Análise do alvo predito da plumieridina em Cryptococcus neoformans
Cryptococcosis is a fungal infection caused by yeast from Cryptococcus spp. The infection starts when desiccated cells or spores are inhaled and reach the lungs. If the disease is not properly treated, the infection can progress, reach the central nervous system and result in meningococcal mening...
Na minha lista:
| Autor principal: | |
|---|---|
| Outros Autores: | |
| Formato: | Dissertação |
| Idioma: | pt_BR |
| Publicado em: |
Universidade Federal do Rio Grande do Norte
|
| Assuntos: | |
| Endereço do item: | https://repositorio.ufrn.br/jspui/handle/123456789/29588 |
| Tags: |
Adicionar Tag
Sem tags, seja o primeiro a adicionar uma tag!
|
| id |
ri-123456789-29588 |
|---|---|
| record_format |
dspace |
| institution |
Repositório Institucional |
| collection |
RI - UFRN |
| language |
pt_BR |
| topic |
Cryptococcus neoformans Plumieridina Antifúngico Bioinformática Triagem virtual Alvo de drogas Quitinase GH18 CNPQ::CIENCIAS BIOLOGICAS |
| spellingShingle |
Cryptococcus neoformans Plumieridina Antifúngico Bioinformática Triagem virtual Alvo de drogas Quitinase GH18 CNPQ::CIENCIAS BIOLOGICAS Souza, Eden Silva e Análise do alvo predito da plumieridina em Cryptococcus neoformans |
| description |
Cryptococcosis is a fungal infection caused by yeast from Cryptococcus spp. The infection
starts when desiccated cells or spores are inhaled and reach the lungs. If the disease is not
properly treated, the infection can progress, reach the central nervous system and result in
meningococcal meningitis and even death. Cryptococcosis treatment is carried out in three
stages and uses three drugs: fluconazole, amphotericin B and 5-flucytosine. Although effective,
the use of these drugs can result in the emergence of fungal resistance, a toxic effect for patients,
and drugs as flucytosine are not commercialized worldwide. Thus, it is proposed to investigate
the mode of action of the antifungal compound plumieridine as well as the identification of its
molecular target in C. neoformans. For this, a series of experiments were carried out in vitro
and in silico. Initially, chromatographic fractions of the aqueous extract of Allamanda
polyantha seeds were subjected to antimicrobial activity tests. The fraction with antifungal
activity was subjected to nuclear magnetic resonance analysis of carbon and hydrogen in order
to identify compounds present in the sample. Antifungal activity, evaluated through antifungal
tests, was 0.250 mg/mL and the major component in the fraction was plumieridine. Through
virtual screening based on ligand’s similarity, chitinase was identified as the molecular target
of plumieridine. Three-dimensional models of chitinases from C. neoformans were created and,
through molecular docking, interaction with residues from the active site was observed.
Chitinolytic activity inhibition assays showed that the activity is significantly reduced in the
secreted fraction and soluble cell fraction. However, chitinolytic activity is little reduced by the
presence of plumieridine in the insoluble cell fraction, where higher concentrations of the
compound are needed. Although plumieridine is able to inhibit chitinolytic activity, the
compound does not appear to be related to the transcriptional levels of chitinases of C.
neoformans, reducing only the transcriptional levels of the CHI22 gene. It was observed that
crude extracts containing chitinases from mouse macrophages, Bacillus subitilis and Tenebrio
molitor are also inhibited in the presence of plumieridine. The treatment with plumieridine still
alters the distribution pattern of the chitooligomers in the cell wall: from a polarized pattern to
a diffuse pattern through the wall. The results validate virtual screening prediction and show
that the inhibition of chitinolytic activity by plumieridine results in incomplete cell division
and, consequently, antifungal activity. Finally, the results indicate that plumieridine inhibits
chitinase and causes death of C. neoformans, however, the inhibition also occurs in other
members of the GH18 family, indicating that this is a potential inhibitor of the GH18 family. |
| author2 |
Vainstein, Marilene Henning |
| author_facet |
Vainstein, Marilene Henning Souza, Eden Silva e |
| format |
masterThesis |
| author |
Souza, Eden Silva e |
| author_sort |
Souza, Eden Silva e |
| title |
Análise do alvo predito da plumieridina em Cryptococcus neoformans |
| title_short |
Análise do alvo predito da plumieridina em Cryptococcus neoformans |
| title_full |
Análise do alvo predito da plumieridina em Cryptococcus neoformans |
| title_fullStr |
Análise do alvo predito da plumieridina em Cryptococcus neoformans |
| title_full_unstemmed |
Análise do alvo predito da plumieridina em Cryptococcus neoformans |
| title_sort |
análise do alvo predito da plumieridina em cryptococcus neoformans |
| publisher |
Universidade Federal do Rio Grande do Norte |
| publishDate |
2020 |
| url |
https://repositorio.ufrn.br/jspui/handle/123456789/29588 |
| work_keys_str_mv |
AT souzaedensilvae analisedoalvopreditodaplumieridinaemcryptococcusneoformans |
| _version_ |
1773962264943001600 |
| spelling |
