Papaína imobilizada em partículas magnéticas: novos modelos de triagem de ligantes
Autor(a) principal: | |
---|---|
Data de Publicação: | 2019 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFSCAR |
Texto Completo: | https://repositorio.ufscar.br/handle/ufscar/11639 |
Resumo: | Cathepsins, human lysosomal enzymes, are associated with the development of various diseases, and cruzaine is an important enzyme in the development of the protozoan Trypanosoma cruzi, causes Chagas' disease. Cysteine proteases, cathepsins and cruzaine, are known as papain-like, due to their structural similarities and catalytic mechanism. Papain is a commercial enzyme, inexpensive and stable, and thus, it has been used as a model for screening assays for cysteine-proteases’ ligands. As the classical assays for these enzymes are spectrophotometrically-based, herein a direct method for measuring the enzymatic product of the reaction using liquid chromatography hyphenated to mass spectrometry is described. For that, new reaction conditions were employed. Under this condition, in solution papain showed a KM of 52.9 ± 3.12 μM. For developing new assay conditions and considering the advantages of use of immobilized enzymes, papain was covalently immobilized on to magnetic particles (P-MB), producing a bioreactor with a KM of 34.9 ± 3.59 μM. For qualifying P-PB screening assay, 10 known cysteine protease inhibitors including acridones, quinolinones, flavonoids, and dipeptidyl nitriles were screened. Maltose was used as a non-binder of papain. With the exception of maltose that showed no inhibitory capacity, in the solution assay (2,1 x 10-3 U) the percentage of inhibition ranged from 13 to 100%, while with P-MBs (1,4 x 10-3 U) the inhibition range was from 34 to 85%. Ligand Fishing assay, papain bioreactors showed affinity for all known binders of cysteine proteases, and no affinity for maltose. Six of them presented expressive affinity only ratio for the active P-MB, with no retention to inactive P-MB control. The other four binders varied their affinity ratio in the range of 2.0 to 3.5. With the ethanolic extract from the stem of Melia azedarach, 11 compounds were fished, among them catechin and epicatechin. The results herein described demonstrates that papain bioreactors can be used for screening cysteine-proteases’ ligands. |
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Leandro, Cleiton SilvaCass, Quezia Bezerrahttp://lattes.cnpq.br/9197210255594409http://lattes.cnpq.br/31853018130802362019-08-07T17:57:39Z2019-08-07T17:57:39Z2019-03-29LEANDRO, Cleiton Silva. Papaína imobilizada em partículas magnéticas: novos modelos de triagem de ligantes. 2019. Dissertação (Mestrado em Química) – Universidade Federal de São Carlos, São Carlos, 2019. Disponível em: https://repositorio.ufscar.br/handle/ufscar/11639.https://repositorio.ufscar.br/handle/ufscar/11639Cathepsins, human lysosomal enzymes, are associated with the development of various diseases, and cruzaine is an important enzyme in the development of the protozoan Trypanosoma cruzi, causes Chagas' disease. Cysteine proteases, cathepsins and cruzaine, are known as papain-like, due to their structural similarities and catalytic mechanism. Papain is a commercial enzyme, inexpensive and stable, and thus, it has been used as a model for screening assays for cysteine-proteases’ ligands. As the classical assays for these enzymes are spectrophotometrically-based, herein a direct method for measuring the enzymatic product of the reaction using liquid chromatography hyphenated to mass spectrometry is described. For that, new reaction conditions were employed. Under this condition, in solution papain showed a KM of 52.9 ± 3.12 μM. For developing new assay conditions and considering the advantages of use of immobilized enzymes, papain was covalently immobilized on to magnetic particles (P-MB), producing a bioreactor with a KM of 34.9 ± 3.59 μM. For qualifying P-PB screening assay, 10 known cysteine protease inhibitors including acridones, quinolinones, flavonoids, and dipeptidyl nitriles were screened. Maltose was used as a non-binder of papain. With the exception of maltose that showed no inhibitory capacity, in the solution assay (2,1 x 10-3 U) the percentage of inhibition ranged from 13 to 100%, while with P-MBs (1,4 x 10-3 U) the inhibition range was from 34 to 85%. Ligand Fishing assay, papain bioreactors showed affinity for all known binders of cysteine proteases, and no affinity for maltose. Six of them presented expressive affinity only ratio for the active P-MB, with no retention to inactive P-MB control. The other four binders varied their affinity ratio in the range of 2.0 to 3.5. With the ethanolic extract from the stem of Melia azedarach, 11 compounds were fished, among them