Ligand screening assay for the enzyme kallikrein immobilized on NHS-activated Sepharose
Autor(a) principal: | |
---|---|
Data de Publicação: | 2021 |
Outros Autores: | , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1016/j.jpba.2021.114026 http://hdl.handle.net/11449/210276 |
Resumo: | Human tissue kallikreins (KLKs) are serine proteases involved in various physiological and pathological conditions, including cancer and neurological disorders. These enzymes constitute attractive drug targets, which has stimulated the search for new KLK inhibitors. In this study, we have covalently immobilized porcine pancreas KLK on an NHS-activated Sepharose matrix, to obtain KLK-Sepharose-NHS. The immobilized enzyme showed high recovered activity and maintained the ability of free KLK to recognize the synthetic substrate Z-Phe-Arg-AMC (K-Mapp = 10.3 +/- 0.9 mu M). As proof of concept, we used leupeptin as a reference inhibitor to perform inhibition studies for KLK-Sepharose-NHS and to determine the halfmaximal inhibitory concentration (IC50 = 0.13 +/- 0.01 mu M), the inhibition constant (K-i = 0.06 mu M), and the leupeptin inhibition mechanism. We evaluated several complex matrixes (plant crude extract) by the same bioassay, to demonstrate their applicability. The species Solanum lycocarpum, Stryphnodendron adstringens, and Psychotria carthagenensis gave the best results. KLK-Sepharose-NHS was fully active after six consecutive reaction cycles and retained about 60 % of its initial activity after being used for at least five months, so the bioassay developed herein is a promising strategy to screen and to identify KLK ligands. (C) 2021 Elsevier B.V. All rights reserved. |
id |
UNSP_71ba11af2d7161473b46ae66f6185f12 |
---|---|
oai_identifier_str |
oai:repositorio.unesp.br:11449/210276 |
network_acronym_str |
UNSP |
network_name_str |
Repositório Institucional da UNESP |
repository_id_str |
2946 |
spelling |
Ligand screening assay for the enzyme kallikrein immobilized on NHS-activated SepharoseKallikreinImmobilized enzymeLigand screeningHuman tissue kallikreins (KLKs) are serine proteases involved in various physiological and pathological conditions, including cancer and neurological disorders. These enzymes constitute attractive drug targets, which has stimulated the search for new KLK inhibitors. In this study, we have covalently immobilized porcine pancreas KLK on an NHS-activated Sepharose matrix, to obtain KLK-Sepharose-NHS. The immobilized enzyme showed high recovered activity and maintained the ability of free KLK to recognize the synthetic substrate Z-Phe-Arg-AMC (K-Mapp = 10.3 +/- 0.9 mu M). As proof of concept, we used leupeptin as a reference inhibitor to perform inhibition studies for KLK-Sepharose-NHS and to determine the halfmaximal inhibitory concentration (IC50 = 0.13 +/- 0.01 mu M), the inhibition constant (K-i = 0.06 mu M), and the leupeptin inhibition mechanism. We evaluated several complex matrixes (plant crude extract) by the same bioassay, to demonstrate their applicability. The species Solanum lycocarpum, Stryphnodendron adstringens, and Psychotria carthagenensis gave the best results. KLK-Sepharose-NHS was fully active after six consecutive reaction cycles and retained about 60 % of its initial activity after being used for at least five months, so the bioassay developed herein is a promising strategy to screen and to identify KLK ligands. (C) 2021 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, Grp Cromatog Bioafinidade & Prod Nat, BR-14040901 Ribeirao Preto, SP, BrazilUniv Estadual Paulista, Fac Ciencias, Dept Quim, BR-17033360 Bauru, SP, BrazilUniv Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Biol, BR-14040901 Ribeirao Preto, SP, BrazilUniv Estadual Paulista, Fac Ciencias, Dept Quim, BR-17033360 Bauru, SP, BrazilFAPESP: 2019/05363-0FAPESP: 2014/50249-8FAPESP: 2016/06260-2CNPq: 141748/2017-6CNPq: 303723/2018-1CNPq: 311969/2019-4CAPES: 001Elsevier B.V.Universidade de São Paulo (USP)Universidade Estadual Paulista (Unesp)Carvalho, Daniella Romano deXimenes, Valdecir Farias [UNESP]Groppo, MiltonCardoso, Carmen Lucia2021-06-25T15:03:24Z2021-06-25T15:03:24Z2021-05-30info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article7http://dx.doi.org/10.1016/j.jpba.2021.114026Journal Of Pharmaceutical And Biomedical Analysis. Amsterdam: Elsevier, v. 199, 7 p., 2021.0731-7085http://hdl.handle.net/11449/21027610.1016/j.jpba.2021.114026WOS:000644480600014Web of Sciencereponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengJournal Of Pharmaceutical And Biomedical Analysisinfo:eu-repo/semantics/openAccess2021-10-23T20:11:45Zoai:repositorio.unesp.br:11449/210276Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462021-10-23T20:11:45Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Ligand screening assay for the enzyme kallikrein immobilized on NHS-activated Sepharose |
