Trilobatin As An HIV-1 Entry Inhibitor Targeting HIV-1 Gp41 Envelope

BACKGROUND:By the end of 2016,36.7 million people had been infected with human immunodeficiency virus/acquired immune deficiency syndrome(HIV/AIDS)and 1 million people had died of AIDS-related illnesses worldwide.At present,the research of safe and effective anti-AIDS drugs are still significant for AIDS prevention and treatment,since there are no major breakthrough in the field of anti-HIV vaccines.Since HIV entry inhibitors can exert antiviral activity in the early stage of virus replication and are resistant to other anti-HIV drug-resistant strains,their development has attracted much attention.The entry of HIV to host cells is regulated by both the envelope protein(s)of HIV and the target cell membrane receptor..First,gp120,the HIV virus envelope glycoprotein,binds to the CD4 receptor on the host cell membrane and the co-receptor CXCR4/CCR5.Then,the conformation of HIV-1 transmembrane protein gp41 changes into a stable 6-Helix Bundle(6-HB),bringing the viral and host cell membranes to sufficient proximity and fusion,which causes the virus to invade the target cell.As a specific envelope protein of conserved sequence virus,gp41 has great advantage as a drug target.The first HIV entry inhibitor approved for clinical use is enfuvirtide(T-20),a 36-mer peptide derived from the C-terminal heptad repeats(CHR)region of gp41,which binds to N-terminal heptad repeats(NHR)and inhibits the formation of six-helix bundle(6-HB).Due to low oral bioavailability,short half-life,high production cost(90 mg twice daily)and drug resistance of T-20,the development of an effective,safe and affordable small molecule compound targeting the gp41 NHR is urgently needed.Currently,many natural polyanionic ingredients or their derivatives with anti-HIV drug activities have aroused concern in the world.Our previous study showed that trilobatin,a new glycosylated dihydrochalcone isolated from the leaves of Lithocarpus polystachyus Rehd,has anti-HIV activity.It is a glycosylated dihydrochalcone compound.Because of its safety and effectiveness,it's possible that trilobatin will be developed into a new kind of anti-HIV drug.OBJECTIVE:The aim of our study was to evaluate the anti-HIV-1 activity and in vitro cytotoxicities of trilobatin,and to further explore its possible mechanism and target for anti-HIV action,in order to provide the necessary theoretical basis for the discovery of a new,safe and effective HIV-1 entry inhibitor.METHODS:1.The antiviral activities of trilobatin in vitro were detected by using different HIV-1 laboratory-adapted strains and HIV-1 clones strains.2.The in vitro cytotoxicities of trilobatin on the target cells(TZM-bl,U87-CD4-CCR5,U87-CD4-CXCR4 and MT-2 cells),HIV effector cells(CHO-WT cells)and PBMCs were evaluated by MTT assay.3.Mechanism study was confirmed by methods such as time of addition assay,cell-cell fusion,the antiviral activities of trilobatin on HIV-1 pseudotyped virus and VSV-G pseudotyped virus,a sandwich ELISA,N-PAGE,CD spectroscopy,cell-based ELISA of CD4 receptor and flow cytometry of CCR5/CXCR4 co-receptor.4.Docking between trilobatin and the surface of the internal trimeric coiled coil of gp41 was analyzed by computational docking.5.The antiviral activity of trilobatin on HIV-1JR-FL mutant viruses was detected and the possible target of trilobatin on envelope protein gp41 was analyzed by means of site-directed mutagenesis analysis.6.The binding affinity of the N36 and trilobatin was detected by the SPR.7.The combination treatment of trilobatin with different kinds of antiviral agents was detected by anti-HIV infection assay.RESULTS:1.Trilobatin had anti-HIV activity on infection by both HIV-1 laboratory-adapted strains and HIV-1 clones strains.2.Trilobatin had low cytotoxicity on the target cells(TZM-bl,U87-CD4-CCR5,U87-CD4-CXCR4 and MT-2 cells)and HIV effector cells(CHO-WT cells).3.The results of time of addition assay,cell fusion assay and HIV-1 pseudovirus experiment suggested that trilobatin inhibited HIV-1 entry by inhibiting the HIV-1 envelope protein and blocking HIV-1 membrane fusion.4.Trilobatin could block HIV-1 entry by inhibiting HIV gp41 six-helix bundle formation,which was shown by a sandwich ELISA,N-PAGE assay,CD spectroscopy and cell-based ELISA of CD4 receptor and flow cytometry of CCR5/CXCR4 co-receptor.5.The computational docking analysis showed that trilobatin could inhibit the formation of HIV-1 JR-FL gp41 core structure through its hydrophobic interaction with Leu559 and Ile564 and its hydrogen bonding with Leu559 and Gln558 in the HIV-1 JR-FL gp41 core..6.Site-directed mutagenesis analysis showed that replacement of Ile 564 in the HIV-1 JR-FL gp41 core with non-conserved alanine(Ala,A)residues resulted in significant decrease in the antiviral activity of trilobatin,which suggested that Ile564 could be the key site of trilobatin.7.The SPR assay also confirmed that trilobatin bound to N36 peptide with a dissociation constant(KD)of 1.73 × 10-7 M.8.Significant synergistic antiviral effects were observed when combining trilobatin with other kinds of antiviral agents.CONCLUSION:Trilobatin had broad anti-HIV activity and low cytotoxicity.The possible mechanism is that it can specifically target gp41 N-terminal heptad repeats regions,thereby inhibiting the entry of HIV.Site-directed mutagenesis analysis suggested that its target is the negatively charged residue(1564)located in gp41 pocket-forming site.Furthermore,trilobatin displayed synergistic effects against HIV-1 infection when combined with other ARV agents.Therefore,trilobatin has an great prospect to be developed as a potential small-molecule HIV-1 entry inhibitor for clinical combination therapy,which helps prevent and treat AIDS and its related diseases and provide theoretical basis for clinical application.