title: Multiple ligand detection and affinity measurement by ultrafiltration and mass spectrometry analysis applied to fragment mixture screening.
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.
authors: 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.
authors: Shanshan
authors: S
authors: Qin
authors: High-throughput Molecular Drug Discovery Center, Tianjin Joint Academy of Biotechnology and Medicine, Tianjin 300457, China.
authors: Yiran
authors: Y
authors: Ren
authors: High-throughput Molecular Drug Discovery Center, Tianjin Joint Academy of Biotechnology and Medicine, Tianjin 300457, China.
authors: Xu
authors: X
authors: Fu
authors: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors: Jie
authors: J
authors: Shen
authors: High-throughput Molecular Drug Discovery Center, Tianjin Joint Academy of Biotechnology and Medicine, Tianjin 300457, China.
authors: Xin
authors: X
authors: Chen
authors: High-throughput Molecular Drug Discovery Center, Tianjin Joint Academy of Biotechnology and Medicine, Tianjin 300457, China.
authors: Quan
authors: Q
authors: Wang
authors: Institute of Elemento-organic Chemistry, Nankai University, Tianjin 300071, China.
authors: Xin
authors: X
authors: Bi
authors: High-throughput Molecular Drug Discovery Center, Tianjin Joint Academy of Biotechnology and Medicine, Tianjin 300457, China.
authors: Wenjing
authors: W
authors: Liu
authors: High-throughput Molecular Drug Discovery Center, Tianjin Joint Academy of Biotechnology and Medicine, Tianjin 300457, China.
authors: Lixin
authors: L
authors: Li
authors: Institute of Elemento-organic Chemistry, Nankai University, Tianjin 300071, China.
authors: Guangxin
authors: G
authors: Liang
authors: High-throughput Molecular Drug Discovery Center, Tianjin Joint Academy of Biotechnology and Medicine, Tianjin 300457, China.
authors: Cheng
authors: C
authors: Yang
authors: 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.
authors: Wenqing
authors: W
authors: Shui
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry* / Q000494:pharmacology*; D002853:Chromatography, Liquid / Q000191:economics / Q000379:methods; D055808:Drug Discovery / Q000191:economics / Q000379:methods*; D016174:Hepacivirus / Q000187:drug effects / Q000378:metabolism*; D006526:Hepatitis C / Q000188:drug therapy / Q000821:virology*; D057166:High-Throughput Screening Assays / Q000191:economics / Q000379:methods; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry / Q000191:economics / Q000379:methods; D011485:Protein Binding; D015203:Reproducibility of Results; D014462:Ultrafiltration / Q000191:economics / Q000379:methods; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D057188:Workflow; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D008024:Ligands; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: Affinity measurement; Fragment library screening; Multiple ligand detection; NS5B; Ultrafiltration-LC/MS