama=0,63 e IS= 38), gerando os derivados Anf B*-TSHa (IC₅₀ama=1,6 µmol L -1 e IS>645), Anf B (RC)-TSHa ( IC₅₀ama=0,31 µmol L -1 e IS>3225), Anf B *-TSHd ( IC₅₀ama=0,95 µmol L -1 e IS>1052), Anf B-Hist 5 (IC₅₀ama=0,4 µmol L -1 e IS>2500) foram sintetizados com alto grau de pureza. Dentre os principais achados, destaca-se a alta seletividade (IS) dos bioconjugados, os quais são pelo menos 600 vezes mais seletivos para o parasita quando comparados à célula do hospedeiro, o que pode ser atribuído a uma possível ação dos peptídeos temporinas TSHa e TSHd na membrana do parasita, quer seja pela formação de poros ou por interações do tipo detergente (carpet like), o que pode estar facilitando a permeação da anfotericina B nos macrófagos, promovendo sua atividade anti-amastigota. Diferentemente para o bioconjugado Anf B-Hist 5, verificou-se que houve menor permeabilização desses na membrana do parasita, isto foi observado através dos ensaios de citometria de fluxo e de microscopia de fluorescência. Os dados de microscopia de fluorescência confocal indicaram uma melhor compartimentalização do peptídeo Anf B-Hist 5 na mitocôndria, sugerindo um mecanismo de ação dual inerente de cada molécula associado à atividade biológica aqui caracterizada: a ação da anfotericina B na membrana do parasita facilitando a entrada do peptídeo Anf B-Hist 5 e assim atividade do mesmo sobre a mitocôndria. Adicionalmente, a fim de sintetizar novos peptídeos leishmanicidas, foi planejado e sintetizado um dímero composto pelos peptídeos TSHa, denominado (TSHa)₂K (IC₅₀ ama=0,58 µmol L -1 e IS>1724), cuja potência e seletividade aumentou cerca de dez vezes em relação ao peptídeo TSHa. Paralelamente, a partir de estudos de modelagem molecular para obtenção da estrutura do canal mitocondrial de cálcio MCU de Leishmania, estudos de docagem molecular foram realizados para planejamento de peptídeos inéditos capazes de interferir seletivamente no influxo de cálcio para a mitocôndria do parasita com base na interação destes com a região do filtro de seletividade DIME, os quais deverão ser sintetizados e validados em estudos no parasita Leishmania.Com a presente pesquisa podemos concluir a bioconjugação com Anf B e o design racional de peptídeos são estratégias interessantes para o desenvolvimento de novas moléculas para o tratamento da leishmaniose, focando no aumento da seletividade e diminuição da toxicidade.
Leishmaniasis is a group of diseases caused by protozoa of the genus Leishmania. The control of leishmaniasis depends on chemotherapeutic intervention, with emphasis on amphotericin B, object of study in this work. Amphotericin B is toxic and causes severe side effects in patients. Within this scenario, part of our objectives is the modification of amphotericin B via coupling of antimicrobial peptides, in a process called therapeutic bioconjugation. In this study, leishmanicidal peptides of the temporine and histatin classes were explored for the development of new bioconjugates. For this, TSHa (IC₅₀ama=5µmol L - 1 and IS>200), TSHd (IC₅₀ama=7.2µmol L -1 and IS=14) and Hist 5 (IC₅₀ama=8 µmol L -1 and IS>125) peptides were functionalized to amphotericin B (IC₅₀ama=0.63 µmol L -1 and IS= 38), generating Anf B*-TSHa derivatives (IC₅₀ama=1.6 µmol L -1 and IS>645), Anf B (RC)-TSHa (IC₅₀ama=0, 31 µmol L -1 and IS>3225), Anf B *-TSHd (IC₅₀ama=0.95 µmol L -1 and IS>1052), Anf B-Hist 5 (IC₅₀ama=0.4 µmol L -1 and IS>2500) were synthesized with a high degree of purity. Among the main findings, the high selectivity (IS) of the bioconjugates stands out, which are at least 600 times more selective for the parasite when compared to the host cell, which can be attributed to a possible action of the temporal peptides TSHa and TSHd in the parasite membrane, either by pore formation or by detergent-like interactions (carpet like), which may be facilitating the permeation of amphotericin B in macrophages, promoting its anti-amastigote activity. Differently for the bioconjugate Anf B-Hist5, it was verified that there was less permeabilization of these in the membrane of the parasite, this was observed through the flow cytometry and fluorescence microscopy assays. Confocal fluorescence microscopy data indicated a better compartmentalization of the peptide Anf B-Hist 5 in the mitochondria, suggesting an inherent dual mechanism of action of each molecule associated with the biological activity characterized here: the actionof amphotericin B on the parasite membrane, facilitating the entry of the parasite. Anf B-Hist 5 peptide and thus its activity on mitochondria. Additionally, in order to synthesize new leishmanicidal peptides, a dimer composed of TSHa peptides, called (TSHa)₂K (IC₅₀ ama=0.58 µmol L -1 and IS>1724) was designed and synthesized, whose potency and selectivity increased by about ten times that of the TSHa peptide. In parallel, from molecular modeling studies to obtain the structure of the mitochondrial calcium channel MCU from Leishmania, molecular docking studies were carried out to design novel peptides capable of selectively interfering with the influx of calcium into the parasite's mitochondria based on the interaction of these with the DIME selectivity filter region, which should be synthesized and validated in studies on the Leishmania parasite. With the present research we can conclude that bioconjugation with Anf B and the rational design of peptides are interesting strategies for the development of new molecules for the treatment of leishmaniasis, focusing on increasing selectivity and decreasing toxicity.
