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

Abstract

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 (IC 50) for ECG was 161.3 ± 1.0 µmol L-1 .

On-flow enzymatic inhibitor screening: The emerging success of liquid chromatography-based assays

“…Different solid supports can be employed for enzyme immobilization in the development of LC-based on-flow screening assays. Silica-based supports (de Moraes et al, 2012Vanzolini et al, 2013;da Silva et al, 2013;Hu et al, 2015;Silva et al, 2015;Vilela et al, 2015Vilela et al, , 2018Calil et al, 2016;Magalhães et al, 2016;Sarria et al, 2016;Ferreira Lopes Vilela and Cardoso, 2017;Cornelio et al, 2018;Lima et al, 2019;Medina et al, 2019;Qian et al, 2019;Seidl et al, 2019Seidl et al, , 2022Ferey et al, 2019;Chapla et al, 2020;Hou et al, 2020;de Castro et al, 2022) present a versatile and largely explored motif due presence of many hydroxyl groups on its surface. For example, those groups are readily reactive with 3aminopropyltriethoxysilane (APTES), furnishing an amine- rich surface that can further react with glutaraldehyde (GA), yielding a terminal reactive aldehyde surface susceptible reactions with nucleophilic groups present on the enzyme surface (Figure 2B).…”

Enzimas calicreína e ciclooxigenase imobilizadas: Estudo de condições para triagem de inibidores

“…Os IMERs também podem ainda ser acoplados a colunas analíticas e utilizados em ensaios on-line bidimensionais através do uso de válvulas de desvio (Figura 6B e 6C). Quando utilizado como pré-coluna, os compostos provenientes da catálise enzimática do IMER são transferidos diretamente para a coluna analítica, onde o excesso de substrato e o produto formado são separados na coluna analítica (CALIL et al, 2016;CORNELIO et al, 2018;LIMA et al, 2019). Em análises em que o IMER é utilizado como pós-coluna os analitos da amostra são inicialmente separados na coluna analítica e, em seguida, os compostos de interesse são transferidos para o biorreator, onde a atividade inibitória é avaliada (YUAN et al, 2020).…”