X-ray crystallographic fragment screening against the human prion protein
Autor(a) principal: | |
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Data de Publicação: | 2021 |
Tipo de documento: | Tese |
Idioma: | eng |
Título da fonte: | Biblioteca Digital de Teses e Dissertações da USP |
Texto Completo: | https://www.teses.usp.br/teses/disponiveis/60/60136/tde-27092021-150052/ |
Resumo: | Prion diseases result from the ordered accumulation of the misfolded conformer of cellular prion protein (PrPC), a glycosyl-phosphatidylinositol (GPI)-anchored protein expressed on the cell surface. The critical event in prion diseases is the conversion of PrPC into the self-propagating conformer scrapie prion protein, PrPSc, with resultant propagation and accumulation resulting in neuronal death and amyloidogenesis. Prognoses are devastating, with an average survival time of approximately one year after the onset of symptoms. Despite the tremendous efforts, PrP physiological function and its mechanism of conversion to PrPSc remain elusive. This research focuses on Xray crystallographic fragment screening technique to map PrP chemical spaces in order to find lead compounds as part of the drug discovery process. Screening against human PrP, currently stigmatized as an \"undruggable\" target, can benefit from the fragment screening strategy. This approach relies on low molecular weight compounds to scan the protein surface in search of binding spots in the protein, enhancing the chances of finding ligands that could offer an alternative route to quest a treatment to prion disease. Any hits could be explored to be used for either i) increase PrPC stabilization, increasing the energy barrier for the protein conversion, ii) destabilization, to induce PrP removal from the cell, thus reducing the quantity of PrP available for conversion, or iii) block protein-protein interaction sites between PrPC and PrPSc , inhibiting the conversion process. We have established a reproducible crystal system for which we collected over 1000 X-ray datasets and screened over 600 fragments. Our data shows two ligands interacting with the prion protein and reveal a pyrazole chemical binding motif for an unprecedented small cavity created by a conformational change of the Lys185 sidechain. The in silico analysis of the collected datasets showed that the globular domain of the PrP is unexpectedly rigid. To overcome the difficulty of finding PrP binder molecules, we performed a second fragment screening assay. The second screening was enabled by achieving a more fragment screening-friendly crystal. This search involved screening for a new crystal system, the use of a PrPspecific nanobody, and PEG-based conditions. Our second screening tested over 100 fragments, with no hits. Together, we believe that our work has the potential to provide structural basis to aid the drug discovery regarding the prion protein while also providing an in-depth analysis that can support other X-ray fragment screening endeavors. |
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X-ray crystallographic fragment screening against the human prion proteinVarredura de fragmentos por cristalografia de raios-X contra a proteína priônica humanaCristalografia de proteínasDescoberta de fármacos baseada em estruturaDoença priônicaFragment screeningFragment-based drug DiscoveryPrion diseasePrion proteinProtein crystallographyProteína priônicaVarredura de fragmentosPrion diseases result from the ordered accumulation of the misfolded conformer of cellular prion protein (PrPC), a glycosyl-phosphatidylinositol (GPI)-anchored protein expressed on the cell surface. The critical event in prion diseases is the conversion of PrPC into the self-propagating conformer scrapie prion protein, PrPSc, with resultant propagation and accumulation resulting in neuronal death and amyloidogenesis. Prognoses are devastating, with an average survival time of approximately one year after the onset of symptoms. Despite the tremendous efforts, PrP physiological function and its mechanism of conversion to PrPSc remain elusive. This research focuses on Xray crystallographic fragment screening technique to map PrP chemical spaces in order to find lead compounds as part of the drug discovery process. Screening against human PrP, currently stigmatized as an \"undruggable\" target, can benefit from the fragment screening strategy. This approach relies on low molecular weight compounds to scan the protein surface in search of binding spots in the protein, enhancing the chances of finding ligands that could offer an alternative route to quest a treatment to prion disease. Any hits could be explored to be used for either i) increase PrPC stabilization, increasing the energy barrier for the protein conversion, ii) destabilization, to induce PrP removal from the cell, thus reducing the quantity of PrP available for conversion, or iii) block protein-protein interaction sites between PrPC and PrPSc , inhibiting the conversion process. We have established a reproducible crystal system for which we collected over 1000 X-ray datasets and screened