Assay And Screening Methods For Fusion Inhibitors Targeting HIV-1gp41

Human immunodeficiency virus type1(HIV-1) fusion inhibitors are designed totarget HIV-1envelope protein as a means of preventing virus-host cell membranefusion, thereby preventing HIV-1infection of host cells. The first peptide fusioninhibitor T20(Fuzeon, enfuvirtide) has been approved for clinical use in the treatmentof HIV-1infections. Various of novel fusion inhibitors are under development as ameans of overcoming shortcomings associated with T20, such as poorpharmacokinetics, ineffectiveness against drug resistant HIV-1isolates, and reduce thecost of the drugs. An accurate, stable and reliable assay is critical for fusion inhibitorsdiscovery and development. Our study employed luciferase reporter gene-basedcell-cell fusion assay and fluorescence resonance energy transfer (FRET)-basedbiochemical assay to screen HIV-1fusion inhibitors and study their mechanisms at thecellular level and molecule level, respectively.Cell-cell fusion assays, mimicking the process of HIV-1infection with itsadvantages of safety and quantification, have been an important means of assessingthe potency of HIV-1fusion inhibitors. Our group carried out cell-cell fusion assay byemploying two kinds of adherent cell lines: TZM-bl cell line and HL2/3cell line.When cell fusion occurred, luciferase was generated and cell fusion signals could bedetected by chemiluminescence. The cell-cell fuison assay in this article wasemployed to test the activities of HIV-1fusion inhibitors, including peptides to testhelical instability, tool peptides used in FRET assay, covalent peptide fusion inhibitorsand surfactants. T20and C34were used as positive control samples in the assay.Diverse classes of fusion inhibitors were assessed and the nanomolar activity ofseveral inhibiors was screened out. The data shows that this assay is repetitive andsensitive to the occurrence of cell fusion. Additionally, experiment conditions werealso optimized.FRET assay was used to screen HIV-1fusion inhibitors from the molecule leveland for mechanisms study, using a peptide contains gp41NHR pocket-formingdomain as receptor and a fluorophore labeled CHR peptide matched to the receptorpeptide sequence as probe, and measured fluorescence signals in the presence of serial dilution of inhibitor solution. This assay was applied to assess the peptides whichtarget the pocket-forming domain in gp41NHR, and obtain two kinds of mechanisms,that is binding with gp41NHR pocket-forming domain or binding with gp41NHRpocket-forming domain and other targets on gp41to inhibit the process of membranefusion. Additionally, small molecule fusion inhibitor SZ-Br which posess the similaractivity to ADSJ-1was also screened by FRET assay.The above methods can be used to evaluate and study the mechanism of HIV-1fusion inhibitors at the cellular level and molecule level, provided a platform forHIV-1fusion inhibitors discovery and development.