On-Flow Ligand Screening Assay Based on Immobilized Nucleoside Diphosphate Kinase B from Homo sapiens

Detalhes bibliográficos
Autor(a) principal: Lima,Juliana M.
Data de Publicação: 2019
Outros Autores: Seidl,Claudia, Cunha,Elise M. F., Oliveira,Arthur H. C., Cardoso,Carmen L.
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Journal of the Brazilian Chemical Society (Online)
Texto Completo: http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0103-50532019001102308
Resumo: We describe an on-flow zonal affinity-based chromatography assay to screen ligands for the nucleoside diphosphate kinase B enzyme (NME2) from Homo sapiens. For the first time, we have covalently immobilized NME2 on the surface of an open fused silica capillary reactor (NME2-ICER) and placed the reactor before the analytical column, which resulted in a two-dimensional liquid chromatography-based system. We evaluated the pH effect on immobilized NME2 activity and carried out steady-state kinetic studies to compare free and immobilized NME2. Steady-state kinetic studies with the substrates adenosine 5’-triphosphate di(tris) salt dihydrate (ATP) and guanosine 5’-diphosphate sodium salt (GDP) resulted in apparent Michaelis-Menten constant values of 1136 and 713 mmol L-1, respectively. The ping-pong catalysis mechanism and substrate specificity were preserved after NME2 immobilization. By employing a reference inhibitor, (-)-epicatechin gallate (ECG), we verified the potential application of this method in NME2 ligand screening and NME2 inhibitor identification. The half maximum inhibitory concentration (IC50) for ECG was 161.3 ± 1.0 μmol L-1.
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spelling On-Flow Ligand Screening Assay Based on Immobilized Nucleoside Diphosphate Kinase B from Homo sapienstwo-dimensional chromatographykinase inhibitors assaytight-bindingping-pong mechanismWe describe an on-flow zonal affinity-based chromatography assay to screen ligands for the nucleoside diphosphate kinase B enzyme (NME2) from Homo sapiens. For the first time, we have covalently immobilized NME2 on the surface of an open fused silica capillary reactor (NME2-ICER) and placed the reactor before the analytical column, which resulted in a two-dimensional liquid chromatography-based system. We evaluated the pH effect on immobilized NME2 activity and carried out steady-state kinetic studies to compare free and immobilized NME2. Steady-state kinetic studies with the substrates adenosine 5’-triphosphate di(tris) salt dihydrate (ATP) and guanosine 5’-diphosphate sodium salt (GDP) resulted in apparent Michaelis-Menten constant values of 1136 and 713 mmol L-1, respectively. The ping-pong catalysis mechanism and substrate specificity were preserved after NME2 immobilization. By employing a reference inhibitor, (-)-epicatechin gallate (ECG), we verified the potential application of this method in NME2 ligand screening and NME2 inhibitor identification. The half maximum inhibitory concentration (IC50) for ECG was 161.3 ± 1.0 μmol L-1.Sociedade Brasileira de Química2019-11-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0103-50532019001102308Journal of the Brazilian Chemical Society v.30 n.11 2019reponame:Journal of the Brazilian Chemical Society (Online)instname:Sociedade Brasileira de Química (SBQ)instacron:SBQ10.21577/0103-5053.20190133info:eu-repo/semantics/openAccessLima,Juliana M.Seidl,ClaudiaCunha,Elise M. F.Oliveira,Arthur H. C.Cardoso,Carmen L.eng2019-10-18T00:00:00Zoai:scielo:S0103-50532019001102308Revistahttp://jbcs.sbq.org.brONGhttps://old.scielo.br/oai/scielo-oai.php||office@jbcs.sbq.org.br1678-47900103-5053opendoar:2019-10-18T00:00Journal of the Brazilian Chemical Society (Online) - Sociedade Brasileira de Química (SBQ)false
dc.title.none.fl_str_mv On-Flow Ligand Screening Assay Based on Immobilized Nucleoside Diphosphate Kinase B from Homo sapiens
title On-Flow Ligand Screening Assay Based on Immobilized Nucleoside Diphosphate Kinase B from Homo sapiens
spellingShingle On-Flow Ligand Screening Assay Based on Immobilized Nucleoside Diphosphate Kinase B from Homo sapiens
Lima,Juliana M.
