The Inhibition Effect And Mechanism Of Oligothiophene And Saponin Derivatives On The Entry Of Influenza Virus Into Target Cells

BACKGROUD:Influenza A is an acute contagious respiratory disease caused by influenza A virus (IAV). Worldwide pandemic of influenza A has broken out for four times in the 20th century, including the 1918 Spanish flu, the number of people died in this pandemic were as many as 20～50 million. Since highly pathogenic H5N1 avian influenza broke out in Hong Kong in 1997, the world health organization (WHO) had reported 694 confirmed cases of H5N1 avian influenza virus infection until January 6th,2015, with 402 deaths, the mortality rate is as high as 57.92%, most of the death were young adults. A Novel influenza A virus (H1N1) had caused worldwide pandemic in 2009, with more than 1.3 million confirmed cases,14000 deaths, brought a great threat to human health. In 2013, many provinces (cities) and districts of China reported a lot of unknown severe pneumonia cases, which are confirmed infected by a novel H7N9 subtype of avian influenza virus,130 cases were reported until May 27, 2013, including 37 deaths, mortality rate close to 30%. The case showed that H5N1 and H7N9 avian influenza virus could overstep the host barrier from avian to infect human, and the existing vaccines were unable to prevent. In 2012, two scientific researches focusing on the transmissibility of H5N1 between mammals has shown that only four site mutations in the hemagglutinin lead influenza virus to be airborne-transmissible between mammal ferrets. Highly pathogenic H5N1 avian influenza virus which mutated to acquire of the airborne-transmission capacity among human would result in great social panic and much high mortality.According to the mechanism of action, currently virus-targeting anti-influenza drugs which are available on market can be divided into two categories:1) the M2 ion channel blockers, such as amantadine and rimantadine, which prevent virus uncoating by blocking the M2 ion channel. However, most influenza A viruses are resistant to both two drugs.2) The neuraminidase inhibitors (NAIs), zanamivir and Oseltamivir (Oseltamivir, tamiflu) approved by FDA, are currently the firstline anti-influenza drugs. NAIs inhibit the hydrolysis of sialic acid receptor by inhibiting the neuraminidase activity; therefore, progeny virus cannot be released. Although most patients infected by H5N1 influenza virus were treated with NAIs, the mortality rate remains undecreased; the NAIs drug-resistant strains are constantly being isolated. It is notable that, in April 2014, Tom Jefferson reported a comprehensive analysis of the clinical data opened by Roche and other clinical data, found that these data can’t prove NAIs can reduce mortality, hospitalization rates and rate of pneumonia in children caused by influenza.Therefore, there is an urgent need to dicover new drugs tareting novel viral targets for prevention and control of influenza. Hemagglutinin (HA) mediates virus entry into host cells at early stage of infection, HA-targeting entry inhibitors are the research hot spot of anti-influenza drugs. It is worthnoting that the virus entry inhibitors can reduce entry of virus into cells even neutralize the infectivy of influenza, and can effectively block the incidence and spread of IAV. Hemagglutinin is encoded by virus gene segment 4, its structure resembles the "mushroom" inlaid on the viral envelope, the spherical head of the "mushroom" is composed of three HA1 subunits, contains antigenic determinant and receptor binding sites, and can combine with sialic acid receptor at the end of glycoprotein on cell membrane surface; stem of "Mushroom" is mainly composed of 3 subunits of HA2, inserted into the envelope of the virus. Under the condition of acidity (pH 5.0-6.0) in endosome, HA1 subunits are rearranged to opened the pocket surrounding the fusion peptide (FP), at the same time, the B ring of HA2 subunits is changed into alpha helix structure, leading FP to pop up from the pocket and insert into membrane of endosome, energy released during the reconformation drags virus particle much closer to endosme, thus mediates membrane fusion, promoting viral neucleocapsid released into the plasma. However, there are many epitopes on HA recognized by host, in order to evade