Discovery Of Novel Anti-Influenza Virus Lead Compounds Via Structure-Based Drug Design And Phenotypic Screening

The seasonal influenza can cause about 500,000 deaths annually in the world and the unpredictable influenza pandemics will bring havoc to human society.In recent years,animal-derived influenza viruses,especially avian influenza viruses,have been able to frequently cross the species barrier to infect human beings,which has dramatically increased the difficulty of influenza prevention and control.In addition,influenza virus can coexist with SARS-CoV-2 in the same host,and the severe case and mortality of co-infection are much higher than that of ones infection.Although three kinds of anti-influenza drugs have been licensed,their widespread application is subject to many factors,and the most serious of which is the drug resistance.Due to the inherent high variability of influenza viruses,mutant strains with drug-resistant properties always appear rapidly after the drugs enter clinical use,such as M2-S31N for amantadine,NA-H274Y for oseltamivir and PA-13 8T for baloxavir.Therefore,developing novel inhibitors is of great significance for the control of influenza.Influenza virus is an RNA virus that can encode at least 12 functional proteins,and blocking the function of these proteins is the basic strategy for the discovering of antiinfluenza drugs.Based on above,in this thesis,we carried out structure-based drug design by targeting the 150-cavity of NA and the PA-PB1 interaction of RNAdependent RNA polymerase to synthesize three classes of 71 novel target compounds and evaluate their anti-influenza activities and preliminary druggable properties.On the other hand,we used cell-based phenotypic screening method to screen the antiinfluenza activities of nearly 100 small molecules in our in-house compound library,and also discussed their mechanism of action.The dissertation is divided into five chapters,mainly containing three parts,which are as follow:1.Discovery of anti-influenza virus lead compounds via targeting the 150-cavity of NANA is a glycoprotein that can catalyze the cleavage of the glycosidic bond between the virus and the host cells and promote the release and spread of offspring virus particles.In order to discover novel NA inhibitors with improved potency,anti-resistant profile and druggability,in this chapter,based on the analysis of NA-oseltamivir crystal complex and the structure-activity relationship summarized by our previous work,we conducted a comprehensive exploration toward the newly discovered 150-cavity in Group-1 NAs,with C-5 amino group of oseltamivir as the modification site.First,with our previously reported compounds 14 and 16 as the lead compounds,we modified their C-5 side chain through using multi-site binding strategy and scaffold hopping strategy to occupy the entrance channel or/and the bottom region of the 150-cavity,and finally synthesized 21 fused-ring-containing oseltamivir derivatives.In the second section,we used substituent decorating strategy to carry out elaborate structural modification around the side chain of compound 14 by comprehensive analyzing the internal space of 150-cavity and the positional relationship between oseltamivir C-5 amino group and 150-cavity,and finally got 29 biphenyl-containing oseltamivir derivatives.Anti-influenza activity evaluation showed that compounds ⅠD-3d,ⅡB-4b and ⅡB-4e exhibited excellent potency at both enzymatic and cellular levels.The inhibitory activity of compound ID-3d(IC50=0.27-0.73 nM)on H1N1-NA,H5N1-NA and H5N8-NA was 14-53 times better than that of OSC(the active form of oseltamivir);the inhibitory activity of compound ⅡB-4b(IC50=0.12-0.91 nM)against these three NAs was 38-86 times better than OSC,and it was 10-69 times better than OSC for compound ⅡB-4e.Compounds ⅡB-4b and ⅡB-4e also showed great inhibition against H274Y-NA,which was the most common and the most serious resistant mutant to oseltamivir.As for against H1N1-H274Y and H5N1-H274Y NAs,the efficacy of ⅡB4b was 34-and 39-fold more potent than OSC,and the efficacy of ⅡB-4e was also 8and 51-fold more potent than OSC.Moreover,compounds ⅠD-3d,ⅡB-4b and ⅡB-4e also exhibited better anti-flu activities than OSC in cellular assays with CC50 values over 250 μM.Importantly,these three compounds displayed high metabolic stability in human liver microsomes and low inhibitory effect on main CYP enzymes,as