over 600 fragments. Our data shows two ligands interacting with the prion protein and reveal a pyrazole chemical binding motif for an unprecedented small cavity created by a conformational change of the Lys185 sidechain. The in silico analysis of the collected datasets showed that the globular domain of the PrP is unexpectedly rigid. To overcome the difficulty of finding PrP binder molecules, we performed a second fragment screening assay. The second screening was enabled by achieving a more fragment screening-friendly crystal. This search involved screening for a new crystal system, the use of a PrPspecific nanobody, and PEG-based conditions. Our second screening tested over 100 fragments, with no hits. Together, we believe that our work has the potential to provide structural basis to aid the drug discovery regarding the prion protein while also providing an in-depth analysis that can support other X-ray fragment screening endeavors.Doenças priônicas resultam do acúmulo de confôrmeros mal enovelados da proteína priônica celular (PrPC), uma proteína de ancoragem glicosilfosfatidilinositol, expressa na superfície de membranas celulares. O evento crítico da doença priônica é a conversão da PrPC em um confôrmero capaz de se automultiplicar, o príon scrapie, cuja propagação e acúmulo resulta em morte neuronal e amiloidogênese. O prognóstico da doença é devastador, com uma média de sobrevida de aproximadamente um ano após o início dos sintomas. Apesar dos tremendos esforços, a função e mecanismo de conversão da PrP ainda são desconhecidas. Esse trabalho teve como foco a utilização da técnica de varredura de fragmentos por cristalografia de raios-X para mapear o espaço químico da PrP para ajudar na busca de compostos de referência para o desenvolvimento de fármacos. A triagem da PrP, que atualmente carrega o estigma de proteína \"não-drogável\", pode se beneficiar da estratégia de empregar fragmentos como fontes de novos hits. Essa abordagem se baseia na utilização de moléculas de baixo peso molecular para rastrear a superfície da proteína na busca de regiões de interação, aumentando as chances de encontrar ligantes que possam oferecer uma rota alternativa para o tratamento da doença. Fragmentos identificados como ligantes podem ser explorados no i) aumento da estabilidade da PrPC, aumentando a barreira de energia para a conversão, ii) redução da estabilidade, induzindo a internalização e degradação da PrPC, iii) bloquear sítios de interação proteína-proteína entre a PrPC e a PrPSc, inibindo o processo de conversão. Nesse trabalho, foi estabelecido um protocolo reprodutível para produção e cristalização da PrP, para a qual mais de 1000 conjuntos de dados foram coletados e mais de 600 fragmentos foram testados. Os dados mostraram dois ligantes interagindo com a PrP, e revelaram o pirazol como motivo químico de interação em uma pequena cavidade gerada pela fechamento da cadeia lateral da Lys185 sobre o fragmento. A análise in silico dos dados coletados também revelaram uma rigidez inesperada para a região globular da PrP. Para superar a dificuldade em encontrar mais ligantes, foi realizada uma segunda varredura de fragmentos, que usou um cristal em condição mais favorável para esse tipo de experimentos. A busca por essa condição envolveu a varredura por uma nova forma cristalina, o uso de nano anticorpos específicos para PrP e condições com PEG. A segunda varredura de fragmentos testou mais de 100 fragmentos, sem positivos. Reunindo os dados gerados, o trabalho tem o potencial de ajudar na busca de fármacos baseado na estrutura da proteína priônica, além de providenciar uma análise profunda sobre varredura de fragmentos que pode auxiliar futuras campanhas.Biblioteca Digitais de Teses e Dissertações da USPEmery, Flavio da SilvaRangel, Victor Lopes2021-08-20info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisapplication/pdfhttps://www.teses.usp.br/teses/disponiveis/60/60136/tde-27092021-150052/reponame:Biblioteca Digital de Teses e Dissertações da USPinstname:Universidade de São Paulo (USP)instacron:USPLiberar o conteúdo para acesso público.info:eu-repo/semantics/openAccesseng2024-10-09T13:16:04Zoai:teses.usp.br:tde-27092021-150052Biblioteca Digital de Teses e Dissertaçõeshttp://www.teses.usp.br/PUBhttp://www.teses.usp.br/cgi-bin/mtd2br.plvirginia@if.usp.br|| atendimento@aguia.usp.br||virginia@if.usp.bropendoar:27212024-10-09T13:16:04Biblioteca Digital de Teses e Dissertações da USP - Universidade de São Paulo (USP)false |
dc.title.none.fl_str_mv |
X-ray crystallographic fragment screening against the human prion protein Varredura de fragmentos por cristalografia de raios-X contra a proteína priônica humana |
title |
X-ray crystallographic fragment screening against the human prion protein |
spellingShingle |
X-ray crystallographic fragment screening against the human prion protein Rangel, Victor Lopes Cristalografia de proteínas Descoberta de fármacos baseada em estrutura Doença priônica Fragment screening Fragment-based drug Discovery Prion disease Prion protein Protein crystallography Proteína priônica Varredura de fragmentos |
title_short |
