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. 2006 Sep 19;103(38):13938-43.
doi: 10.1073/pnas.0601036103. Epub 2006 Sep 8.

Small molecules that bind the inner core of gp41 and inhibit HIV envelope-mediated fusion

Gary Frey  1 , Sophia Rits-Volloch, X-Q Zhang, Robert T Schooley, Bing Chen, Stephen C Harrison
Affiliations

Small molecules that bind the inner core of gp41 and inhibit HIV envelope-mediated fusion

Gary Frey et al. Proc Natl Acad Sci U S A. .

Abstract

HIV-1 enters cells by membrane fusion, mediated by the trimeric viral envelope glycoprotein gp160, which is processed by a single proteolytic cleavage into stably associated gp120 and gp41. The gp120/gp41 trimer can be triggered to undergo an irreversible conformational change. Using a protein-based assay designed to mimic the gp41 conformational change, we screened for small molecules that prevent the formation of postfusion gp41. Several compounds were identified. One set of structurally related molecules inhibited formation of a postfusion-like assembly with an IC50 of approximately 5 microM. The compounds also inhibited envelope-mediated membrane fusion in both cell-cell fusion and viral infectivity assays. Thus, our screen identifies effective fusion inhibitors. Tested against a panel of envelope proteins from primary HIV-1 isolates, the compounds inhibited fusion across a broad range of clades, including both M and T tropic strains. They bind in a highly conserved, hydrophobic pocket on the inner core of the gp41 trimer, a region previously identified as a potential inhibitor site.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Model for fusion by the HIV envelope glycoprotein. Binding of gp120 to CD4 and coreceptor (first panel) triggers a conformational change that releases the grip of gp120 (red) on gp41 (blue). The latter extends (second panel) so that its fusion peptide (green) inserts into the target-cell membrane (green bar at top). As the outer-layer region of gp41 (dark blue) zips up along the outside of the inner layer (light blue: a three-chain, α-helical coiled coil), the viral membrane (tan) and cell membrane (green) are drawn together (third and fourth panels). It is this step that is inhibited by T-20/enfuvirtide. The formation of a hemifusion stalk (fourth panel) and a fusion pore (fifth panel) completes the membrane-fusion reaction.
Fig. 2.
Fig. 2.
Association of an outer-layer peptide with gp41-5 as a screening assay. (A) The sequence of a single-chain polypeptide that can fold into a model for five of the six helices in the postfusion form of the gp41 ectodomain. (B) Diagram illustrating that a fluoresceinated, outer-layer peptide can bind to gp41-5. N, N terminus; C, C terminus. (C) Fluorescence anisotropy binding curve for the association of the outer-layer peptide with gp41-5. The fraction bound, f, is plotted as a function of gp41-5 concentration (in nM), at a constant concentration of peptide (5 nM).
Fig. 3.
Fig. 3.
Results of high-throughput screen. (A) Compounds identified as strong inhibitors of outer-layer peptide binding (5M038, 5M030, 5M041, and S2986), as well as compounds in the chemical library related to compound 5M038. (B) Inhibition of peptide binding for five of the compounds shown in A. The fraction bound, θ, is plotted as a function of inhibitor concentration. Symbols correspond to those beneath the various compounds in A. The curves show optimal sigmoidal fits to the data for 5M030 and 5M038, the only two compounds that were sufficiently soluble to yield reliable plots. Compound S2986 had an IC50 of ≈5 μM in this assay (data not shown).
Fig. 4.
Fig. 4.
Inhibition of cell–cell fusion by 5M038 (A), 5M041 (B), and 6M007 (C). The fractional degree of fusion, determined by the luciferase assay described in Materials and Methods, is plotted as a function of inhibitor concentration (diamonds). Squares show an experiment in which luciferase was expressed directly in the target cells, independent of fusion, as a control for toxicity and other side effects of the inhibitors. Curves are fractional inhibition, normalized for these toxicity effects. All points are the average of three experiments (error bars).
Fig. 5.
Fig. 5.
Effect of 5M038 during the time course of HIV infection. MT-2 cells were infected with HIV in the presence or absence of either 30 μM (blue) or 50 μM (red) 5M038. Time points were taken at 3, 5, and 7 days, and viral infection was measured by the amount of p24 antigen (p24 Ag) produced. Enfuvirtide, a potent peptidic viral entry inhibitor, was also tested (11 nM) and used for comparison (green). All points are the average of three experiments (bars represent standard error). The control (no added compound) is in black.
Fig. 6.
Fig. 6.
NMR measurements of the association of 5M038 with three-chain coiled coils containing segments of the inner core of postfusion gp41. (A) Chemical diagram showing the various protons detected in the spectra. (B) Spectrum of free 5M038. (C–E) Spectra of 5M038 in the presence of coiled coils containing residues 34–50 of gp41 (C), residues 41–57 (D), and residues 54–70 (E). There was significant broadening and shifting of the resonance peaks only in the last of the four spectra, showing that 5M038 associates selectively with the part of the gp41 inner core formed by residues 54–70.
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