On-Flow Ligand Screening Assay Based on Immobilized Nucleoside Diphosphate Kinase B from Homo sapiens
AUTOR(ES)
Lima, Juliana M.
FONTE
J. Braz. Chem. Soc.
DATA DE PUBLICAÇÃO
24/10/2019
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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