Leishmaniasis is a group of diseases caused by protozoa of the genus Leishmania. The control of leishmaniasis depends on chemotherapeutic intervention, with emphasis on amphotericin B, object of study in this work. Amphotericin B is toxic and causes severe side effects in patients. Within this scenario, part of our objectives is the modification of amphotericin B via coupling of antimicrobial peptides, in a process called therapeutic bioconjugation. In this study, leishmanicidal peptides of the temporine and histatin classes were explored for the development of new bioconjugates. For this, TSHa (IC₅₀ama=5µmol L - 1 and IS>200), TSHd (IC₅₀ama=7.2µmol L -1 and IS=14) and Hist 5 (IC₅₀ama=8 µmol L -1 and IS>125) peptides were functionalized to amphotericin B (IC₅₀ama=0.63 µmol L -1 and IS= 38), generating Anf B*-TSHa derivatives (IC₅₀ama=1.6 µmol L -1 and IS>645), Anf B (RC)-TSHa (IC₅₀ama=0, 31 µmol L -1 and IS>3225), Anf B *-TSHd (IC₅₀ama=0.95 µmol L -1 and IS>1052), Anf B-Hist 5 (IC₅₀ama=0.4 µmol L -1 and IS>2500) were synthesized with a high degree of purity. Among the main findings, the high selectivity (IS) of the bioconjugates stands out, which are at least 600 times more selective for the parasite when compared to the host cell, which can be attributed to a possible action of the temporal peptides TSHa and TSHd in the parasite membrane, either by pore formation or by detergent-like interactions (carpet like), which may be facilitating the permeation of amphotericin B in macrophages, promoting its anti-amastigote activity. Differently for the bioconjugate Anf B-Hist5, it was verified that there was less permeabilization of these in the membrane of the parasite, this was observed through the flow cytometry and fluorescence microscopy assays. Confocal fluorescence microscopy data indicated a better compartmentalization of the peptide Anf B-Hist 5 in the mitochondria, suggesting an inherent dual mechanism of action of each molecule associated with the biological activity characterized here: the actionof amphotericin B on the parasite membrane, facilitating the entry of the parasite. Anf B-Hist 5 peptide and thus its activity on mitochondria. Additionally, in order to synthesize new leishmanicidal peptides, a dimer composed of TSHa peptides, called (TSHa)₂K (IC₅₀ ama=0.58 µmol L -1 and IS>1724) was designed and synthesized, whose potency and selectivity increased by about ten times that of the TSHa peptide. In parallel, from molecular modeling studies to obtain the structure of the mitochondrial calcium channel MCU from Leishmania, molecular docking studies were carried out to design novel peptides capable of selectively interfering with the influx of calcium into the parasite's mitochondria based on the interaction of these with the DIME selectivity filter region, which should be synthesized and validated in studies on the Leishmania parasite. With the present research we can conclude that bioconjugation with Anf B and the rational design of peptides are interesting strategies for the development of new molecules for the treatment of leishmaniasis, focusing on increasing selectivity and decreasing toxicity.
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Palavras-chave
Bioconjugação, Peptídeos antimicrobianos, Leishmanioses, danos na membrana, design de moléculas