Establishment of an at-line nanofractionation-based screening platform for rapid identification of influenza PA_N/PA_N 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_NIs) 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_NIs from plant extracts with good sensitivity (baloxavir at the half maximal inhibitory concentration (IC₅₀) 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_NIs 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_NIs screening platform, but also proved the feasibility to discover PA_NIs from complex TCMs, which has a great potential in future anti-influenza drug discovery.