catechin and epicatechin. The results herein described demonstrates that papain bioreactors can be used for screening cysteine-proteases’ ligands.As catepsinas, enzimas lisossomais humanas, estão associadas ao desenvolvimento de várias doenças, e a cruzaína é uma enzima importante no desenvolvimento do protozoário Trypanossoma Cruzi, causador da doença de Chagas. As catepsinas e a cruzaína são cisteíno proteases, conhecidas como papain-like, devido suas similaridades estruturais e mecanismo catalítico com a papaína. A papaína é uma enzima comercial, de baixo custo, estável, e foi utilizada como modelo no desenvolvimento de métodos de triagem para ligantes de cisteíno proteases. Uma vez que os métodos clássicos de triagem são espectrofotométricos, o ensaio aqui descrito mesura diretamente o produto da reação enzimática por cromatografia liquida acoplada a espectrometria de massa (LC-MS/MS). Neste contexto, novas condições reacionais foram determinadas. A papaína em solução apresentou um KM de 52,9 ± 3,12 μM. A fim de desenvolver um novo modelo de triagem, um biorreator de papaína, P-MB (KM de 34,9 ± 3,59 μM), foi produzido pela imobilização covalente da enzima em partículas magnéticas. Para qualificação do ensaio de prospecção de ligantes usando a P-MB, 10 inibidores conhecidos de cisteíno proteases (acridonas, quinolinonas, flavonoides e dipeptidil nitrilas) foram ensaiados com a papaína. Com exceção da maltose, com valor de inibição em torno de 3%, a porcentagem de inibição da papaína em solução (2,1 x 10-3 U), variou de 13 a 100%, enquanto com as P-MBs (1,4 x 10-3 U) a inibição variou entre 34 e 85%. No ensaio de Ligand Fishing, os biorreatores de papaína apresentaram afinidade por todos os ligantes conhecidos de cisteíno proteases, e não-afinidade com a maltose. Seis deles apresentaram expressivos valores de afinidade pela P-MB ativa, sem nenhuma retenção com a P-MB controle. Os outros quatro ligantes mostraram razão de afinidade entre 2,0 a 3,5. Com o extrato etanólico do caule de Melia azedarach foram bioconjugados 11 compostos entre eles a catequina e a epicatequina. Os resultados descritos nesse trabalho inferem que biorreatores de papaína podem ser usados em ensaios de triagem para ligantes de cisteíno proteases.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq: 132831/2017-1porUniversidade Federal de São CarlosCâmpus São CarlosPrograma de Pós-Graduação em Química - PPGQUFSCarPapaínaInibidoresLigand fishingCIENCIAS EXATAS E DA TERRA::QUIMICAPapaína imobilizada em partículas magnéticas: novos modelos de triagem de ligantesImmobilized papain on magnetic particles: new models of ligand screeninginfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesis12 meses após a data da defesainfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINALDissertação_CleitonSilvaLeandro.pdfDissertação_CleitonSilvaLeandro.pdfapplication/pdf1959605https://{{ getenv "DSPACE_HOST" "repositorio.ufscar.br" }}/bitstream/ufscar/11639/1/Disserta%c3%a7%c3%a3o_CleitonSilvaLeandro.pdff4107569606ffc8d8d93d64af13f8814MD51LICENSElicense.txtlicense.txttext/plain; charset=utf-81957https://{{ getenv "DSPACE_HOST" "repositorio.ufscar.br" }}/bitstream/ufscar/11639/3/license.txtae0398b6f8b235e40ad82cba6c50031dMD53TEXTDissertação_CleitonSilvaLeandro.pdf.txtDissertação_CleitonSilvaLeandro.pdf.txtExtracted texttext/plain154772https://{{ getenv "DSPACE_HOST" "repositorio.ufscar.br" }}/bitstream/ufscar/11639/4/Disserta%c3%a7%c3%a3o_CleitonSilvaLeandro.pdf.txtddb7d3b415b781831176d83e2a76ac66MD54THUMBNAILDissertação_CleitonSilvaLeandro.pdf.jpgDissertação_CleitonSilvaLeandro.pdf.jpgIM Thumbnailimage/jpeg8822https://{{ getenv "DSPACE_HOST" "repositorio.ufscar.br" }}/bitstream/ufscar/11639/5/Disserta%c3%a7%c3%a3o_CleitonSilvaLeandro.pdf.jpgf5bfd01dbb1a11a3e76250c52bdbd413MD55ufscar/116392020-08-05 22:33:23.536oai:repositorio.ufscar.br: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Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222020-08-05T22:33:23Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false |
dc.title.por.fl_str_mv |
Papaína imobilizada em partículas magnéticas: novos modelos de triagem de ligantes |
dc.title.alternative.eng.fl_str_mv |
Immobilized papain on magnetic particles: new models of ligand screening |
title |
Papaína imobilizada em partículas magnéticas: novos modelos de triagem de ligantes |
spellingShingle |
Papaína imobilizada em partículas magnéticas: novos modelos de triagem de ligantes Leandro, Cleiton Silva Papaína Inibidores Ligand fishing CIENCIAS EXATAS E DA TERRA::QUIMICA |
title_short |
Papaína imobilizada em partículas magnéticas: novos modelos de triagem de ligantes |
title_full |
Papaína imobilizada em partículas magnéticas: novos modelos de triagem de ligantes |
title_fullStr |