well as low oral toxicity in Kunmign mice;and the pharmacokinetic studies showed that the oral bioavailability of ⅠD-3d,ⅡB-4b and ⅡB-4e in rats were 13.7%,11.8%and 3.9%respectively,which was better than or equivalent to OSC.Of note,in the embryonated egg model,ⅠD-3d,ⅡB-4b and ⅡB-4e displayed potent antiviral efficacy that could greatly improve the survival rate and prolong the survival time of the experimental animals.In a word,ⅠD-3d,ⅡB-4b and ⅡB-4e appear to be promising drug candidates for the treatment of influenza.2.Discovery of small molecule inhibitors via targeting the polymerase PA-PB1 interactionThis heterotrimer of RdRp is stabilized by the interactions between the PA,PB1 and PB2,and interfering the interactions can effectively destroy the activity of RdRp.In this chapter,we putted our attention on such protein-protein interactions and selected PA-PB1 interaction as the target for drug design.According to literature researches,we used the cycloheptathiophene-2-amino-3-carboxamide(cHTC),a privileged fragment with strong binding affinity with PA-PB 1 interaction interface,as the core structure,and introduced the favorable 2-pyridine moiety into its carboxamide side chain;meanwhile,we carry out elaborate structural modification on its amino side chain with the aim to occupy the hydrophobic interface of protein-protein interaction to the greatest extent.Finally,21 cHTC derivatives were synthesized.The ELISA assay indicated that 13 compounds in this series had a certain inhibitory effect on the PA-PB 1 interaction,and the most active compounds Ⅲ-6a and Ⅲ-6q had IC50 values of 75 μM and 85 μM.Moreover,Ⅲ-6a and Ⅲ-6q exerted broad-spectrum antiviral activity with EC50 values of 14.6-69.8 μM;although they were less potent than OSC,their potency was very close to compound 40,which was the most effective anti-influenza inhibitor with cHTC skeleton.In addition,Ⅲ-6a and Ⅲ-6q exhibited no cytotoxicity,and exerted certain anti-influenza virus activity in chicken embryo model.3.Discovery of novel anti-influenza virus lead compounds based on phenotypic screeningPhenotypic screening is an effective method to discover novel small molecules with specific pharmacological activities,of which cell-based phenotypic screening has attracted much more attention due to the efficiency,high-speed and convenience.In this part,we performed cell-based phenotypic screening to assess the anti-influenza activity of diarylpyrimidine(DAPY)derivatives and chalcone derivatives in our in-house compound library,and finally found that DAPY derivative A4 and chalcone derivative B23 possessed promising antiviral potency in cellular level.Mechanism of action studies showed that compound A4 could inhibit viral replication by promoting the nonfunctional aggregation of NP and blocking the nuclear import of NP,while compound B23 could exert antiviral effect by inhibiting the nuclear export of NP.Besides,the preliminary drugability evaluation indicated that A4 and B23 had favorable in vitro human plasma stability,low acute toxicity in mice,and acceptale inhibitory effects on several major CYP enzymes.Overall,compounds A4 and B23 not only displayed remarkable antiviral potency and drug-like properties,but also possessed brand new chemical scaffold and mechanism of action from approved drugs,so they could serve as anti-influenza virus lead compounds for further investigation.In summary,in order to discover novel,efficient and anti-resistant anti-influenza virus lead compounds,we carried out rational drug design by targeting the 150-cavity of NA and the PA-PB1 interaction of RdRp to synthesize three classes of 71 novel target compounds.Through biological evaluation and preliminary druggability assessment,three NA inhibitors(ⅠD-3d,ⅡB-4b and ⅡB-4e)with excellent antiviral efficacy and satisfactory druggability were found.On the other hand,DAPY derivative A4 and chalcone derivative B23 with promising anti-influenza virus activity were found through cell-based phenotype screening,and the mechanism studies showed that they could inhibit virus proliferation by inhibiting the nuclear transport of NP.All in all,in this thesis,we not only discovered promising anti-influenza drug candidates,but also obtained anti-influenza lead compounds with novel chemical scaffolds and mechanism of actions,and this laid a solid foundation and provided novel directions for the development of anti-influenza drugs.