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Analytical and Bioanalytical Chemistry

Analytical and Bioanalytical Chemistry

IF: 3.8
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Development of a UHPLC-MS method for inhibitor screening against α-L-1,3-fucosidase

Published:1 February 2019 DOI: 10.1007/s00216-019-01575-x PMID: 30706074
Tangrong Liu, Ruonan Liu, Li Zhu, Xuan Zou, Huashi Guan, Zhe Xu

Abstract

α-L-Fucosidase (AFU) is a promising therapeutic target for the treatment of inflammation, cancer, cystic fibrosis, and fucosidosis. Some of the existing analytical methods for the assessment of AFU activity are lacking in sensitivity and selectivity, since most of them are based on spectrofluorimetric methods. More recently, mass spectrometry (MS) has evolved as a key technology for enzyme assays and inhibitor screening as it enables accurate monitoring of the conversion of substrate to product in enzymatic reactions. In this study, UHPLC-MS has been utilized to develop a simple, sensitive, and accurate assay for enzyme kinetics and inhibition studies of AFU3, a member of the AFU family. A reported method for analyzing saccharide involving a porous graphitic carbon column, combined with reduction by NaBH4/CH3OH, was used to improve sensitivity. The conversion of saccharide into alditol could reach nearly 100% in the NaBH4 reduction reaction. In addition, the bioanalytical quantitative screening method was validated according to US-FDA guidance, including selectivity, linearity, precision, accuracy, stability, and matrix effect. The developed method displayed a good accuracy, high sensitivity (LOD?=?0.05?mg?L?1), and good reproducibility (RSD <?15%). The assay accurately measured an IC50 value of 0.40?μM for the known AFU inhibitor, deoxyfuconojirimycin, which was consistent with results reported in the literature. Further validation of the assay was achieved through the determination of a high Z′-factor value of 0.89. The assay was applied to screen a marine-derived chemical library against AFU3, which revealed two marine-oriented pyrimidine alkaloids as potential AFU3 inhibitors.

Substances (8)

Materials
Procduct Name CAS Molecular Formula Supplier Price
1-Deoxynojirimycin 19130-96-2 C6H13NO4 479 suppliers $12.00-$3093.75
1-Deoxynojirimycin 19130-96-2 C6H13NO4 479 suppliers $12.00-$3093.75
1-Deoxynojirimycin 19130-96-2 C6H13NO4 479 suppliers $12.00-$3093.75
1-Deoxynojirimycin 19130-96-2 C6H13NO4 479 suppliers $12.00-$3093.75
Miglitol 72432-03-2 C8H17NO5 361 suppliers $9.00-$1504.50
Miglitol 72432-03-2 C8H17NO5 361 suppliers $9.00-$1504.50
Miglitol 72432-03-2 C8H17NO5 361 suppliers $9.00-$1504.50
Miglitol 72432-03-2 C8H17NO5 361 suppliers $9.00-$1504.50

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