A Novel Fluorescence Intensity Screening Assay Identifies New Low-Molecular-Weight Inhibitors of the gp41 Coiled-Coil Domain of Human Immunodeficiency Virus Type 1

Abstract

A metallopeptide-based fluorescence assay has been designed for the detection of small-molecule inhibitors of human immunodeficiency virus type 1 gp41, the viral protein involved in membrane fusion. The assay involves two peptides representing the inner N-terminal-heptad-repeat (HR1) coiled coil and the outer Cterminal-heptad-repeat (HR2) helical domains of the gp41 six-helix bundle which forms prior to fusion. The two peptides span a hydrophobic pocket previously defined in the literature. The HR1 peptide is modified with a metal-ligated dye complex, which maintains structural integrity and permits association with a fluorophorelabeled HR2 peptide to be followed by fluorescence quenching. Compounds able to disrupt six-helix bundle formation can act as fusion inhibitors, and we show that they can be detected in the assay from an increase in the fluorescence that is correlated with the potency of the compound. Assay optimization and validation have resulted in a simple quantitative competitive inhibition assay for fusion inhibitors that bind in the hydrophobic pocket. The assay has an assay quality factor (Z) of 0.88 and can rank order inhibitors at 10 M concentration with K i s in the range of 0.2 M to 30 M, an ideal range for drug discovery. Screening of a small peptidomimetic library has yielded three new low-molecular-weight gp41 inhibitors. In vitro syncytium inhibition assays confirmed that the compounds inhibited cell-cell fusion in the low micromolar range. These lead compounds provide a new molecular scaffold for the development of fusion inhibitors.

High-Throughput HIV–Cell Fusion Assay for Discovery of Virus Entry Inhibitors

“…In vitro screening has identified competitive inhibitors of assembly of the gp41 HR1-and HR2-derived peptides into the 6HB. [13][14][15][16][17][18][19][20][21][22][23][24] HTS for smallmolecule inhibitors competing with the chemokine (RANTES) binding to CCR5 has led to the identification of identified coreceptor antagonists that effectively blocked fusion of CCR5-tropic viruses: maraviroc, Sch-C, and TAK-779. [10][11][12] These narrowly focused readouts provide a powerful means to identify specific inhibitors of a given step of the virus entry, but exclude all other targets for inhibition of HIV-1 fusion.…”

“…In the final 6-helix bundle structure (6HB), three HR1 and three HR2 coalesce forming a highly stable antiparallel helical bundle. A number of small-molecule inhibitors of HIV-1 fusion that interfere with CD4-induced conformational changes in gp120, 8,9 coreceptor binding, [10][11][12] and the gp41 6HB formation [13][14][15][16][17][18][19][20][21][22][23][24] have been identified by high-throughput screening (HTS). Currently, only two HIV-1 fusion inhibitors (enfuvirtide and maraviroc) have been approved for clinical use.…”

Peptides and Peptidomimetics as Tools to Probe Protein-Protein Interactions – Disruption of HIV-1 gp41 Fusion Core and Fusion Inhibitor Design

“…This enables direct determination of binding by using a fluorophore labeled C-peptide as the probe. Compounds which are able to bind to the NHR target and displace the probe can be measured with a competitive inhibition assay by following the recovery of probe fluorescence intensity (Cai & Gochin 2007). The BPY-metal complex FRET strategy is generally applicable to different interacting peptide pairs, as long as the two peptide sequences are matched.…”

“…Env5.0 contains the whole groove, and it has been used to identify ligands that interact with the range of the groove outside of the deep pocket by designing suitable probes ). In addition, Env2.0 has been successfully used as a target for a screening assay to identify small molecule fusion inhibitors (Cai & Gochin 2007;Zhou et al 2010). In summary, peptides have been used to construct the HIV-1 gp41 fusion core, which is a 6-HB.…”

“…Addition of physicochemical restraints in N-peptides has been shown to be an efficient way to construct a stable and discrete NHR trimer. Typical NHR constructs include: IQN17 (3) and IZN17 (4) , 5-helix (Root et al 2001;Frey et al 2006), and Env2.0 (5) and Env5.0 (6) (Cai & Gochin 2007;Cai et al 2009). These stable NHR-trimers can be efficient targets for fusion inhibitor discovery and development.…”