ri-123456789-295882020-07-19T07:45:19Z Análise do alvo predito da plumieridina em Cryptococcus neoformans Souza, Eden Silva e Vainstein, Marilene Henning Lima, João Paulo Matos Santos Barbosa, Euzébio Guimarães Souza, Gustavo Antonio de Staats, Charley Christian Cryptococcus neoformans Plumieridina Antifúngico Bioinformática Triagem virtual Alvo de drogas Quitinase GH18 CNPQ::CIENCIAS BIOLOGICAS Cryptococcosis is a fungal infection caused by yeast from Cryptococcus spp. The infection starts when desiccated cells or spores are inhaled and reach the lungs. If the disease is not properly treated, the infection can progress, reach the central nervous system and result in meningococcal meningitis and even death. Cryptococcosis treatment is carried out in three stages and uses three drugs: fluconazole, amphotericin B and 5-flucytosine. Although effective, the use of these drugs can result in the emergence of fungal resistance, a toxic effect for patients, and drugs as flucytosine are not commercialized worldwide. Thus, it is proposed to investigate the mode of action of the antifungal compound plumieridine as well as the identification of its molecular target in C. neoformans. For this, a series of experiments were carried out in vitro and in silico. Initially, chromatographic fractions of the aqueous extract of Allamanda polyantha seeds were subjected to antimicrobial activity tests. The fraction with antifungal activity was subjected to nuclear magnetic resonance analysis of carbon and hydrogen in order to identify compounds present in the sample. Antifungal activity, evaluated through antifungal tests, was 0.250 mg/mL and the major component in the fraction was plumieridine. Through virtual screening based on ligand’s similarity, chitinase was identified as the molecular target of plumieridine. Three-dimensional models of chitinases from C. neoformans were created and, through molecular docking, interaction with residues from the active site was observed. Chitinolytic activity inhibition assays showed that the activity is significantly reduced in the secreted fraction and soluble cell fraction. However, chitinolytic activity is little reduced by the presence of plumieridine in the insoluble cell fraction, where higher concentrations of the compound are needed. Although plumieridine is able to inhibit chitinolytic activity, the compound does not appear to be related to the transcriptional levels of chitinases of C. neoformans, reducing only the transcriptional levels of the CHI22 gene. It was observed that crude extracts containing chitinases from mouse macrophages, Bacillus subitilis and Tenebrio molitor are also inhibited in the presence of plumieridine. The treatment with plumieridine still alters the distribution pattern of the chitooligomers in the cell wall: from a polarized pattern to a diffuse pattern through the wall. The results validate virtual screening prediction and show that the inhibition of chitinolytic activity by plumieridine results in incomplete cell division and, consequently, antifungal activity. Finally, the results indicate that plumieridine inhibits chitinase and causes death of C. neoformans, however, the inhibition also occurs in other members of the GH18 family, indicating that this is a potential inhibitor of the GH18 family. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES Criptococose é uma infecção fúngica causada por leveduras de Cryptococcus spp. A infecção inicia-se quando células dessecadas ou esporos são inalados e chegam aos pulmões. Se a doença não for propriamente tratada, a infecção pode evoluir, atingir o sistema nervoso central e resultar em meningite meningocócica e até em óbito. O tratamento da criptococose é realizado em três estágios e faz uso de três drogas: fluconazol, anfotericina B e 5-flucitosina. Embora eficaz, o uso destas drogas pode resultar no surgimento de resistência fúngica, efeito tóxico para os pacientes e fármacos com a flucitosina não são comerciados em todos os países. Desta forma, propõe-se investigar o modo de ação do composto antifúngico plumieridina bem como a identificação do seu alvo molecular em C. neoformans. Para isso, realizou-se uma série de experimentos in vitro e in silico. Inicialmente, frações cromatográficas do extrato aquoso das sementes de Allamanda polyantha foram submetidas a ensaios de atividade antimicrobiana. Submeteu-se a fração com atividade antifúngica a análise de ressonância magnética nuclear de carbono e hidrogênio afim de se identificar compostos presentes na amostra. Atividade antifúngica, avaliada através de ensaios antifúngicos, foi de 0.250 mg/mL e o componente majoritário na fração foi a plumieridina. Através da triagem virtual baseada na similaridade do ligante, quitinase foi identificada como alvo molecular da plumieridina. Modelos tridimensionais das quitinases de C. neoformans foram criados e, através do atracamento molecular, observou-se a interação com resíduos do sítio ativo. Ensaios de inibição da atividade quitinolítica mostraram que a atividade é significativamente reduzida na fração secretada e fração celular solúvel. No entanto, a atividade quitinolítica é pouco reduzida pela presença de plumieridina na fração celular insolúvel, onde são necessárias maiores concentrações do composto. Embora plumieridina seja capaz de inibir a atividade quitinolítica, o composto não parece estar relacionado aos níveis transcricionais das quitinases de C. neoformans, reduzindo apenas os níveis transcricionais do gene CHI22. Observou-se que extratos contendo quitinases de macrófagos de camundongo, Bacillus subitilis e de Tenebrio molitor também são inibidos na presença de plumieridina. O tratamento com plumieridina ainda altera o padrão de distribuição dos quitooligômeros na parece celular: de um padrão polarizado para um padrão difuso pela parede. Os resultados validam a predição da triagem virtual e mostram que a inibição da atividade quitinolítica pela plumieridina resulta em divisão celular incompleta e, consequente, atividade antifúngica. Finalmente, os resultados indicam que a plumieridina inibe quitinase e cause morte de C. neoformans, entrentanto, a inibição também ocorre em outros membros da família GH18, indicando que este é um potencial inibidor de GH18. 2020-07-13T22:40:33Z 2020-07-13T22:40:33Z 2020-02-28 masterThesis SOUZA, Eden Silva e. Análise do alvo predito da plumieridina em Cryptococcus neoformans. 2020. 56f. Dissertação (Mestrado em Bioinformática) - Instituto Metrópole Digital, Universidade Federal do Rio Grande do Norte, Natal, 2020. https://repositorio.ufrn.br/jspui/handle/123456789/29588 pt_BR Acesso Aberto application/pdf Universidade Federal do Rio Grande do Norte Brasil UFRN PROGRAMA DE PÓS-GRADUAÇÃO EM BIOINFORMÁTICA |