Virus Entry Inhibitors Targeting The Envelope Glycoprotein Transmembrane Subunits Of HIV And AIV

Background:Acquiredimmunodeficiency syndrome(AIDS) and influenza are the major threats to public health worldwide.Similar to the SARS coronavirus(SARS-CoV), human immunodeficiency virus(HIV) and avian influenza virus(AIV) are also classâ… enveloped virus.Classâ… enveloped viruses share a similar virus-host cell membrane fusion mechanism:the envelope glycoprotein(Env) binds to the host cell receptor and undergoes conformational changes which trigger the membrane fusion.Using HIV and influenza virus as examples,the membrane fusion is mediated by the Envs of influenza virus(hemagglutinin,HA0) and of HIV(gp160).Both envelope proteins consist of two associated parts:the surface subunit(HA1/gp120) responsible for binding receptor and the transmembrane subunit(HA2/gp41) medaiteing membrane fusion.A peptide HIV entry inhibitor derived from the HIV-1 gp41 can bind to its conterpart in gp41,thereby preventing further conformation change of gp41 and interfering with the virus fusion,which provide new ideas and strategies to the treatment of viral diseases.However,the peptide derived from the HA2 of AIV exhibits no inhibitory activity against influenza virus.This is because AIV has a fusogenic mechanism different from that mediated by the HIV gp41.Fusion of the AIV envelope with the host cell membranes takes place in endosomal compartments, whilethese initial stages of virus replication are dependent on the acidification of endosomes by cellular proton pumps.The reduction in endosomal pH triggers the conformational changes in HA that lead to membrane fusion.Most of the active antiviral peptides derived from the virus envelope proteins contain more 30 amino acid residues.However,the peptide with large molecular size peptides is difficult to enter through the cell membrane to the intracellular compartments to interact with the influenza virus HA2.Even a peptide is able to enter the endosome,it may be inactivated by low pH(about 5) in the acidic environment.In contrast,many small molecular compounds can enter into the cell through the cellular membrane. Therefore,a small molecule compound that can prevent the influenza virus HA2 conformational change may be effective ininhibiting influenza virus infection.HIV,a lentivirus,is the causative agent of AIDS,a disease that progressively reduces the effectiveness of human immune system,leading to life-threatening opportunistic infections and tumors.HIV primarily infects vital cells in the human immune system such as CD4⁺ T helper(Th) cells.To develop effective therapeutics and vaccines for treatement and prevention of AIDS,it is critical to understand pathogenesis of HIV infection and the interaction between virus and immune cells, especially T-cell activation mediated by viral proteins.T-cell activation is a double-edged sword in the pathogenesis of HIV/AIDS. On the one hand,activation of CD4⁺ T cells is required to eliminate the infected viruses because the activated CD4⁺ Th cells can mediate humoral and cellular immune responses against HIV-1.On the other hand,HIV-1 preferentially infects the activated CD4⁺ T cells,especially those in mucosa.Abnormal CD4⁺ T-cell activation caused by HIV-1 infection contributes to activation-induced cell apoptosis and precedes the host immune system crash.Accordingly,proper regulation of CD4⁺ T-cell activation may be applied as a therapeutic or prophylactic strategy for treatment or prevention of HIV-1 infection.Munch et al have reported that a 20-mer peptide derived from the C-proximal subfragment of al-antitrypsin(al-AT), designated Virus Inhibitory Peptide(VIRIP),is able to interact with the HIV-1 gp41 FP and inhibit HIV-1 Env-mediated cell-cell fusion.Its analog peptide,VIR576,is about two orders of magnitude more potent than VIRIP in inhibiting HIV-1 replication.Given that FP could effectively downregulate the T-cell activation as proven by Shai and colleagues,I thus hypothesized that the VIRIP may also affect T cell activation and may be applied as a dual functional anti-HIV agent.In the present study,I focused on two projects:(1) Identification of influenza virus entry inhibitors targeting H5N1 Env hemagglutinin HA2 subunit;and(2) Down-regulation of antigen-specific T cell activation by a peptide HIV-1 fusion inhibitor via interaction with T cell receptor.For the first project,I intended to establish:(â…°) an FLISA-based primary HTS assay for screening compounds that can block six-helix bundle formation between the NHR and CHR domains and that can inhibit the formation of the N-terminal cap structure in HA2 of