Multiple ligand detection and affinity measurement by ultrafiltration and mass spectrometry analysis applied to fragment mixture screening |
| |
Authors: | Shanshan Qin Yiran Ren Xu Fu Jie Shen Xin Chen Quan Wang Xin Bi Wenjing Liu Lixin Li Guangxin Liang Cheng Yang Wenqing Shui |
| |
Institution: | 1. Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China;2. High-throughput Molecular Drug Discovery Center, Tianjin Joint Academy of Biotechnology and Medicine, Tianjin 300457, China;3. Institute of Elemento-organic Chemistry, Nankai University, Tianjin 300071, China |
| |
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 Kd determination through UF-LC/MS by comparison with classical ITC measurement. A single-point Kd 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: | Ultrafiltration-LC/MS Multiple ligand detection Affinity measurement Fragment library screening NS5B |
本文献已被 ScienceDirect 等数据库收录! |
|