Structural and Functional Characterization of a New Bacterial Target Against Tuberculosis: The Phosphatase PtpA
Autor(a) principal: | |
---|---|
Data de Publicação: | 2019 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10362/88726 |
Resumo: | Tuberculosis (TB) is one of the top causes of death remaining a major public health problem worldwide. Mycobacterium tuberculosis is the agent of TB, infecting the human respiratory tract. Its remarkable pathogenicity hinges upon the ability to challenge the immune system of the host by secreting phosphatases into macrophages. Among them, Protein Tyrosine Phosphatase A (PtpA) plays a key role on the infection process, preventing the phagosome-lysosome fusion and promoting the inhibition of phagosome acidification. Thus, PtpA becomes a promising target for the development of new anti-TB drugs. The aim of this work is to contribute to find new structure-based drug design approaches against TB, studying the inhibitory properties of three different families of compounds towards PtpA – chalcones, thiosemicarbazones and azaindoles. The protein was overexpressed in E. coli – final yield of 20 mg protein/ liter of culture – and successfully purified using affinity chromatography. To provide new insights into the binding mode of the studied compounds, molecular docking studies were performed suggesting thiosemicarbazones as non-competitive inhibitors and the chalcones and azaindoles with a preferential active site binding. The protein was also biophysically characterized. The oligomeric state was confirmed by SEC, proving that PtpA is a monomer in solution. The protein stability was assessed through TSA revealing that, with 10% glycerol, PtpA resists to the effects of 10% DMSO. TSA was also used to find a suitable protein storage condition (-80°C) and to confirm PEG400 as an alternative solvent for the inhibitors. In addition, distinct biophysical approaches – TSA, MST and urea-gel electrophoresis – were implemented to detect protein-ligand interactions but definitive evidence were not obtained. Ligand-free and co-crystallization assays were extensively explored and several crystals were tested at the ESRF, Diamond and MAX IV. Two crystal structures were obtained: a co-crystallization PtpA-Lap11 structure at 3.6 Å resolution and a soaking PtpA-C33 structure at 2.8 Å resolution. Despite the low/medium resolution obtained, both structures reveal the potential binding of the inhibitors with suspicious density blobs near His120B for Lap11 and at the active site for C33. The ligands were preliminarily modelled but further refinement cycles are required to elucidate the respective binding. |
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Structural and Functional Characterization of a New Bacterial Target Against Tuberculosis: The Phosphatase PtpATuberculosisMycobacterium tuberculosisProtein Tyrosine Phosphatase A (PtpA)Chalcones and ThiosemicarbazonesChalcones and ThiosemicarbazonesIn silico studiesDomínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e TecnologiasTuberculosis (TB) is one of the top causes of death remaining a major public health problem worldwide. Mycobacterium tuberculosis is the agent of TB, infecting the human respiratory tract. Its remarkable pathogenicity hinges upon the ability to challenge the immune system of the host by secreting phosphatases into macrophages. Among them, Protein Tyrosine Phosphatase A (PtpA) plays a key role on the infection process, preventing the phagosome-lysosome fusion and promoting the inhibition of phagosome acidification. Thus, PtpA becomes a promising target for the development of new anti-TB drugs. The aim of this work is to contribute to find new structure-based drug design approaches against TB, studying the inhibitory properties of three different families of compounds towards PtpA – chalcones, thiosemicarbazones and azaindoles. The protein was overexpressed in E. coli – final yield of 20 mg protein/ liter of culture – and successfully purified using affinity chromatography. To provide new insights into the binding mode of the studied compounds, molecular docking studies were performed suggesting thiosemicarbazones as non-competitive inhibitors and the chalcones and azaindoles with a preferential active site binding. The protein was also biophysically characterized. The oligomeric state was confirmed by SEC, proving that PtpA is a monomer in solution. The protein stability was assessed through TSA revealing that, with 10% glycerol, PtpA resists to the effects of 10% DMSO. TSA was also used to find a suitable protein storage condition (-80°C) and to confirm PEG400 as an alternative solvent for the inhibitors. In addition, distinct biophysical approaches – TSA, MST and urea-gel electrophoresis – were implemented to detect protein-ligand interactions but definitive evidence were not obtained. Ligand-free and co-crystallization assays were extensively explored and several crystals were tested at the ESRF, Diamond and MAX IV. Two crystal structures were obtained: a co-crystallization PtpA-Lap11 structure at 3.6 Å resolution and a soaking PtpA-C33 structure at 2.8 Å resolution. Despite the low/medium resolution obtained, both structures reveal the potential binding of the inhibitors with suspicious density blobs near His120B for Lap11 and at the active site for C33. The ligands were preliminarily modelled but further refinement cycles are required to elucidate the respective binding.Silva, TeresaMarques, Maria ManuelRUNFernandes, Andreia Catarina Fernandes Pinto2021-09-01T00:30:21Z2019-11-1520192019-11-15T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/88726enginfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T04:39:25Zoai:run.unl.pt:10362/88726Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:36:52.808363Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Structural and Functional Characterization of a New Bacterial Target Against Tuberculosis: The Phosphatase PtpA |
