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Previous issue date: 2016-02-23 / A doença de Chagas (DC), causada pelo protozoário Trypanosoma cruzi, é uma doença negligenciada endêmica em diferentes regiões empobrecidas da América Latina. O tratamento, baseado em dois nitroderivados, Nifurtimox e Benzonidazol (Bz), é insatisfatório, demandando a busca de novos fármacos com ação tripanocida que sejam mais seletivos e eficazes. Nesse âmbito, o presente trabalho busca a identificação de novos agentes antiparasitários para a DC, explorando a avaliação fenotípica de novas amidinas aromáticas sintéticas in vitro incluindo ensaios de combinação entre estes compostos. Dez novas amidinas foram testadas sobre tripomastigotas sanguíneos e amastigotas de diferentes cepas do T. cruzi (Y e Tulahuen) e também sobre células de mamífero hospedeiras (linhagem L929 e células cardíacas) para determinar seu perfil eficácia e de toxicidade, respectivamente. Dentre as moléculas testadas (apresentando um ou dois grupamentos catiônicos terminais), cinco foram mais ativas sobre tripomastigotas sanguíneos que o fármaco de referência (Bz), sendo uma delas, a DB2267 (molécula dicatiônica) a mais eficaz, exibindo EC50 de 0,23 \03BCM e um índice de seletividade (IS) de 417. Esta diamidina foi 28 vezes mais ativa e cerca de três vezes mais seletiva que Bz
Para determinar se a combinação de duas amidinas teria um efeito tripanocida superior ao seu uso em monoterapia, tripomastigotas sanguíneos foram incubados com DB2267 e DB2236 em proporções fixas e os resultados mostraram apenas um efeito aditivo com \01A9FIC<4. Interessantemente, quando formas intracelulares foram expostas à DB2267, sua atividade foi relacionada à cepa do parasito, sendo eficaz (EC50 = 0.87 ± 0.05 \03BCM) contra DTU II (cepa Y), mas não contra um representante da DTU VI (Tulahuen), mesmo quando utilizamos veículo diferente do DMSO (\03B2-ciclodextrina). Esta divergência pode estar relacionada a diferenças inerentes aos alvos e/ou rotas metabólicas das cepas do parasito e que merecem ser mais investigadas visando conhecer melhor a relação entre as características estruturais e ação universal das moléculas sobre DTUs e formas do T. cruzi para o desenho de compostos mais promissores. Devido à fluorescência intrínseca apresentada pelas amidinas, a captação de duas delas foi avaliada. Os dados obtidos com a amidina não ativa (DB2236) e com a outra ativa (DB2267) sobre amastigotas da cepa Y revelaram que ambas foram localizadas intracelularmente, mas em compartimentos distintos: DB2236 no citoplasma, enquanto DB2267 no núcleo. Nossos dados são encorajadores a respeito da atividade antiparasitária das amidinas aromáticas, no tocante a futuras investigações de novos agentes promissores para o tratamento da DC / Chagas disease (CD), caused by the protozoan
Trypanosoma cruzi,
is
a neglected
disease endemic in different poor areas of Latin America. The treatment, based on
two nitroderivatives, Nifurtimox and Benznidazole (Bz), is unsatisfactory, demanding
the screening of new potential trypanocidal drugs more selective and potent.
In this
scope, the present work deals with the identification of new anti
-
parasitic agents for
CD, exploring the phenotypic screening of novel synthetic aromatic amidines
in vitro
and also
combination assays between these
compounds.
The novel
ten amidines
were tested against bloodstream trypomastigotes (BT) and amastigote forms of
different
T.cruzi
strains (Y and Tulahuen) and
were also
evaluated on mammalian
host cells (L929 cells and cardiac cells) to check their toxicity profile. Among these
molecules,
five
were more active against BT than the reference drug (Bz), being
one
of them, the
DB2267
(a dicationic molecule)
the most potent, exhibiting an EC
50
value of 0.23 μM and a se
lectivity index (SI) of 417. This
diamidine was 28
-
fold more
active and about
3 times more selective than Bz. To ascertain if the combination of
two amidines could improve their trypanocidal activity, BT were incubated with
DB2267 and DB2236 in fixed
-
ratio proportions and the data showed only an additive
effect with ƩFIC<4. Interest
ingly, when intracellular forms were exposed to DB2267,
its activity was related to the parasite strain, being effective (EC
50
= 0.87 ± 0.05 μM)
against DTU II (Y strain) but not against one representative of DTU VI (Tulahuen)
even using different vehicles
(β
-
cyclodextrins and DMSO).
