Related Experiment Video
Updated: Jan 17, 2026
High-throughput Detection Method for Influenza Virus
Published on: February 4, 2012
Multi-detector based at-line nanofractionation strategy accelerates discovery of influenza PA endonuclease inhibitors
Jiaming Yuan1, Yuexiang Chang1, Rongkai Gu2
1Institute of Pharmaceutical Analysis, College of Pharmacy/State Key Laboratory of Bioactive Molecules and Druggability Assessment/International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Ministry of Education (MOE) of China, Jinan University, Guangzhou 510632, China; KU Leuven, University of Leuven, Pharmaceutical Analysis, Department of Pharmaceutical and Pharmacological Sciences, O&N2, PB 923, Herestraat 49, 3000 Leuven, Belgium.
Abstract:
The highly conserved N-terminal domain of polymerase acidic endonuclease (PAN) makes it an attractive target for the development of anti-influenza virus drugs. This study developed an innovative multi-detector based at-line nanofractionation platform by coupling an HPLC system containing triple-detector (diode array detector (DAD), charged aerosol detector (CAD) and MS) with fluorescence resonance energy transfer (FRET)-based bioassays to systematically identify potential PAN inhibitors from traditional Chinese medicine (TCM). A preliminary screening of 230 TCM extracts revealed an exceptional PAN inhibition by Terminalia chebula Retz. (IC50 = 0.88 μg/mL). A time-resolved FRET bioassay in 384 well-plates with parallelized MS/MS analysis enabled real-time acquisition of both biological activity profiles and chemical information from Terminalia chebula Retz. extracts. HPLC-DAD-CAD analysis further elucidated the correlation between the content and bioactivity of the identified potential PAN-inhibitory compounds, addressing the limitations of traditional single UV-based detection. Remarkably, 31 bioactive compounds (including gallotannins, ellagitannins, and phenolic acids) were identified through this approach, with 75.9% exhibiting low abundance (CAD response < 1 pA·min), highlighting the unique capability of this platform in discovering low-abundant bioactive entities. Five verified compounds (ellagic acid, chebulinic acid, punicalagin, chebulagic acid, and 1,3,6-tri-O-galloyl-β-D-glucose) exhibited superior PAN inhibition (IC50 = 0.52-1.00 μM) in comparison to baloxavir marboxil (IC50 = 10.83 ± 1.46 μM). A molecular docking study elucidated their crucial PAN-binding interactions and fundamental mechanisms. Overall, this study not only identified several potential PAN inhibitors from Terminalia chebula Retz., but also offered a scalable at-line nanofractionation strategy to reveal the content-activity correlation of complex TCM extracts.