title |
Structural and Functional Characterization of a New Bacterial Target Against Tuberculosis: The Phosphatase PtpA |
spellingShingle |
Structural and Functional Characterization of a New Bacterial Target Against Tuberculosis: The Phosphatase PtpA Fernandes, Andreia Catarina Fernandes Pinto Tuberculosis Mycobacterium tuberculosis Protein Tyrosine Phosphatase A (PtpA) Chalcones and Thiosemicarbazones Chalcones and Thiosemicarbazones In silico studies Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
title_short |
Structural and Functional Characterization of a New Bacterial Target Against Tuberculosis: The Phosphatase PtpA |
title_full |
Structural and Functional Characterization of a New Bacterial Target Against Tuberculosis: The Phosphatase PtpA |
title_fullStr |
Structural and Functional Characterization of a New Bacterial Target Against Tuberculosis: The Phosphatase PtpA |
title_full_unstemmed |
Structural and Functional Characterization of a New Bacterial Target Against Tuberculosis: The Phosphatase PtpA |
title_sort |
Structural and Functional Characterization of a New Bacterial Target Against Tuberculosis: The Phosphatase PtpA |
author |
Fernandes, Andreia Catarina Fernandes Pinto |
author_facet |
Fernandes, Andreia Catarina Fernandes Pinto |
author_role |
author |
dc.contributor.none.fl_str_mv |
Silva, Teresa Marques, Maria Manuel RUN |
dc.contributor.author.fl_str_mv |
Fernandes, Andreia Catarina Fernandes Pinto |
dc.subject.por.fl_str_mv |
Tuberculosis Mycobacterium tuberculosis Protein Tyrosine Phosphatase A (PtpA) Chalcones and Thiosemicarbazones Chalcones and Thiosemicarbazones In silico studies Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
topic |
Tuberculosis Mycobacterium tuberculosis Protein Tyrosine Phosphatase A (PtpA) Chalcones and Thiosemicarbazones Chalcones and Thiosemicarbazones In silico studies Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
description |
Tuberculosis (TB) is one of the top causes of death remaining a major public health problem worldwide. Mycobacterium tuberculosis is the agent of TB, infecting the human respiratory tract. Its remarkable pathogenicity hinges upon the ability to challenge the immune system of the host by secreting phosphatases into macrophages. Among them, Protein Tyrosine Phosphatase A (PtpA) plays a key role on the infection process, preventing the phagosome-lysosome fusion and promoting the inhibition of phagosome acidification. Thus, PtpA becomes a promising target for the development of new anti-TB drugs. The aim of this work is to contribute to find new structure-based drug design approaches against TB, studying the inhibitory properties of three different families of compounds towards PtpA – chalcones, thiosemicarbazones and azaindoles. The protein was overexpressed in E. coli – final yield of 20 mg protein/ liter of culture – and successfully purified using affinity chromatography. To provide new insights into the binding mode of the studied compounds, molecular docking studies were performed suggesting thiosemicarbazones as non-competitive inhibitors and the chalcones and azaindoles with a preferential active site binding. The protein was also biophysically characterized. The oligomeric state was confirmed by SEC, proving that PtpA is a monomer in solution. The protein stability was assessed through TSA revealing that, with 10% glycerol, PtpA resists to the effects of 10% DMSO. TSA was also used to find a suitable protein storage condition (-80°C) and to confirm PEG400 as an alternative solvent for the inhibitors. In addition, distinct biophysical approaches – TSA, MST and urea-gel electrophoresis – were implemented to detect protein-ligand interactions but definitive evidence were not obtained. Ligand-free and co-crystallization assays were extensively explored and several crystals were tested at the ESRF, Diamond and MAX IV. Two crystal structures were obtained: a co-crystallization PtpA-Lap11 structure at 3.6 Å resolution and a soaking PtpA-C33 structure at 2.8 Å resolution. Despite the low/medium resolution obtained, both structures reveal the potential binding of the inhibitors with suspicious density blobs near His120B for Lap11 and at the active site for C33. The ligands were preliminarily modelled but further refinement cycles are required to elucidate the respective binding. |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019-11-15 2019 2019-11-15T00:00:00Z 2021-09-01T00:30:21Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10362/88726 |
url |
http://hdl.handle.net/10362/88726 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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