the host immune recognition, the HA1 subunits mutate extensively, only HA2 subunits are very conservative. The HA2-targeting fusion inhibitors may broadly inhibit influenza virus, thus reduces the possibility of evolution of drug-resistant strains.The main measures for prevention and control of influenza virus epidemic is to eliminate the source of infection, so application of virucidal agent during influenza pademic is very important. If the influenza virus entry inhibitors irreversibly combined with virus hemagglutinin, interfering hemagglutinin-mediated membrane fusion, may also inactivated influenza viruses. Disinfectants which can inactivate influenza virus can be divided into:surfactant, alkali, acid, chloride and its derivatives, the oxidant, aldehydes, phenolic compounds, alcohols and quaternary ammonium compounds (QACs). Most of disinfectants can inactivate virus only at temperature of 20 ℃ or above, and also are toxic or irritant to human body.These charicteristics greatly limit their application. In addition, the extensive existence of organic matters in environment greatly abolished the virucidal effect of alkali, oxidant, amine, aldehyde, chloride and its derivatives. Therefore, organic matters must be cleaned by surface cleaning to guarantee the virucidal effect of such disinectants. HA-targeting virucidals, however, of high specificity, probably do not require for strict temperature and concentration of organic matter in environment.On the research of virus entry inhibitors, currently HA-targeting pseudovirus screening system is used to dicover specific virus entry inhibitors. Applying H5N1 pseudovirus based high-throughput screening models and methods of molecular docking, our group has found two series of small molecular compounds which can specifically inhibit HA2 subunits, thus inhibiting viral entry into host cell. (1) Compound CL-385319 inhibits entry of H5N1 avian influenza virus through its benzene ring π-π covalently bonding with aromatic amino acid residues of HA2 subuint. Based on this target pocket, novel designed compound II with thiophene ring possesses significantly strongerπ-π covalently bonding force than CL385319, thus increased the inhibition activity of the H5N1 virus, indicates thiophene ring of compound II which π-π covalently bonding with HA is critical for the inhibition activity. (2) A series of small molecule saponins has been synthesized as entry inhibitors of H5N1 avian influenza virus, compound 3 (methyl ursolate saponin) is of the highest acitivity, further study of structure-activity relationship found that the chacotriose and aglycon moieties of compounds 1-3 both are indispensible for the inhibition activity.OBJECTIVES:hemagglutinin was exploited as a novel anti-influenza target, and a H5N1 pseudovirus-based high throughput screening method was establish in this study. Based on molecular skeletons of the virus entry inhibitors found in our laboratory, small molecular compounds were rational designed to be screened as influenza entry inhibitors. The mechanisms of active compounds were further researched; Due to the frequent mutating nature of hemagglutinin, the active compounds with confirmed mechanism were further screened against HlNl influenza virus. Specific compounds and design of screening library are as follows:1. Based on the mechanisim ofπ-πcovalently binding with target pocket of HA, we design the oligothiophene derivatives, the induction of oligothiophene may produce greaterπ-πcovalent bonding with HA, and the activity is likely to increase.2. Based on the influenza virus entry inhibitor compound 3, the structure-activity relationship by substitutions of aglycons or amidation and acylation surrounding aglycons is observer; Due to strict-dependency on oligosaccharide of the inhibitory activity,3-OH of aglycons was only changed from β conformation to a conformation.METHODS:1. The inhibitory activity of small compounds was screened with H5N1 pseudo virus screening modle:the plasmid HA which expresses hemagglutinin of influenza strain A/Thailand/Kan353/2004(H5N1) is critical for producing pseudovirus, thus was identified by sequencing; backbone plasmid pNL4.3 R-E-, envelope plasmid HA and NA were tansfected into 293T cells with PEI to produce H5N1 influenza virus; After infection of pseudovirus for 