Papaína imobilizada em partículas magnéticas: novos modelos de triagem de ligantes |
title_full_unstemmed |
Papaína imobilizada em partículas magnéticas: novos modelos de triagem de ligantes |
title_sort |
Papaína imobilizada em partículas magnéticas: novos modelos de triagem de ligantes |
author |
Leandro, Cleiton Silva |
author_facet |
Leandro, Cleiton Silva |
author_role |
author |
dc.contributor.authorlattes.por.fl_str_mv |
http://lattes.cnpq.br/3185301813080236 |
dc.contributor.author.fl_str_mv |
Leandro, Cleiton Silva |
dc.contributor.advisor1.fl_str_mv |
Cass, Quezia Bezerra |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/9197210255594409 |
contributor_str_mv |
Cass, Quezia Bezerra |
dc.subject.por.fl_str_mv |
Papaína Inibidores |
topic |
Papaína Inibidores Ligand fishing CIENCIAS EXATAS E DA TERRA::QUIMICA |
dc.subject.eng.fl_str_mv |
Ligand fishing |
dc.subject.cnpq.fl_str_mv |
CIENCIAS EXATAS E DA TERRA::QUIMICA |
description |
Cathepsins, human lysosomal enzymes, are associated with the development of various diseases, and cruzaine is an important enzyme in the development of the protozoan Trypanosoma cruzi, causes Chagas' disease. Cysteine proteases, cathepsins and cruzaine, are known as papain-like, due to their structural similarities and catalytic mechanism. Papain is a commercial enzyme, inexpensive and stable, and thus, it has been used as a model for screening assays for cysteine-proteases’ ligands. As the classical assays for these enzymes are spectrophotometrically-based, herein a direct method for measuring the enzymatic product of the reaction using liquid chromatography hyphenated to mass spectrometry is described. For that, new reaction conditions were employed. Under this condition, in solution papain showed a KM of 52.9 ± 3.12 μM. For developing new assay conditions and considering the advantages of use of immobilized enzymes, papain was covalently immobilized on to magnetic particles (P-MB), producing a bioreactor with a KM of 34.9 ± 3.59 μM. For qualifying P-PB screening assay, 10 known cysteine protease inhibitors including acridones, quinolinones, flavonoids, and dipeptidyl nitriles were screened. Maltose was used as a non-binder of papain. With the exception of maltose that showed no inhibitory capacity, in the solution assay (2,1 x 10-3 U) the percentage of inhibition ranged from 13 to 100%, while with P-MBs (1,4 x 10-3 U) the inhibition range was from 34 to 85%. Ligand Fishing assay, papain bioreactors showed affinity for all known binders of cysteine proteases, and no affinity for maltose. Six of them presented expressive affinity only ratio for the active P-MB, with no retention to inactive P-MB control. The other four binders varied their affinity ratio in the range of 2.0 to 3.5. With the ethanolic extract from the stem of Melia azedarach, 11 compounds were fished, among them catechin and epicatechin. The results herein described demonstrates that papain bioreactors can be used for screening cysteine-proteases’ ligands. |
publishDate |
2019 |
dc.date.accessioned.fl_str_mv |
2019-08-07T17:57:39Z |
dc.date.available.fl_str_mv |
2019-08-07T17:57:39Z |
dc.date.issued.fl_str_mv |
2019-03-29 |
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.citation.fl_str_mv |
LEANDRO, Cleiton Silva. Papaína imobilizada em partículas magnéticas: novos modelos de triagem de ligantes. 2019. Dissertação (Mestrado em Química) – Universidade Federal de São Carlos, São Carlos, 2019. Disponível em: https://repositorio.ufscar.br/handle/ufscar/11639. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufscar.br/handle/ufscar/11639 |
identifier_str_mv |
LEANDRO, Cleiton Silva. Papaína imobilizada em partículas magnéticas: novos modelos de triagem de ligantes. 2019. Dissertação (Mestrado em Química) – Universidade Federal de São Carlos, São Carlos, 2019. Disponível em: https://repositorio.ufscar.br/handle/ufscar/11639. |
url |
https://repositorio.ufscar.br/handle/ufscar/11639 |
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por |
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por |
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info:eu-repo/semantics/openAccess |
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openAccess |
dc.publisher.none.fl_str_mv |
Universidade Federal de São Carlos Câmpus São Carlos |
dc.publisher.program.fl_str_mv |
Programa de Pós-Graduação em Química - PPGQ |
dc.publisher.initials.fl_str_mv |
UFSCar |
publisher.none.fl_str_mv |
Universidade Federal de São Carlos Câmpus São Carlos |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UFSCAR instname:Universidade Federal de São Carlos (UFSCAR) instacron:UFSCAR |
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Repositório Institucional da UFSCAR |
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