Neuraminidase Enzyme Microreactor As A Drug Discovery Tool For Screening Anti-influenza Virus Agents

Influenza viruses cause annual epidemics and occasional pandemics that have claimed the lives of millions.Influenza neuraminidase(NA),a key enzyme in viral replication,spread,and pathogenesis,is considered to be one of the most promising targets for combating influenza.However,only three of these drugs are currently on the market and sudden changes in NAI susceptibility revealed the urgent need in the discovery of novel inhibitors.Natural product extracts are a rich source of bioactive compounds for target-oriented drug discovery in the battle against this highly contagious pathogen.Unfortunately,traditional screening approach of natural products for biologically active compounds is laborious,time consuming and costly.The screening of biological matrices for the identification of novel active components is nevertheless still challenging.Herein,we reported for the first time the synthesis and characterization of NA coated magnetic nanoparticles and their application as a drug discovery tool for the screening of compound libraries and plant extracts.Based on the enzymatic activity(hydrolysis ratio),the inhibitory activities of 12 compounds were screened.The results not only presented high enzymatic activity,good reusability and satisfactory stability of this tool,but also confirmed the desired anti-NA activity of flavonoids.Meanwhile,coupled with LC-MS/MS,a modified ligand fishing assay to isolate NA ligands from natural extracts was first developed.The mixture containing NA ligand(oseltamivir)and nonligands(lycorine,matrine)was used to testify the efficiency of the proposed fishing assay.Subsequently,four ligands(luteolin-7-O-β-D-glucoside,3,5-di-O-caffeoylquinic acid,3,4-di-O-caffeoylquinic acid and luteolin)were fished out from Flos Lonicerae which demonstrates the antiviral activity.This faster and reliable approach overcame the limitations of the conventional bioassay in natural extract screening,such as labor intensiveness,time consuming and large amount of sample consumption.Moreover,natural extracts are rich source of bioactive compounds and new drug lead molecules,this fishing techniques also profound significance under the essential situation that need for novel compounds targeting neuraminidase.This proposed protocol provides a facile and efficient neuraminidase inhibitor discovery tool.Further work can be focused on the identification of anti-influenza components from nature samples,and adapted to other essential targeting enzymes.