Structure-based identification and neutralization mechanism of tyrosine sulfate mimetics that inhibit HIV-1 entry

Abstract

Tyrosine sulfate-mediated interactions play an important role in HIV-1 entry. After engaging the CD4 receptor at the cell surface, the HIV-1 gp120 glycoprotein binds to the CCR5 co-receptor via an interaction that requires two tyrosine sulfates, at positions 10 and 14 in the CCR5-N terminus. Building on previous structure determinations of this interaction, here we report the targeting of these tyrosine sulfate binding sites for drug design through in silico screening of small molecule libraries, identification of lead compounds, and characterization of biological activity. A class of tyrosine sulfate-mimicking small molecules containing a "phenyl sulfonate-linker-aromatic" motif was identified that specifically inhibited binding of gp120 to the CCR5-N terminus as well as to sulfated antibodies that recognize the co-receptor binding region on gp120. The most potent of these compounds bound gp120 with low micromolar affinity and its CD4-induced conformation with K(D)'s as tight as ∼50 nM. Neutralization experiments suggested the targeted site to be conformationally inaccessible prior to CD4 engagement. Primary HIV-1 isolates were weakly neutralized, preincubation with soluble CD4 enhanced neutralization, and engineered isolates with increased dependence on the N terminus of CCR5 or with reduced conformational barriers were neutralized with IC(50) values as low as ∼1 μM. These results reveal the potential of targeting the tyrosine sulfate interactions of HIV-1 and provide insight into how mechanistic barriers, evolved by HIV-1 to evade antibody recognition, also restrict small-molecule-mediated neutralization.

Figure 1

Target site. A critical step of HIV entry involves interaction with sulfated tyrosine residues in the CCR5 co-receptor, which the antibody 412d mimics to neutralize HIV-1. a) gp120-CCR5-N terminus interaction. HIV-1 gp120 (grey) interacts with its cell surface receptor CD4 (yellow) and co-receptor CCR5 (magenta). CCR5-N terminus binds in an α-helical conformation at the base of the V3 loop (orange) of gp120. Two sulfated tyrosine residues (stick representation) are critical for binding of the CCR5-N terminus to gp120. b) Mimicry of gp120-CCR5-N terminus interaction by CDR H3 loop of 412d antibody. The CDR H3 loop (red) of antibody 412d (green) is a functional mimic of the CCR5-N terminus with two sulfated tyrosine residues (sticks) dominating its interaction with gp120.

Figure 2

Inhibitor identification flowchart. Starting from atomic level co-ordinates of gp120-CD4-CCR5-N terminus and gp120-CD4-412d Fab, two methods of in silico screening were used in combination with an ELISA-based screen to identify binding chemotypes. Selected compounds were characterized further for their gp120 binding and HIV-1 neutralization properties.

Figure 3

Tyrosine-sulfate mimicry in inhibitor identification. The critical role of two N terminal tyrosine-sulfate residues in CCR5 binding and their unique chemistry suggested the utility of the tyrosine-sulfate binding sites as drug targets. a) Tyrosine-sulfate binding sites on gp120. Sequences of CCR5-N terminus and 412d CDR H3 loop are shown above with the tyrosine residues that are sulfated and critical for binding colored red. The CCR5-N terminus (magenta) and the CDR H3 loop (red) of 412d antibody interact with the base of the V3 loop (orange) of gp120 (grey). The region surrounding the tyrosine-sulfate residues was targeted for small molecule screening. b) Ligand-based probes for in silico screening. Structure-based probes (shown as sticks) centered around the tyrosine-sulfate moieties were used for shape based screening of the ZINC small molecule library. c) Tyrosine-sulfate analogs. The rank order of binding affinity of CCR5 peptides containing modified tyrosines at positions 10 and 14 are shown. Compounds containing phenyl sulfonate and phenyl sulfonamide groups were screened in this study.

Figure 4

Small molecule inhibitors of CCR5-N terminus binding to gp120. Small molecules were selected on the basis of their ability to inhibit binding of gp120 to a CCR5-N terminus peptide analog. All inhibitors contained a phenyl sulfonate moiety, which retains the anionic and aromatic properties of tyrosine-sulfate. The two classes of compounds shown were selected for further analyses.

Figure 5

gp120 binding. a) Direct binding to gp120. Binding of compounds 1, 2, 3 and 7 to gp120 in the unliganded (top panel) and in the CD4-bound conformation (bottom panel) was measured by SPR and affinity calculated by steady state analysis. b) Inhibition of binding of CD4i antibody 412d to gp120 in the CD4-bound conformation by the compounds.

Figure 6

Inhibition of HIV entry. a) Neutralization profiles of compounds 1, 2 and monoclonal antibody 17b depicted with 29-isolate neutralization dendrograms. SIV and MuLV were used as controls. IC₅₀ values are listed in Supplementary Table 1. Insets show neutralization of two HIV-2 isolates tested. b) Inhibition of entry in presence of two-domain CD4 (sCD4) or the small molecule NBD-556. The p-values indicate statistical significance for effect of triggerring with sCD4 or NBD-556 compared to untreated virus calculated using ANOVA. c) Left: inhibition of entry of HIV-1 ADA S199A, and right, inhibition of CCR5-mediated entry (circles) and of CXCR4-mediated entry (squares) of CEMAX.