AIV;(â…±) a cell-based secondary HTS assay for screening compounds that inhibit infection of MDCK cells by H5N1 pseudotyped viruses;and(â…²) a mouse model for evaluating the in vivo efficacy and toxicity of the potential anit-AIV compounds.For the second project,I intended to study:(â…°) the effect of VIR576 on the proliferation of T cells in response to specific and non-specific antigen stimuation;(â…±) the effect of VIR576 on TCR-TMD-mediated hemolysis;and(â…²) whether VIR576 is able to colocalize with CD4 molecule in the CD4⁺ T cell membrane to interact with TCR.Partâ… .Identification of influenza virus entry inhibitors targeting H5N1 Env hemagglutiniu HA2 subunitOBJECTIVE:To establish the high-throughput screening methods for screening the compounds inhibited formation of the six-helix bundle and the N-terminal cap structure in the HA2 domain of AIV,and to develop a cell-based pharmacological model by using the H5N1 pseudotyped viral system,a MDCK cell infected model for in vitro infection by live H5N1 virus,and a mouse model for in vivo infection by live H5N1 virus,and to screen compounds which can specifically inhibit the virus entry.METHODS: 1.To synthesize a series of peptides derived from the HA2 domain of the highly pathogenic H5N1 avian influenza virus,including an N-peptide(N29, aa77～aa105),a C-peptide(C19,aa110～aa128) and an FITC-labeled C-peptide,a biotin-labeled N-terminal cap peptide(N8,aa34～aa37),and an FITC-labeled C-terminal cap peptide(C8,aa173～aa176).The potential interactions between the N- and C-peptides and between N- and C-terimal cap peptides were studies by using FLISA,natural fluorescence gel electrophoresis,SE-HPLC methods,in order to establish the high-throughput screening methods for screening of compounds that can inhibit formation of the six-helix bundle formation and the N-terminal cap structure in HA2 of AIV.2.To establish a cell-based pharmacological model for compounds screening by using the H5N1 pseudotyped viral system.The plasmids encoding the AIV Envs HA and NA and the pNL43Luc.RE-(with a luciferase reporter gene and a HIV-1 core gene) were transfected into 293T cells for expression of HA and NA on pseudotyped virus containing the HIV-1 core.This system would be used for screening of anti-AIV compounds.3.To develop in vitro H5N1 AIV infection model using MDCK cells and in vivo H5N1 AIV infection model using the Balb/c mice for evaluation of the in vitro and in vivo efficacy of anti-AIV compounds.4.To establish an XTT assay and a mouse model for evaluating the in vitro and in vivo toxicity,respectively,of the selected anti-AIV compounds.5.To establish an HPLC method for studying the pharmacokinetics of ARC-36 in mice.RESULTS:1.The results from FLISA,natural fluorescence gel electrophoresis,SE-HPLC analysis showed that there were no detectable interactions between N29 and C19 and between N8 and C8 peptides derived from the HA2 domain of H5N1 AIV.2.The HA-NA/HIV pseudovirus could infect 293T cells and MDCK cells,and ARC-36 could inhibit the AIV pseudovirus infection with IC₅₀ value of 4.00±0.38μM. 3.ARC-36 also inhibited live H5N1 virus infection of MDCK cells with IC50 of 27.03±2.54μM.It had now toxicity to MDCK cells with CC₅₀ of 525.4±63.22μM.Intranasal administration of ARC-36 before the intranasal infection protected mice from H5N1 AIV challenge,with the average survival time of 8.8 days,while 7.4 days for the control normal mice,a significant differences(p＜0.01).4.As analyszed by HPLC(A phase:100%methanol,B phase:0.5%glacial acetic acid),the retention time of ARC-36 was 6.3 min with a standard curve equation of Y= 5354.2X+7106.5,r = 0.9993(n = 5).The Ct curve is two-compartment model after intraperitoneal injection of ARC-36.The elimination half-life(t1/2)_βis 31.6min.The LD50 of ARC-36 by intraperitoneal injection is 149.2 mg·kg^-1 (7.5mg·ml^-1,211.8μM),and is about 50-fold of the IC₅₀ value of ARC-36 determed in pseudovirus assay(4.00±0.38μM).CONCLUSION:1.Because of no detectable interactions between N29 and C19 and between N8 and C8,we failed to develop these peptides-based high-throughput screening methods for screening of the compounds that inhibit formation of six-helix bundle and the N-terminal cap structure in HA2 of H5N1 AIV.2.I successfully developed the HA-NA/HIV pseudovirus infection system using 293T cells and MDCK cells for screening of anti-AIV compounds and using this system,we found that ARC-36 was effective in inhibiting AIV infection,but was infective when it was added 24 hours post infection.3.I established the Balb/c mouse AIV challenge model and MDCK cell H5N1 AIV infection model for evaluating the in vivo and