On-Flow Ligand Screening Assay Based on Immobilized Nucleoside Diphosphate Kinase B from Homo sapiens
Autor(a) principal: | |
---|---|
Data de Publicação: | 2019 |
Outros Autores: | , , , |
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. |
id |
SBQ-2_ae28ff1a5a3aa96f9f00b554b57be150 |
---|---|
oai_identifier_str |
oai:scielo:S0103-50532019001102308 |
network_acronym_str |
SBQ-2 |
network_name_str |
Journal of the Brazilian Chemical Society (Online) |
repository_id_str |
|
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 |
_version_ |
1750318182197886976 |