title |
Ligand screening assay for the enzyme kallikrein immobilized on NHS-activated Sepharose |
spellingShingle |
Ligand screening assay for the enzyme kallikrein immobilized on NHS-activated Sepharose Carvalho, Daniella Romano de Kallikrein Immobilized enzyme Ligand screening |
title_short |
Ligand screening assay for the enzyme kallikrein immobilized on NHS-activated Sepharose |
title_full |
Ligand screening assay for the enzyme kallikrein immobilized on NHS-activated Sepharose |
title_fullStr |
Ligand screening assay for the enzyme kallikrein immobilized on NHS-activated Sepharose |
title_full_unstemmed |
Ligand screening assay for the enzyme kallikrein immobilized on NHS-activated Sepharose |
title_sort |
Ligand screening assay for the enzyme kallikrein immobilized on NHS-activated Sepharose |
author |
Carvalho, Daniella Romano de |
author_facet |
Carvalho, Daniella Romano de Ximenes, Valdecir Farias [UNESP] Groppo, Milton Cardoso, Carmen Lucia |
author_role |
author |
author2 |
Ximenes, Valdecir Farias [UNESP] Groppo, Milton Cardoso, Carmen Lucia |
author2_role |
author author author |
dc.contributor.none.fl_str_mv |
Universidade de São Paulo (USP) Universidade Estadual Paulista (Unesp) |
dc.contributor.author.fl_str_mv |
Carvalho, Daniella Romano de Ximenes, Valdecir Farias [UNESP] Groppo, Milton Cardoso, Carmen Lucia |
dc.subject.por.fl_str_mv |
Kallikrein Immobilized enzyme Ligand screening |
topic |
Kallikrein Immobilized enzyme Ligand screening |
description |
Human tissue kallikreins (KLKs) are serine proteases involved in various physiological and pathological conditions, including cancer and neurological disorders. These enzymes constitute attractive drug targets, which has stimulated the search for new KLK inhibitors. In this study, we have covalently immobilized porcine pancreas KLK on an NHS-activated Sepharose matrix, to obtain KLK-Sepharose-NHS. The immobilized enzyme showed high recovered activity and maintained the ability of free KLK to recognize the synthetic substrate Z-Phe-Arg-AMC (K-Mapp = 10.3 +/- 0.9 mu M). As proof of concept, we used leupeptin as a reference inhibitor to perform inhibition studies for KLK-Sepharose-NHS and to determine the halfmaximal inhibitory concentration (IC50 = 0.13 +/- 0.01 mu M), the inhibition constant (K-i = 0.06 mu M), and the leupeptin inhibition mechanism. We evaluated several complex matrixes (plant crude extract) by the same bioassay, to demonstrate their applicability. The species Solanum lycocarpum, Stryphnodendron adstringens, and Psychotria carthagenensis gave the best results. KLK-Sepharose-NHS was fully active after six consecutive reaction cycles and retained about 60 % of its initial activity after being used for at least five months, so the bioassay developed herein is a promising strategy to screen and to identify KLK ligands. (C) 2021 Elsevier B.V. All rights reserved. |
publishDate |
2021 |
dc.date.none.fl_str_mv |
2021-06-25T15:03:24Z 2021-06-25T15:03:24Z 2021-05-30 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1016/j.jpba.2021.114026 Journal Of Pharmaceutical And Biomedical Analysis. Amsterdam: Elsevier, v. 199, 7 p., 2021. 0731-7085 http://hdl.handle.net/11449/210276 10.1016/j.jpba.2021.114026 WOS:000644480600014 |
url |
http://dx.doi.org/10.1016/j.jpba.2021.114026 http://hdl.handle.net/11449/210276 |
identifier_str_mv |
Journal Of Pharmaceutical And Biomedical Analysis. Amsterdam: Elsevier, v. 199, 7 p., 2021. 0731-7085 10.1016/j.jpba.2021.114026 WOS:000644480600014 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Journal Of Pharmaceutical And Biomedical Analysis |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
7 |
dc.publisher.none.fl_str_mv |
Elsevier B.V. |
publisher.none.fl_str_mv |
Elsevier B.V. |
dc.source.none.fl_str_mv |
Web of Science reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
_version_ |
1797790307638575104 |