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

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.

Evolution of potent and stable placental-growth-factor-1-targeting CovX-bodies from phage display peptide discovery.

Novel phage-derived peptides are the first reported molecules specifically targeting human placental growth factor 1 (PlGF-1). Phage data enabled peptide modifications that decreased IC(50) values in PlGF-1/VEGFR-1 competition ELISA from 100 to 1 µM. Peptides exhibiting enhanced potency were bioconjugated to the CovX antibody scaffold 1 (CVX-2000), generating bivalent CovX-Bodies with 2 nM K(D) against PlGF-1. In vitro and in vivo peptide cleavage mapping studies enabled the identification of proteolytic hotspots that were subsequently chemically modified. These changes decreased IC(50) to 0.4 nM and increased compound stability from 5% remaining at 6 h after injection to 35% remaining at 24 h with a ß phase half-life of 75 h in mice. In cynomolgus monkey, a 78 h ß half-life was observed for lead compound 2. The pharmacological properties of 2 are currently being explored.

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

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.

Structures of the CCR5 N terminus and of a tyrosine-sulfated antibody with HIV-1 gp120 and CD4.

The CCR5 co-receptor binds to the HIV-1 gp120 envelope glycoprotein and facilitates HIV-1 entry into cells. Its N terminus is tyrosine-sulfated, as are many antibodies that react with the co-receptor binding site on gp120. We applied nuclear magnetic resonance and crystallographic techniques to analyze the structure of the CCR5 N terminus and that of the tyrosine-sulfated antibody 412d in complex with gp120 and CD4. The conformations of tyrosine-sulfated regions of CCR5 (alpha-helix) and 412d (extended loop) are surprisingly different. Nonetheless, a critical sulfotyrosine on CCR5 and on 412d induces similar structural rearrangements in gp120. These results now provide a framework for understanding HIV-1 interactions with the CCR5 N terminus during viral entry and define a conserved site on gp120, whose recognition of sulfotyrosine engenders posttranslational mimicry by the immune system.

A monoclonal Fab derived from a human nonimmune phage library reveals a new epitope on gp41 and neutralizes diverse human immunodeficiency virus type 1 strains.

A monoclonal Fab (Fab 3674) selected from a human nonimmune phage library by panning against the chimeric construct N(CCG)-gp41 (which comprises an exposed coiled-coil trimer of gp41 N helices fused in the helical phase onto the minimal thermostable ectodomain of gp41) is described. Fab 3674 is shown to neutralize diverse laboratory-adapted B strains of human immunodeficiency virus type 1 (HIV-1) and primary isolates of subtypes A, B, and C in an Env-pseudotyped-virus neutralization assay, albeit with reduced potency (approximately 25-fold) compared to that of 2F5 and 4E10. Alanine scanning mutagenesis maps a novel epitope to a shallow groove on the N helices of gp41 that is exposed between two C helices in the fusogenic six-helix bundle conformation of gp41. Bivalent Fab 3674 and the C34 peptide (a potent fusion inhibitor derived from the C helix of gp41) are shown to act at similar stages of the fusion reaction and to neutralize HIV-1 synergistically, providing additional evidence that the epitope of Fab 3674 is new and distinct from the binding site of C34.

Efficient synthesis of Man2, Man3, and Man5 oligosaccharides, using mannosyl iodide donors.

[reaction: see text] A highly efficient protocol for making Man(3) and Man(5) oligosaccharides with use of orthogonally protected glycosyl iodide donors has been developed. Glycosylation of a C-2-O-acetyl mannosyl iodide donor in the presence of silver triflate at -40 degrees C initially gave a mixture of the desired alpha-linked mannoside and an orthoacetate resulting from attack at the C-2 acetate. However, upon warming to room temperature the orthoacetate quantitatively rearranged to the desired oligosaccharide. Employing a 3,6-dihydroxy acceptor and subjecting it to double glycosidation quickly afforded high mannose sugars in nearly quantitative yields. Glycosyl iodide donors offer advantages over previously reported chloride donors as the reactions are faster, proceed in higher yields, and are not diminished in higher order constructs. These studies continue to dispel the notion that glycosyl iodides are too reactive to be of synthetic utility.

Glycal scavenging in the synthesis of disaccharides using mannosyl iodide donors.

[reaction: see text] High mannose glycans composed of alpha (1-->2) and alpha (1-->6) branched sugars are important components of the HIV-associated envelope glycoprotein, gp120. These substructures can be efficiently prepared in solution from glycosyl iodide precursors requiring only a slight excess of the iodide donor, which offers advantages over solid-phase methods that require more than 5 equiv of donor. During the reaction, excess iodide is converted to a glycal that is not easily separated from the desired disaccharide. To overcome this difficulty, a phase-trafficking methodology that relies upon nucleophilic interception of the 1,2 anhydrosugar resulting from oxidation of the glycal has been developed.

Efficient route to 2-deoxy beta-O-aryl-d-glycosides via direct displacement of glycosyl iodides.

[reaction: see text]. The conversion of glycals to 2-deoxy glycosyl acetates followed by reaction with trimethylsilyl iodide affords the corresponding glycosyl iodides, which readily undergo substitution with aryl alkoxy anions to provide 2-deoxy-beta-O-aryl glycosides. Direct displacement of the anomeric iodide alleviates the need to introduce temporary C-2 stereodirecting groups that require subsequent removal. The only observable byproducts from the glycosylations result from elimination of HI giving the starting glycals, which can be recycled through the reaction sequence.

Solution- and solid-phase oligosaccharide synthesis using glucosyl iodides: a comparative study.

Glycosyl iodide donors have been used in both solid- and solution-phase syntheses yielding alpha-(1 --> 6)-linked glucosyl oligomers in highly efficient protocols. While the solid-phase strategy offers advantages in terms of ease of purification, it requires a total of 7.5 equiv of donor and approximately 12 h to complete the incorporation of one monosaccharide unit. In contrast, solution-phase methods require only 2.5 equiv of donor and 2-3 h reaction time per glycosylation. Moreover, since the reactions are virtually quantitative (> 90%) column chromatography of the material is facile. The overall advantages of solution-phase oligosaccharide synthesis were further illustrated in the convergent synthesis of a hexamer (methoxycarbonylmethyl 6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl-(1 --> 6)-tetrakis-(2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl-(1 --> 6))-2,3,4-tri-O-benzyl-1-thio-alpha-D-glucopyranoside) that was constructed from dimer donor iodides in a two-plus-two and a two-plus-four fashion.

Solution-phase hexasaccharide synthesis using glucosyl iodides.

[reaction: see text] Oligosaccharides composed of 1,6-glucosyl residues have been prepared from glucosyl iodides. The reactions are highly stereoselective, giving the alpha-glycosides as the only isolated products in yields ranging from 84% to 94%. Oligomer synthesis can take place in an iterative 1 + 1 + 1 fashion or in a convergent manner where dimer iodides serve as donors for higher order acceptors.