Rapid and Automated Fluorescence-Linked Immunosorbent Assay for High-Throughput Screening of Hiv-1 Fusion Inhibitors Targeting gp41

Abstract

The human immuno deficiency virus type 1 (HIV-1) envelope glycoprotein gp41 plays animportant role in the virus entry. During the process of fusion between the viral and target cell membranes, the N-and C-terminal heptad repeat (HR) regions of the gp41 extracellular domain associate to form a 6-helical bundle, corresponding to the fusion-active gp41 core. Any compound that blocks the gp41 6-helix bundle formation between the N- and C-peptides, which are derived from the N- and C-terminal HR regions, respectively, may inhibit HIV-1 mediated membrane fusion. Based on this principle, we previously established a sandwich enzyme-linked immunosorbent assay (ELISA) for drug screening by using the N-peptide N36 and the C-peptide C34 and a monoclonal antibody (NC-1) which specifically recognizes the gp41 6-helix bundle. In the present study, a fluorescence-linked immunosorbent assay (FLISA) was developed by using fluorescein isothiocyanate (FITC)-conjugated C34 to replace C34 and by directly detecting fluorescence intensity instead of more complicated enzymatic reaction. Compared with the sandwich ELISA, this FLISA has similar sensitivity and specificity, but it is much more rapid, economic and convenient. Using an Integrated Robotic Sample Processing System, this assay has been applied for high-throughput screening of organic compounds on a large scale for HIV-1 fusion inhibitors targeting gp41.

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

“…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.…”

“…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.…”

Computational study of bindings of olive leaf extract (OLE) to HIV‐1 fusion protein gp41

“…Gp41 is an important target for developing AIDS drugs besides RT (reverse transcriptase), protease and integrase. Inhibition of gp41 can effectively block HIV-1 entry into human cells and thus prevent new infection of AIDS virus [7][8][9][10].…”

HIV gp41 C-terminal Heptad Repeat Contains Multifunctional Domains

“…they interact with the viral NHR to form heterologous 6-HB and block the formation of the homologous 6-HB core between the viral NHR and CHR, thereby inhibiting HIV fusion. Indeed, C34 is very effective in inhibiting the gp41 6-HB formation (11,12). However, T20 is a weak inhibitor of the gp41 core formation, suggesting that T20 and C34, although they are derived from the same region, may have different mechanisms of action (13).…”