48h, the entry of pseudovirus was quantified by luminescence catalyzed by reporter luciferase expressed in infected MDCK cells.2. Cytotoxicity and virus-induced cytopathic effect (CPE) were detected by MTT assay:MDCK cells treated with small molecules for 48h, and the cytotoxicity was detected, and the concentration causing half cytotoxicity (CC50) was caiculated by Calccusyn software; MDCK cells were infected by influenza virus for 48h in the presence or absence of compounds, then CPE was detected and the concentration causing half reduction of CPE (IC50) was calculated with Reed-Muench method.3. Detection of bioactivity and titres of influenze virus based on envelope protein: Base on hemagglutinin, hemagglutinin titers of influenza virus and pseudovirus was detected, H5 subtye HA and its antiserum bought from Harbin veterinary research institute were used as control; Based on neuraminidase, neraminidase inhibitor screening kit or neurominidase activity assay provided by WHO Standard Operating Procedure were used respectively to detect neuraminidase activity. The neurominidase actitivy of cuture supernatant was quantified to reflect the level of virus.4. The specificity of HA-targeting was primarily confirmed with pseudovirus system:VSV pesudovirus with same backbone but different envelop asVSV-G was produced, if compound specifically inhibit H5N1 influenza virus rather than VSV pseudovirus than it probably target the specific envelope of influenza, if compound did not inhibit neuraminidase acitiviy, then it probably target HA rather than HA.5. The interaction with HA2 subunit was observed:interaction with HA1 subunit was observed with hemagglutination inhibition assay, compounds which did not inhibit hemagglutination was further explore for its interaction with HA protein and HA2 subunit by surface plasmon resonance (SPR).6. Observation of inhibitory effect and mechanism of active compounds against HIN1 influenza virus A/Puerto Rico/8/34(HlNl):viruses were propagated in chicken embryos, the TCID50 titers was calculated with Reed-Muench method; Inhibitory activity of compounds against H1N1 influenza viruses were observed in three drug delivery mode:drug presented in whole process, before adsorpption or after adsorption. Time of addition assay was used to explore wether the inhibition is restricted at early stage of infection; Viral proteins were detected with immunofluorescence assay.7. Blockade of viral infection to host was observed in mice modle.8. Data were analyzed with SPSS 19.0 software. Oneway ANOVA analysis, homogeneity of variance was analyzed with Levene t test, the difference between each two groups with homogeneity are compare LSD test, the difference between each two groups without homogeneity are compare Dunnet T3 test, and data were presented as "Mean±SD". Survival analysis was detected with the life table method; Weight-time curve data was analyzed with ANOVA analysis for repeated measurement data.RESULTS:1. Oligothiophene derivatives based on the π-πcovalently binding pocket in HA, derivatives of the virus entry inhibitor compound 3 inhibited H5N1 influenza pseudovirus, the IC50 of their inhibitory activity were 0.029-74 μM,0.98-22.5 μM repectively; The cytotoxivity of these compounds are very low, their selective index against H5N1 influenza pseudovirus were 2.08-1169.59,3.5-162.6 respectively2. Structure-acticity relationship analysis of oliogothiophene derivatives demonstated that, derivatives with 4 carbon linker chain were much more potent on inhibition of H5N1 pseudovirus than their counterparts with 3 carbon linker chain, the activity increased for 10-100 fold (3sf> 3sc,3sk> 3sh,3si> 3sg, 3se>3sb,2sd> 2sb); the number of thiophene cycles in the structure of most active compounds (ICso<1μM) such as 2sd,3sf,3si,3sk,4sc are no less than 2, indicated that oligothiophene is the basic scaffold of these derivative series.3. Structure-activity relationship analysis of saponin compounds revealed that amidation or acylation at 17-COOH of aglycons or conformational change of 3-OH of aglycons from β to a significantly improve the cytotoxicity and selectivity.4. Oligothiophene derivatives with 3 carbon linker chain (represented by 4sc) and saponin derivatives of compound 3 (represented by 15) was primarily speculated in pseudovirus