Peptide Triazole Inactivators of HIV-1 Utilize a Conserved Two-Cavity Binding Site at the Junction of the Inner and Outer Domains of Env gp120

Abstract

We used coordinated mutagenesis, synthetic design, and flexible docking to investigate the structural mechanism of Env gp120 encounter by peptide triazole (PT) inactivators of HIV-1. Prior results demonstrated that the PT class of inhibitors suppresses binding at both CD4 and coreceptor sites on Env and triggers gp120 shedding, leading to cell-independent irreversible virus inactivation. Despite these enticing anti-HIV-1 phenotypes, structural understanding of the PT-gp120 binding mechanism has been incomplete. Here we found that PT engages two inhibitor ring moieties at the junction between the inner and outer domains of the gp120 protein. The results demonstrate how combined occupancy of two gp120 cavities can coordinately suppress both receptor and coreceptor binding and conformationally entrap the protein in a destabilized state. The two-cavity model has common features with small molecule gp120 inhibitor binding sites and provides a guide for further design of peptidomimetic HIV-1 inactivators based on the PT pharmacophore.

Figure 1

Env gp120 residues studied in the context of the mAb F105-bound gp120 crystal structure. Crystal structure of HIV-1 core monomeric gp120_YU-2 in the F105 bound conformation shows the open cavity between the inner and outer domain (generated from the PDB file 3HI1). Residues comprising site 1 (Phe43 cavity) and site 2 (gp120 inner domain cavity) included in this study are shown as CPK.

Figure 2

Direct binding of wt gp120_YU-2/mutants to sCD4 and mAb 17b binding using SPR. Representative SPR sensograms show concentration-dependent binding of wt gp120, S375W, and W112A to immobilized CD4 (left panel) and 17b binding (right panel) on the CM5 sensor chip surface. Each curve represents the average response from two runs. Numbers on the right show the wt gp120/mutant concentrations in nM.

Figure 3

Inhibition and antiviral potencies of peptide triazoles. (Left) Inhibition potencies of peptide 1 (■), peptide 2 (●), peptide 3 (▲), and peptide 4 (▼) for wt gp120_YU-2 binding to sCD4 immobilized on CM5 sensor chip. Normalized binding responses (RUs) were measured at 5 s before the end of association and plotted. Binding signal obtained from binding of peptide alone was subtracted, and resulting signals were plotted versus peptide concentration. Fits obtained from applying a four-parameter sigmoidal logistic equation to the data points are overlaid. (Right) Antiviral potencies of peptides using single round cell infection assays. Recombinant HIV-1 BaL.01 virus was preincubated with serial dilutions of peptide 1 (■), peptide 2 (●), peptide 3 (▲), and peptide 4 (▼) for 30 min at 37 °C. The virus–inhibitor mixture was then added to HOS.CD4.CCR5 cells for 48 h. Infection was determined based on luciferase activity. IC₅₀ values were obtained by fitting data points to a simple sigmoidal inhibition model using the Origin software package to derive the best-fit lines. Data represent a minimum of three repeats.

Figure 4

Inhibition of binding of gp120 site 1 mutants to immobilized sCD4 by SPR. Varying concentrations of peptide 1 (left panel) and peptide 2 (right panel) were mixed with a constant concentration of wt gp120, S375W, S375H, and T257R and injected over the covalently immobilized sCD4 on the CM5 sensor surface. Each curve represents the average response from two runs. Numbers on the right show the concentrations of peptides in μM.

Figure 5

Inhibition of binding of gp120 site 2 mutant W112A to immobilized sCD4 by SPR SPR sensograms are shown for binding of fixed concentrations of W112A (analyte) to CD4 on the SPR chip surface in the presence of increasing concentrations of peptide 1 (left) and peptide 2 (center). Numbers on the right show the concentrations of peptides in μM. (Right) Inhibition potential of peptide 1 (■), peptide 2 (●), and peptide 3 (▲) for W112A binding to sCD4 immobilized on CM5 sensor chip. Normalized binding responses (RUs) were measured at 5 s before the end of association and plotted. Binding signal obtained from binding of peptide alone was subtracted, and resulting signals were plotted versus peptide concentration. Fits obtained from applying a four-parameter sigmoidal logistic equation to the data points are overlaid. Results are an average of three data sets.

Figure 6

Selected binding modes of the four peptides 1–4. The peptide residue 6 side chains are located in the Phe43 site (site 1, shown in yellow), whereas the ferrocene moiety penetrates through the inner domain cavity (site 2, shown in orange). Autodock 4 was used for docking and calculation of the binding energy (ΔG_binding). Modeling was performed on the crystal structure of F105-bound gp120 (pdb: 3HI1) based on the previously developed PT model.

Figure 7

Refined two-cavity model of PT binding to gp120. (A) 3D representation of the binding mode of peptide 5 in complex with gp120 (surface shown in gray). Hot spots Trp112, Thr257, and Asp474 are shown as CPK in red. Peptide 5 Trp residue rests in site 1, whereas the phenylethyltriazole moiety is buried in site 2. (B) 2D representation of the binding mode of peptide 5 showing possible interactions with residues in site 1 and site 2. H-bonds are shown as purple arrows and π–π interactions shown as green lines.

Scheme 1. Solid-Phase Synthesis of Peptide Triazoles 1–6^a

^aR is the alkyne derivative used for the click reaction. X is the peptide residue 6 side-chain aromatic ring.