X-ray crystallographic fragment screening against the human prion protein |
title_full |
X-ray crystallographic fragment screening against the human prion protein |
title_fullStr |
X-ray crystallographic fragment screening against the human prion protein |
title_full_unstemmed |
X-ray crystallographic fragment screening against the human prion protein |
title_sort |
X-ray crystallographic fragment screening against the human prion protein |
author |
Rangel, Victor Lopes |
author_facet |
Rangel, Victor Lopes |
author_role |
author |
dc.contributor.none.fl_str_mv |
Emery, Flavio da Silva |
dc.contributor.author.fl_str_mv |
Rangel, Victor Lopes |
dc.subject.por.fl_str_mv |
Cristalografia de proteínas Descoberta de fármacos baseada em estrutura Doença priônica Fragment screening Fragment-based drug Discovery Prion disease Prion protein Protein crystallography Proteína priônica Varredura de fragmentos |
topic |
Cristalografia de proteínas Descoberta de fármacos baseada em estrutura Doença priônica Fragment screening Fragment-based drug Discovery Prion disease Prion protein Protein crystallography Proteína priônica Varredura de fragmentos |
description |
Prion diseases result from the ordered accumulation of the misfolded conformer of cellular prion protein (PrPC), a glycosyl-phosphatidylinositol (GPI)-anchored protein expressed on the cell surface. The critical event in prion diseases is the conversion of PrPC into the self-propagating conformer scrapie prion protein, PrPSc, with resultant propagation and accumulation resulting in neuronal death and amyloidogenesis. Prognoses are devastating, with an average survival time of approximately one year after the onset of symptoms. Despite the tremendous efforts, PrP physiological function and its mechanism of conversion to PrPSc remain elusive. This research focuses on Xray crystallographic fragment screening technique to map PrP chemical spaces in order to find lead compounds as part of the drug discovery process. Screening against human PrP, currently stigmatized as an \"undruggable\" target, can benefit from the fragment screening strategy. This approach relies on low molecular weight compounds to scan the protein surface in search of binding spots in the protein, enhancing the chances of finding ligands that could offer an alternative route to quest a treatment to prion disease. Any hits could be explored to be used for either i) increase PrPC stabilization, increasing the energy barrier for the protein conversion, ii) destabilization, to induce PrP removal from the cell, thus reducing the quantity of PrP available for conversion, or iii) block protein-protein interaction sites between PrPC and PrPSc , inhibiting the conversion process. We have established a reproducible crystal system for which we collected over 1000 X-ray datasets and screened over 600 fragments. Our data shows two ligands interacting with the prion protein and reveal a pyrazole chemical binding motif for an unprecedented small cavity created by a conformational change of the Lys185 sidechain. The in silico analysis of the collected datasets showed that the globular domain of the PrP is unexpectedly rigid. To overcome the difficulty of finding PrP binder molecules, we performed a second fragment screening assay. The second screening was enabled by achieving a more fragment screening-friendly crystal. This search involved screening for a new crystal system, the use of a PrPspecific nanobody, and PEG-based conditions. Our second screening tested over 100 fragments, with no hits. Together, we believe that our work has the potential to provide structural basis to aid the drug discovery regarding the prion protein while also providing an in-depth analysis that can support other X-ray fragment screening endeavors. |
publishDate |
2021 |
dc.date.none.fl_str_mv |
2021-08-20 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://www.teses.usp.br/teses/disponiveis/60/60136/tde-27092021-150052/ |
url |
https://www.teses.usp.br/teses/disponiveis/60/60136/tde-27092021-150052/ |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
|
dc.rights.driver.fl_str_mv |
Liberar o conteúdo para acesso público. info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Liberar o conteúdo para acesso público. |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.coverage.none.fl_str_mv |
|
dc.publisher.none.fl_str_mv |
Biblioteca Digitais de Teses e Dissertações da USP |
publisher.none.fl_str_mv |
Biblioteca Digitais de Teses e Dissertações da USP |
dc.source.none.fl_str_mv |
reponame:Biblioteca Digital de Teses e Dissertações da USP instname:Universidade de São Paulo (USP) instacron:USP |
instname_str |
Universidade de São Paulo (USP) |
instacron_str |
USP |
institution |
USP |
reponame_str |
Biblioteca Digital de Teses e Dissertações da USP |
collection |
Biblioteca Digital de Teses e Dissertações da USP |
repository.name.fl_str_mv |
Biblioteca Digital de Teses e Dissertações da USP - Universidade de São Paulo (USP) |
repository.mail.fl_str_mv |
virginia@if.usp.br|| atendimento@aguia.usp.br||virginia@if.usp.br |
_version_ |
1815256539777204224 |