| Influenza is an acute respiratory infectious disease caused by influenza virus.Seasonal influenza causes serious economic losses and social burdens worldwide every year.Today,influenza and COVID-19 coinfection increased the rate of severe illness and the risk of death.Although vaccines are the most effective way to prevent and control influenza,rapid mutation and unpredictable changes in antigens lead to a lag in vaccine prediction and production,which still requires the intervention of chemical drugs.Although a variety of small molecule drugs have been approved for the treatment of influenza,the rapid mutation of influenza virus has led to the emergence of drug resistance.For example,the M2-S31N mutant,NA-H274Y mutant and PA-13 8T mutant were resistant to Amantadine(M2 ion channel inhibitor),Oseltamivir(Neuraminidase inhibitor)and Baloxavir(PA inhibitor),respectively.Therefore,it is particularly urgent to use new technologies to discover new influenza virus inhibitors with new mechanisms and new skeletons.In our previous study,we first constructed a phenotypic screening method based on a cell model to screen our in-house compound library.This is the first time that ligustrazine-chalcone compounds have been found to have anti-influenza virus activity.The results of Time-of-Addition assay and immunofluorescence experiments showed that the anti-influenza mechanism of these compounds was different from that of the existing marketed drugs—they can interfere with viral replication by blocking the nuclear export of influenza viruses.This finding provides important clues for obtaining new skeletons and new mechanisms of influenza virus inhibitors.In the evaluation of antiviral activity at the cellular level,A9 containing 2,6-dimethoxyphenyl had the best activity(the EC50 values for H1N1 PR8,H1N1 pdm09,H3N2,Yamagata and Victoria strains were 3.51 μM,1.34 μM,3.69 μM,2.47μM and 3.87 μM,respectively),which was comparable to the positive control oseltamivir(the EC50 values for H1N1 PR8,H1N1 pdm09,H3N2,Yamagata and Victoria strains were 3.08 μM,>100 μM,1.08 μM,0.98μM and 2.43 μM,respectively).However,A9 has obvious cytotoxicity(CC50＝41.46 μM),which limits its further development.Unfortunately,the target and binding mode of these compounds have not been determined,which restricts target-based rational optimization of lead compounds.Therefore,to find more active influenza virus inhibitors with low toxicity,this thesis conducts multidimensional structural modification of lead compound A9.According to the preliminary structure-activity relationship obtained by phenotypic screening,this thesis retained the privileged skeleton 2,6-dimethoxyphenyl.The miscellaneous structure α,β-unsaturated ketone was modified by bioisosterism and scaffold-hoping strategy.Finally,we synthesized 25 novel target compounds in 5 classes of chemical types.In the subsequent cell-level activity test,most of the compounds had reduced cytotoxicity compared with A9.Among them,the compounds with linker as 1,4-diene-3-ones showed lower overall cytotoxicity(cell viability was greater than 95%at 50 μM).However,these compounds lost some antiviral activity,and the cell protection rate was only 70%at 50 μM,which still needs further optimization.Based on the results of the first round of optimization and activity test,2,6-dimethoxyphenyl and 1,4-diene-3-ones linker were regarded as privileged skeletons,so they were retained in the second round of optimization.In addition,the scaffold-hoping strategy was used to replace the tetramethylpyrazine ring with an insignificant contribution.We introduce alkyl chains with different lengths,five-and six-membered aliphatic heterocycles and aromatic heterocycles,which aims to enrich the structure-activity relationships,improve activity and reduce toxicity.Finally,we synthesized 23 novel target compounds in 4 classes of chemical types.The results of the cell level activity test showed that most of the compounds had no obvious cytotoxicity(cell survival rate was greater than 95%at 50 μM),and the anti-influenza activity was improved compared with the first series of compounds.Among them,morpholine ring-bearing compound IIB-2 showed micromolar activity against a variety of highly pathogenic strains(EC50 values for H1N1 PR8,H1N1 pdm09,H5N8 and Yamagata strains were 7.31 μM,4.56 μM,9.60 pM,and 4.82 μM,respectively).Although the activity was slightly weaker than that of A9,the toxicity was significantly reduced(CC50>100 μM),which lays a foundation for subsequent research and development.Based on the low toxicity and high efficiency of IIB-2,we localized and quantified influenza nucleoproteins by immunofluorescence and WB experiments.The results showed that compared with the blank control group,the content of nucleoproteins in the administration group was significantly reduced,and most of them aggregated in the host nucleus.Since the nucleoproteins have been assembled into a ribonucleoprotein complex(RNP)with the influenza RNA and polymerase during nuclear export.Therefore,the experimental results show that ⅡB-2 can inhibit the nuclear export of RNP.Subsequently,an immunofluorescence model based on cytidine deaminase was established to explore whether ⅡB-2 inhibits host-related nuclear export pathways.The results showed that ⅡB-2 did not affect the nuclear export of cytidine deaminase,ruling out the possibility that ⅡB-2 hindered the host nuclear export pathway and locked the target to influenza virus.Finally,surface plasmon resonance experiments showed that the affinity-dissociation trend of ⅡB-2 and influenza nucleoprotein changed in a concentration-dependent manner,which was consistent with the principle of steady-state affinity binding(Kd=0.2392 μM).This indicates that there is a specific binding between ⅡB-2 and nucleoprotein,and its target may be located in influenza nucleoprotein.In summary,this thesis takes A9 with new mechanism and new skeleton as the lead compound for further optimization.Although A9 exhibits comparable anti-influenza activity to oseltamivir,its obvious cytotoxicity seriously hinders subsequent drug development.Therefore,this study carried out two rounds of iterative optimization of A9 via bioisosterism and scaffold-hoping strategy.Subsequently,we obtained the target compound ⅡB-2 with slightly weaker activity than A9 but significantly reduced toxicity.Finally,we constructed relevant immunofluorescence,WB and surface plasmon resonance experiments to explore the preliminary mechanism and finally speculated that these compounds may act on the nucleoprotein of influenza virus.The work of this thesis provides lead compounds for the discovery of new anti-influenza drugs. |
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