two-dimensional chromatography
kinase inhibitors assay
tight-binding
ping-pong mechanism
title_short On-Flow Ligand Screening Assay Based on Immobilized Nucleoside Diphosphate Kinase B from Homo sapiens
title_full On-Flow Ligand Screening Assay Based on Immobilized Nucleoside Diphosphate Kinase B from Homo sapiens
title_fullStr On-Flow Ligand Screening Assay Based on Immobilized Nucleoside Diphosphate Kinase B from Homo sapiens
title_full_unstemmed On-Flow Ligand Screening Assay Based on Immobilized Nucleoside Diphosphate Kinase B from Homo sapiens
title_sort On-Flow Ligand Screening Assay Based on Immobilized Nucleoside Diphosphate Kinase B from Homo sapiens
author Lima,Juliana M.
author_facet Lima,Juliana M.
Seidl,Claudia
Cunha,Elise M. F.
Oliveira,Arthur H. C.
Cardoso,Carmen L.
author_role author
author2 Seidl,Claudia
Cunha,Elise M. F.
Oliveira,Arthur H. C.
Cardoso,Carmen L.
author2_role author
author
author
author
dc.contributor.author.fl_str_mv Lima,Juliana M.
Seidl,Claudia
Cunha,Elise M. F.
Oliveira,Arthur H. C.
Cardoso,Carmen L.
dc.subject.por.fl_str_mv two-dimensional chromatography
kinase inhibitors assay
tight-binding
ping-pong mechanism
topic two-dimensional chromatography
kinase inhibitors assay
tight-binding
ping-pong mechanism
description We describe an on-flow zonal affinity-based chromatography assay to screen ligands for the nucleoside diphosphate kinase B enzyme (NME2) from Homo sapiens. For the first time, we have covalently immobilized NME2 on the surface of an open fused silica capillary reactor (NME2-ICER) and placed the reactor before the analytical column, which resulted in a two-dimensional liquid chromatography-based system. We evaluated the pH effect on immobilized NME2 activity and carried out steady-state kinetic studies to compare free and immobilized NME2. Steady-state kinetic studies with the substrates adenosine 5’-triphosphate di(tris) salt dihydrate (ATP) and guanosine 5’-diphosphate sodium salt (GDP) resulted in apparent Michaelis-Menten constant values of 1136 and 713 mmol L-1, respectively. The ping-pong catalysis mechanism and substrate specificity were preserved after NME2 immobilization. By employing a reference inhibitor, (-)-epicatechin gallate (ECG), we verified the potential application of this method in NME2 ligand screening and NME2 inhibitor identification. The half maximum inhibitory concentration (IC50) for ECG was 161.3 ± 1.0 μmol L-1.
publishDate 2019
dc.date.none.fl_str_mv 2019-11-01
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0103-50532019001102308
url http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0103-50532019001102308
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 10.21577/0103-5053.20190133
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv text/html
dc.publisher.none.fl_str_mv Sociedade Brasileira de Química
publisher.none.fl_str_mv Sociedade Brasileira de Química
dc.source.none.fl_str_mv Journal of the Brazilian Chemical Society v.30 n.11 2019
reponame:Journal of the Brazilian Chemical Society (Online)
instname:Sociedade Brasileira de Química (SBQ)
instacron:SBQ
instname_str Sociedade Brasileira de Química (SBQ)
instacron_str SBQ
institution SBQ
reponame_str Journal of the Brazilian Chemical Society (Online)
collection Journal of the Brazilian Chemical Society (Online)
repository.name.fl_str_mv Journal of the Brazilian Chemical Society (Online) - Sociedade Brasileira de Química (SBQ)
repository.mail.fl_str_mv ||office@jbcs.sbq.org.br
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