Analytica Chimica Acta 2015-07-30

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

Shanshan Qin, Yiran Ren, Xu Fu, Jie Shen, Xin Chen, Quan Wang, Xin Bi, Wenjing Liu, Lixin Li, Guangxin Liang, Cheng Yang, Wenqing Shui

Index: Anal. Chim. Acta 886 , 98-106, (2015)

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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.Copyright © 2015 Elsevier B.V. All rights reserved.