Tyrosine-sulfate isosteres of CCR5 N-terminus as tools for studying HIV-1 entry

Abstract

The HIV-1 co-receptor CCR5 possesses sulfo-tyrosine (TYS) residues at its N-terminus (Nt) that are required for binding HIV-1 gp120 and mediating viral entry. By using a 14-residue fragment of CCR5 Nt containing two TYS residues, we recently showed that CCR5 Nt binds gp120 through a conserved region specific for TYS moieties and suggested that this site may represent a target for inhibitors and probes of HIV-1 entry. As peptides containing sulfo-tyrosines are difficult to synthesize and handle due to limited stability of the sulfo-ester moiety, we have now incorporated TYS isosteres into CCR5 Nt analogs and assessed their binding to a complex of gp120-CD4 using saturation transfer difference (STD) NMR and surface plasmon resonance (SPR). STD enhancements for CCR5 Nt peptides containing tyrosine sulfonate (TYSN) in complex with gp120-CD4 were very similar to those observed for sulfated CCR5 Nt peptides indicating comparable modes of binding. STD enhancements for phosphotyrosine-containing CCR5 Nt analogs were greatly diminished consistent with earlier findings showing sulfo-tyrosine to be essential for CCR5 Nt binding to gp120. Tyrosine sulfonate-containing CCR5 peptides exhibited reduced water solubility, limiting their use in assay and probe development. To improve solubility, we designed, synthesized, and incorporated in CCR5 Nt peptide analogs an orthogonally functionalized azido tris(ethylenoxy) l-alanine (l-ate-Ala) residue. Through NMR and SPR experiments, we show a 19-residue TYSN-containing peptide to be a functional, hydrolytically stable CCR5 Nt isostere that was in turn used to develop both SPR-based and ELISA assays to screen for inhibitors of CCR5 binding to gp120-CD4.

Figure 1

CCR5 Nt peptides and analogs.

Figure 2

Close up of model of CCR5 Nt binding to gp120. Tyrosine residues 10, 14 and 15 are labeled. Surfaces for gp120 atoms within 5 Å of TYS10, TYS14 and TYR15 are colored blue, beige and green, respectively.

Figure 3

Aromatic region of ¹H STD-NMR spectra of 14-residue CCR5 Nt^2–15 (1) and analogs 2–4. (a) CCR5 Nt^2–15 (1) containing TYS10 and TYS14; (b) analog 2 containing TYS10 and TYP14; (c) analog 3 containing TYP10 and TYS14; (d) analog 4 containing TYSN10 and TYSN14. Spectra were recorded at 298 K on samples containing 20 mM gp120-CD4 in the presence of 800 mM peptide at pH 6.8.

Figure 4

Expansions of NMR spectra of 17-residue CCR5 Nt (2–18) and analogs. ¹H NMR spectra of free and bound peptides are shown in black and blue, respectively, and ¹H STD spectra of complexes are red. Number and position of TYS and TYSN residues are shown above respective spectra and in Figure 1 and Figure 5.

Figure 5

Surface plasmon resonance experiments of CCR5 Nt peptides binding to gp120-CD4. Doubly referenced sensorgrams for (a) doubly sulfated Nt peptide 1 and (b) triply sulfonated Nt peptide 11 binding to CD4-captured HIV-1 gp120 with peptide concentrations ranging from 3.1 to 50mM. R_max plots for immobilized peptide 12 binding to (c) gp120-CD4 (complex), CD4, and gp120, with protein concentrations ranging from 39 nM to 5.0 mM; and (d) gp120-CD4 in the presence of 10-fold serial dilutions of 412d ranging from 100 nM through 0.001 nM. Response units (RUs) are shown on the y-axis with doubly referenced sensorgrams plotted on the same scale for panels a and b.

Scheme 1

Synthesis of the alpha amino acid azido tris(ethylenoxy) L-alanine (L-ate-Ala). (a) NaN₃, DMF, quant (b) PBr₃, THF, 85% (c) N-Boc L-Ser, NaH, DMF, 75% (d) TFA, TIS, CH₂Cl₂ (e) FmocCl, DIPEA, H₂O/dioxane, 75% 2 steps (f) SPPS (g) Cu(II)SO₄, Na ascorbate, propargyl biotin, H₂O/MeOH.