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 (IC50) for ECG was 161.3 ± 1.0 μmol L-1.
Highlights
The success of a drug-discovery project depends upon having a well-characterized target and a good compound library, and efficient and appropriate screening technologies to identify valuable chemical starting points.[1]Nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) is an ubiquitous enzyme that catalyzes γ-phosphoryl transfer from a triphosphate nucleotide (NTP) to a diphosphate nucleotide (NDP), including 2’-desoxynucleoside.[2]
We report on successful covalent NME2 immobilization on open tubular silica capillary
The activities were evaluated by using 50 mmol L-1 citrate-phosphate buffer and 50 mmol L-1 Tris buffer. Method qualification Both the two-dimensional on-flow zonal affinity-based chromatography assay and the free NME2 activity assay were qualified according to Food and Drug Administration (FDA) bioanalytical guidelines.[34]
Summary
The success of a drug-discovery project depends upon having a well-characterized target and a good compound library, and efficient and appropriate screening technologies to identify valuable chemical starting points.[1]Nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) is an ubiquitous enzyme that catalyzes γ-phosphoryl transfer from a triphosphate nucleotide (NTP) to a diphosphate nucleotide (NDP), including 2’-desoxynucleoside.[2]. In vitro and in vivo studies have shown higher NME2 protein expression in several kinds of human cancers such as hepatocellular carcinoma, breast cancer, colorectal cancer, pancreatic cancer, lung cancer, endometrial cancer, and liver cell tumor.[7,9,10,11,12,13] More recently, NME2 inhibition by stauprimide has been demonstrated to down-regulate MYC transcription with consequent tumor growth inhibition in an animal model.[14] in vivo studies on NME2 blockade impact have revealed decreased tumor growth and metastasis in breast cancer.[15] NME2 knockdown has been shown to reduce cell survival and to increase cell apoptosis in colorectal cancer cells.[16] finding a small molecule that can inhibit NME2 activity may aid cancer therapy
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