Publicação
Drug screening of Bisnaphthalimidopropyl derivatives on Trypanosoma brucei
| Resumo: | African trypanosomiasis (AT) is a parasitic vector-borne endemic disease in sub-Saharan Africa and approximately 60 million of people are currently at risk. It is transmitted by the tsetse fly bite. Despite the efforts made by the World Health Organization (WHO) this disease is a continuing problem in Africa. The causative agent is the extracellular protozoan parasite Trypanosoma brucei (T. brucei) which is sub-divided in three subspecies: T. brucei brucei, T. brucei gambiense and T. brucei rhodesiense. The two latter subspecies are responsible for Human African Trypanosomiasis (HAT) also known as sleeping sickness disease, which evolve to the slow onset chronic disease or to the fast onset acute disease, respectively [1, 2]. The current state of drug therapy is limited to five drugs. Suramin and pentamidine are applied against the early phase of the disease denominated as the hemo-lymphatic stage, and melarsoprol, eflornithine and the combination therapy nifurtimox/eflornithine are used against the late meningo-encephalitic stage [3]. However, some drawbacks are associated with these compounds such as their general toxicity to human organism, expensive cost and difficulty in their administration [3-5]. Thus, there is an urgent need for new drugs that are effective against T. brucei, with lower cost, easy to administer, and safer to humans. The anti-parasitic activity on Leishmania infantum (member of the Trypanosomatid family) of bisnaphthalimidopropyl (BNIP) derivative compounds has been described [6-10]. These are composed by two naphthalimidopropyl groups linked by a carbonated chain, varying in size and in the presence of amines, oxygens, cyclohexanes and/or benzene rings. The present work had the objective to evaluate a small library of BNIP-derivative compounds as therapeutic drugs in the treatment of infections caused by T. brucei. At first, an in vitro optimization of two methods to assess parasite growth inhibition [resazurin-based assay and tetrazole 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay] was performed to defined the cell-based drug screening assay towards the bloodstream form T. b. brucei strain Lister 427. Mouse fibroblast L929 cells were used as a counter-screen for non-selective inhibitors determined by the MTT assay. The most potent compound against T. brucei was chosen to determine its reversibility and time kill action, in vivo efficacy on T. brucei-infected BALB/c mice, identification of its putative protein(s) target(s) and development of polyethylene glycol (PEG) – coated poly (lactic acid) (PLA) nanoparticles. In this project, the current used drugs in therapy, pentamidine and suramin, exhibited IC50 (half maximal inhibitory concentration) values against T. brucei of ~4 nM and 70 nM, respectively, in accordance with available literature [11]. From the small 15 BNIP library, 12 compounds exhibited an IC50 in the nanomolar range and it was demonstrated that the presence of two cyclohexane or benzene rings in the linker chain hampers their anti-T. brucei activity. Also it was verified a decrease in the anti-parasitic activity with the increment on the linker chain of the BNIP derivative compounds. BNIPDabut was the most potent compound with ~3 nM activity, similar to the IC50 of pentamidine and with a selectivity index for T. brucei over fibroblasts of about 8000-fold higher. Also, it was characterized as an in vitro trypanocidal compound dependent of concentration and time that is also able to reduce parasite quantity in vivo. Moreover, it exhibits a specific interaction with some T. brucei proteins, in particular with fructose-1,6-bisphosphate aldolase. In addition, BNIPDabut PEG-PLA nanoparticles were successfully produced and are effective in vitro towards T. brucei at the nanomolar range. In conclusion, BNIP-derivative compounds showed promising toxic potential against T. brucei pathogen at low concentrations which can be applied in the future for HAT treatment. |
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| Autores principais: | Martins, Patrícia Varela Oliveira,1988- |
| Assunto: | Tripanossomíase africana Trypanosoma Parasitas Fármacos Doença do sono Teses de mestrado - 2012 |
| Ano: | 2012 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | African trypanosomiasis (AT) is a parasitic vector-borne endemic disease in sub-Saharan Africa and approximately 60 million of people are currently at risk. It is transmitted by the tsetse fly bite. Despite the efforts made by the World Health Organization (WHO) this disease is a continuing problem in Africa. The causative agent is the extracellular protozoan parasite Trypanosoma brucei (T. brucei) which is sub-divided in three subspecies: T. brucei brucei, T. brucei gambiense and T. brucei rhodesiense. The two latter subspecies are responsible for Human African Trypanosomiasis (HAT) also known as sleeping sickness disease, which evolve to the slow onset chronic disease or to the fast onset acute disease, respectively [1, 2]. The current state of drug therapy is limited to five drugs. Suramin and pentamidine are applied against the early phase of the disease denominated as the hemo-lymphatic stage, and melarsoprol, eflornithine and the combination therapy nifurtimox/eflornithine are used against the late meningo-encephalitic stage [3]. However, some drawbacks are associated with these compounds such as their general toxicity to human organism, expensive cost and difficulty in their administration [3-5]. Thus, there is an urgent need for new drugs that are effective against T. brucei, with lower cost, easy to administer, and safer to humans. The anti-parasitic activity on Leishmania infantum (member of the Trypanosomatid family) of bisnaphthalimidopropyl (BNIP) derivative compounds has been described [6-10]. These are composed by two naphthalimidopropyl groups linked by a carbonated chain, varying in size and in the presence of amines, oxygens, cyclohexanes and/or benzene rings. The present work had the objective to evaluate a small library of BNIP-derivative compounds as therapeutic drugs in the treatment of infections caused by T. brucei. At first, an in vitro optimization of two methods to assess parasite growth inhibition [resazurin-based assay and tetrazole 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay] was performed to defined the cell-based drug screening assay towards the bloodstream form T. b. brucei strain Lister 427. Mouse fibroblast L929 cells were used as a counter-screen for non-selective inhibitors determined by the MTT assay. The most potent compound against T. brucei was chosen to determine its reversibility and time kill action, in vivo efficacy on T. brucei-infected BALB/c mice, identification of its putative protein(s) target(s) and development of polyethylene glycol (PEG) – coated poly (lactic acid) (PLA) nanoparticles. In this project, the current used drugs in therapy, pentamidine and suramin, exhibited IC50 (half maximal inhibitory concentration) values against T. brucei of ~4 nM and 70 nM, respectively, in accordance with available literature [11]. From the small 15 BNIP library, 12 compounds exhibited an IC50 in the nanomolar range and it was demonstrated that the presence of two cyclohexane or benzene rings in the linker chain hampers their anti-T. brucei activity. Also it was verified a decrease in the anti-parasitic activity with the increment on the linker chain of the BNIP derivative compounds. BNIPDabut was the most potent compound with ~3 nM activity, similar to the IC50 of pentamidine and with a selectivity index for T. brucei over fibroblasts of about 8000-fold higher. Also, it was characterized as an in vitro trypanocidal compound dependent of concentration and time that is also able to reduce parasite quantity in vivo. Moreover, it exhibits a specific interaction with some T. brucei proteins, in particular with fructose-1,6-bisphosphate aldolase. In addition, BNIPDabut PEG-PLA nanoparticles were successfully produced and are effective in vitro towards T. brucei at the nanomolar range. In conclusion, BNIP-derivative compounds showed promising toxic potential against T. brucei pathogen at low concentrations which can be applied in the future for HAT treatment. |
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