in vitro efficacy of the selected anti-AIV compounds.We confirmed that ARC-36 is effective in inhibiting H5N1 AIVinfection in MDCK cells.Intranasal administration of ARC-36 before the intranasal infection resulted in prolonged average survival time of mice.4.I established the HPLC method for quantitative analysis of ARC-36 level in vivo. I found that he Ct curve is two-compartment model after intraperitoneal injection of ARC-36,and the elimination half-life(t1/2)_βis 31.6 min,owing to the quick metabolism.The LD50 of ARC-36 by intraperitoneal injection is 50-fold of the IC50 value determined in pseudovirus assay,suggesting that ARC-36 is relatively safe.PARTâ…¡.Down-regulation of antigen-specific T cell activation by a peptide HIV-1 fusion inhibitor via interaction with T cell receptorOBJECTIVE:To study the effect of VIR576 on antigen-specific T cell(Ab2 cells,CD4⁺ CD25T cells) activation and non-antigen-specific T cell activation;To analyze the membrane distribution of VIR576 in T cell using the fluorescent confocal microscope and the colocalization of VIR576 with TCR;To study the interaction of VIR576 and the synthetic peptide derived from TCR-TMD,the CP sequence(aa 253-261),using hemolysis test,hemolysis inhibition assay,FRET,FLIA,and competitive FLISA.METHODS:1.To investigate the potential effect of FP16,VIR576,and the mixture of FP16 and VIR576 on the proliferation of A2b cells in response to MOG 35-55 at 10μg/ml, to test the effect of FP16,VIR576 and their mixture on proliferation of splenocytes of OVA-specific DO 11.10 Tg mice in response to chicken OVA,and to test the proliferation activity of non-stimulated and Con A-stimulated splenocytes from BALB/c.2.To establish TCR-TMD-mediated hemolysis assay,and using this assay,to study the effect of VIR576 on the hemolytic activity of TCR-TMD.3.To develop a simple and rapid fluorescence-based binding assay using Rhodamine(Rho)-conjugated VIR576 to evaluate the potential interaction between VIR576 and TCR-TMD,to conduct a competitive assay to confirm the specific interaction between TCR-TMD and Rho-VIR576 using unlabeled-VIR576 and VIRIP scramble peptide,and to identify the critical binding site of VIR576 in TCR-TMD.4.4.To study whether VIR576 could colocalize with CD4 molecule in the CD4⁺ T cell membrane to interact with TCR. RESULTS:1.VIR576 alone suppressed the proliferation of A2b T cells in responses to MOG 35-55,and it could also block FP16-mediated inhibition of A2b cell activation (p＜0.05).VIR576 itself also inhibited splenocytes proliferation in responses to OVA,and it could block FP16-mediated inhibition of splenocytes activation (p＜0.05).Both splenocytes and CD4⁺CD25^-T cells of DO11.10 mice showed a dose-dependent strong proliferative in response to the stimulation by OVA,while the addition of VIR576 resulted in significant inhibition of proliferation of splenocytes and the primary CD4⁺CD25^- T cells of DO11.10 mice.The proliferation of non-stimulated and Con A-stimulated splenocytes was not affected by addition of VIR576 at the concentration as high as 50μg/ml. Consistently,VIR576 had no significant effect on proliferation of DO11.10 mouse splenocytes in the absence or presence of Con A or anti-CD3 antibody.2.VIR576 interacted with CP since incubation of human erythrocytes with TCR-TMD resulted in significant hemolysis in a dose-dependant manner with IC₅₀ of 1.23±0.16μM,whereas the VIRIP scramble peptide had no significant inhibition on TCR-TMD-mediated hemolysis(p＜0.05).Rho-VIR576 bound to TCR-TMD in a dose-dependant manner,resulting in a significant increase of the fluorescence intensity.A significant decrease in fluorescence intensity when mixture of unlabeled-VIR576 and Rho-VIR576 were added to the immobilized TCR-TMD,while the unlabeled VIRIP scramble peptide had no effect on the binding of Rho-VIR576 to TCR-TMD(p＜0.05).When Rho-VIR576(final concentration 0.04-0.12μM) was added to a mixture of NBD-CP(0.04μM) and PC LUV liposome(100μM),a dose-dependent quenching of the donor's emission,which is consistent with energy transfer,was observed.3.Rho-VIR576 did not bind to the unstimulated lymphocytes,but strongly interacted with the CD4⁺ T cells stimulated with anti-CD3 antibody.Only a part of activated T cell membrane was stained by Alexa-labeled anti-CD4 antibody.CONCLUSION:1.VIR576 was effective in suppressing the antigen-specific T cell activation,but has no effect on non-specific T cell proliferation.2.VIR576 had the ability to downregulate the antigen-specific T cell activation by interaction with TCR transmembrane domain.3.Rho-VIR576 strongly interacted with the CD4⁺ T cells stimulated with anti-CD3 antibody.