This discrepancy might be
related to differences concerning the targets and/or metabolic pathways of the
parasite that deserve to be more investigated aiming to better understand the
relationship between the structural characte
ristics and universal action of the
molecules against DTUs and forms of the
T.cruzi
for the design of more promising
compounds
.
Due to the intrinsic fluorescence presented by the amidines, the uptake
of two of them was assessed.
The data obtained with the
non
-
active amidine
(DB2236)
and the other active one
(DB2267)
against amastigotes of the Y strain
showed that both were localized intracellular
ly
, but in distinct compartments
: DB2236
in the cytoplasm
,
while
DB2267
in the nucleus
.
Our results are encouragi
ng
regarding the antiparasitic activity of the a
romatic
amidines
,
referring to future
investigations of new promising agents for the treatment of CD / Chagas disease (CD), caused by the protozoan
Trypanosoma cruzi,
is
a neglected
disease endemic in different poor areas of Latin America. The treatment, based on
two nitroderivatives, Nifurtimox and Benznidazole (Bz), is unsatisfactory, demanding
the screening of new potential trypanocidal drugs more selective and potent.
In this
scope, the present work deals with the identification of new anti
-
parasitic agents for
CD, exploring the phenotypic screening of novel synthetic aromatic amidines
in vitro
and also
combination assays between these
compounds.
The novel
ten amidines
were tested against bloodstream trypomastigotes (BT) and amastigote forms of
different
T.cruzi
strains (Y and Tulahuen) and
were also
evaluated on mammalian
host cells (L929 cells and cardiac cells) to check their toxicity profile. Among these
molecules,
five
were more active against BT than the reference drug (Bz), being
one
of them, the
DB2267
(a dicationic molecule)
the most potent, exhibiting an EC
50
value of 0.23 μM and a se
lectivity index (SI) of 417. This
diamidine was 28
-
fold more
active and about
3 times more selective than Bz. To ascertain if the combination of
two amidines could improve their trypanocidal activity, BT were incubated with
DB2267 and DB2236 in fixed
-
ratio proportions and the data showed only an additive
effect with ƩFIC<4. Interest
ingly, when intracellular forms were exposed to DB2267,
its activity was related to the parasite strain, being effective (EC
50
= 0.87 ± 0.05 μM)
against DTU II (Y strain) but not against one representative of DTU VI (Tulahuen)
even using different vehicles
(β
-
cyclodextrins and DMSO).
This discrepancy might be
related to differences concerning the targets and/or metabolic pathways of the
parasite that deserve to be more investigated aiming to better understand the
relationship between the structural characte
ristics and universal action of the
molecules against DTUs and forms of the
T.cruzi
for the design of more promising
compounds
.
Due to the intrinsic fluorescence presented by the amidines, the uptake
of two of them was assessed.
The data obtained with the
non
-
active amidine
(DB2236)
and the other active one
(DB2267)
against amastigotes of the Y strain
showed that both were localized intracellular
ly
, but in distinct compartments
: DB2236
in the cytoplasm
,
while
DB2267
in the nucleus
.
Our results are encouragi
ng
regarding the antiparasitic activity of the a
romatic
amidines
,
referring to future
investigations of new promising agents for the treatment of CD
| Identifer | oai:union.ndltd.org:IBICT/oai:www.arca.fiocruz.br:icict/12859 |
| Date | January 2015 |
| Creators | Silva, Marianne Rocha Simões |
| Contributors | Santos, Eduardo Caio Torres dos, Cardoso, André Gustavo Tempone, Calvet, Cláudia, Boechat, Nubia, Soeiro, Maria de Nazaré Correia |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Source | reponame:Repositório Institucional da FIOCRUZ, instname:Fundação Oswaldo Cruz, instacron:FIOCRUZ |
| Rights | info:eu-repo/semantics/openAccess |
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