system, as compounds 4sc and 15 did not inhibit VSV pseudovirus and NA activity.5. compounds 4sc and 15 did not inhibit hemagglutination induced by HA, indicated that they did not inhibit virus apsorption to cell receptor, thus may inhibt fusion mediated by HA2; molecular docking revealed that compound 15 bind with Lys-26、 Asn-50、 Asn-53、 Asp-57 in HA2 subunit.6. SPR detection revealed that ologothiophene compounds 4sc,3sc and saponin compounds 14,15 specifically binded with hemagglutinin, further study showed that 4sc、3sc、14、15 binded with HA2 subunit. Surprisingly, nonspecific entry inhibitor 3sf also binded with HA2 subunit.7. The anti-influenza spectrum of active compounds is expanded to H1N1 subtype influenza virus PR8:to be consistant with the compounds deliver mode in pseudovirus system, whole process deliver mode was applied, virus was pretreated with indicated compounds for 30 min, then was adsorbed to MDCK cells, the compounds was maintained after viral adsorption; under this deliver mode, oligothiophene compounds 3sf,3sk,3sc,4sc potently inhibited the progeny production and cytopathic effect of PR8 virus; however, representative saponin compounds 15 could not inhibited PR8 virus.8. The inhibition activity of oligothiophene compounds on influenza virus may be virucidal effect:under theraputical mode, oligothiophene compounds 3sf,3sc,4sc can not inhibited progeny production of PR8 virus; therefore direct deliver mode was applied, PR8 viruses pretreated with compounds 3sf,3sc,4sc for 30 min were adsorbed to MDCK cells for 1h, then culture medium was discarded and MDCK cells were washed twice after adsorption. The results showed that the infectivity of PR8 virus did not recovered after elution of compounds 3sf,3sc,4sc, indicated that the virucidal effect is not reversed by elution.9. HA-targeting compounds 4sc possesses the highest activity and selectivity of against influenza virus, was therefore further studied. Time of addition assay revealed that HA-targeting oligothiophene compound 4sc potently neutralized virus,4s could inhibited PR8 virus if only PR8 virus was pretreated with 4se, which was not effective even when added immediately after viral adsorption.10. The specificity of 4sc targeting virus was observed:owing to the treatment of 4sc on MDCK cells during the viral adsorption process, MDCK cells were pretreated with 4sc for 1h then washed twice with PBS, whereas 4sc-pretreated MDCK cells was not immune from virus infection; on the other hand, PR8 virus pretreated with 4sc could not recover infectivity after 4sc was diluted to ineffective concentrations.11. The influence of temperature and organic substance on virucidal effect of 4sc was observerd:the virus titer in allantoic fluid with aboundant organinc substance was decreased for io5.06±0.26 folds if incubated with 10 μM 4sc for 15 min; the concentration-effect curve of 4sc at 4 ℃ was close to the curve of 4sc at 37 ℃; Fetus bovine serum at concentration of 10% in the H5N1 pseudovirus system did not affect virucidal effct of 4sc.12. Mice were intranasally inoculated with PR8 virus at a dose of 5 LD50, and all died at day 7-10 after infection, the median survival time is 8.5 days; however 5 LD50 PR8 virus pretreated with 20 μM or 2 μM 4sc did not damage the mice significantly, no symptoms of infection was observed, no death was observed in day 12 after infection, and the bodyweight of mice increased normally.CONCLUSSION:1. Oligothiophene derivative 4sc based on the π-π covalently binding pocket and saponin derivatives of compound 3 inhibited H5N1 influenza pseudovirus through targeting HA2 subunit of hemagglutinin, the IC50 of the highest activity reached low nM and μM, repectively;2. Oligothiophene compound 4sc potently neutralized H1N1 influenza virus PR8, and the neutralization is not influenced by temperature and organic concentration, the cytocoxicity of 4sc is very low, therefore 4sc can be developed as special virucidal compound;3. The inhibitory activity of saponin derivatives of compound 3 are comparable to parent compound; However, amidation and acylation at 17-COOH of aglycons or conformational change of 3-OH of aglycons from β to a significantly improve the cytotoxicity and selectivity, compound 15 binds with Lys-26、Asn-50、Asn-53、 Asp-57 in HA2 subunit through hydrogen bonds.