Establishment of an at-line nanofractionation-based screening platform for rapid identification of influenza PAN/PAN I38T inhibitors from Artemisiae Argyi

The N -terminal domain of influenza viral polymerase (PA N ), a highly conserved region with critical catalytic function related to viral RNA replication and transcription, is considered as a very promising anti-influenza drug target. There is an urgent need for highly efficient and rapid screening methods to identify potential PA N inhibitors (PA N Is) from complex matrices. In this work, a novel high-throughput screening (HTS) platform was established by coupling high performance liquid chromatography and high-resolution mass spectrometry (HPLC-HRMS) with a fluorescence resonance energy transfer (FRET)-based endonuclease activity assay through an at-line nanofractionation system (ANF). The proposed screening platform could rapidly identify potential PA N Is from plant extracts with good sensitivity (baloxavir at the half maximal inhibitory concentration (IC 50 ) could be detected) and reliability ( Z ’ factor of 0.77). This platform was then successfully applied to the screening of potential inhibitors against PA N /PA N I38T from an aqueous extract of Artemisiae Argyi and 17 potential PA N Is were identified. Among them, three compounds (cynarine, isochlorogenic acid B and isochlorogenic acid C) showed comparable inhibitory activity against PA N and even better activity against PA N I38T compared to baloxavir. This study not only established a novel high-throughput ANF-based PA N Is screening platform, but also proved the feasibility to discover PA N Is from complex TCMs, which has a great potential in future anti-influenza drug discovery.