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Anal Chim Acta. 2015 Jul 30;886:98-106. doi: 10.1016/j.aca.2015.06.017. Epub 2015 Jul 9.

Multiple ligand detection and affinity measurement by ultrafiltration and mass spectrometry analysis applied to fragment mixture screening.

Author information

1
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; High-throughput Molecular Drug Discovery Center, Tianjin Joint Academy of Biotechnology and Medicine, Tianjin 300457, China.
2
High-throughput Molecular Drug Discovery Center, Tianjin Joint Academy of Biotechnology and Medicine, Tianjin 300457, China.
3
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
4
Institute of Elemento-organic Chemistry, Nankai University, Tianjin 300071, China.
5
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. Electronic address: shui_wq@tib.cas.cn.

Abstract

Binding affinity of a small molecule drug candidate to a therapeutically relevant biomolecular target is regarded the first determinant of the candidate's efficacy. Although the ultrafiltration-LC/MS (UF-LC/MS) assay enables efficient ligand discovery for a specific target from a mixed pool of compounds, most previous analysis allowed for relative affinity ranking of different ligands. Moreover, the reliability of affinity measurement for multiple ligands with UF-LC/MS has hardly been strictly evaluated. In this study, we examined the accuracy of K(d) determination through UF-LC/MS by comparison with classical ITC measurement. A single-point K(d) calculation method was found to be suitable for affinity measurement of multiple ligands bound to the same target when binding competition is minimized. A second workflow based on analysis of the unbound fraction of compounds was then developed, which simplified sample preparation as well as warranted reliable ligand discovery. The new workflow implemented in a fragment mixture screen afforded rapid and sensitive detection of low-affinity ligands selectively bound to the RNA polymerase NS5B of hepatitis C virus. More importantly, ligand identification and affinity measurement for mixture-based fragment screens by UF-LC/MS were in good accordance with single ligand evaluation by conventional SPR analysis. This new approach is expected to become a valuable addition to the arsenal of high-throughput screening techniques for fragment-based drug discovery.

KEYWORDS:

Affinity measurement; Fragment library screening; Multiple ligand detection; NS5B; Ultrafiltration-LC/MS

PMID:
26320641
DOI:
10.1016/j.aca.2015.06.017
[Indexed for MEDLINE]

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