Binding Site Geometry and Subdomain Valency Control Effects of Neutralizing Lectins on HIV‑1 Viral Particles

Carbohydrate binding proteins such as griffithsin, cyanovirin-N, and BanLec are potent HIV entry inhibitors and promising microbicides. Each binds to high-mannose glycans on the surface envelope glycoprotein gp120, yet the mechanisms by which they engage viral spikes and exhibit inhibition constants ranging from nanomolar to picomolar are not understood. To determine the structural and mechanistic basis for recognition and potency, we selected a panel of lectins possessing different valencies per subunit, oligomeric states, and relative orientations of carbohydrate binding sites to systematically probe their contributions to inhibiting viral entry. Cryo-electron micrographs and immuno gold staining of lectin-treated viral particles revealed two distinct effectsnamely, viral aggregation or clustering of the HIV-1 envelope on the viral membranethat were dictated by carbohydrate binding site geometry and valency. “Sandwich” surface plasmon resonance experiments revealed that a second binding event occurs only for those lectins that could aggregate viral particles. Furthermore, picomolar <i>K</i>_d values were observed for the second binding event, providing a mechanism by which picomolar IC₅₀ values are achieved. We suggest that these binding and aggregation phenomena translate to neutralization potency

Discovery and Synthesis of Namalide Reveals a New Anabaenopeptin Scaffold and Peptidase Inhibitor

The discovery, structure elucidation, and solid-phase synthesis of namalide, a marine natural product, are described. Namalide is a cyclic tetrapeptide; its macrocycle is formed by only three amino acids, with an exocyclic ureido phenylalanine moiety at its C-terminus. The absolute configuration of namalide was established, and analogs were generated through Fmoc-based solid phase peptide synthesis. We found that only natural namalide and not its analogs containing l-Lys or l-<i>allo</i>-Ile inhibited carboxypeptidase A at submicromolar concentrations. In parallel, an inverse virtual screening approach aimed at identifying protein targets of namalide selected carboxypeptidase A as the third highest scoring hit. Namalide represents a new anabaenopeptin-type scaffold, and its protease inhibitory activity demonstrates that the 13-membered macrolactam can exhibit similar activity as the more common hexapeptides

Design of HIV Coreceptor Derived Peptides That Inhibit Viral Entry at Submicromolar Concentrations

HIV/AIDS continues to pose an enormous burden on global health. Current HIV therapeutics include inhibitors that target the enzymes HIV protease, reverse transcriptase, and integrase, along with viral entry inhibitors that block the initial steps of HIV infection by preventing membrane fusion or virus–coreceptor interactions. With regard to the latter, peptides derived from the HIV coreceptor CCR5 were previously shown to modestly inhibit entry of CCR5-tropic HIV strains, with a peptide containing residues 178–191 of the second extracellular loop (peptide <b>2C</b>) showing the strongest inhibition. Here we use an iterative approach of amino acid scanning at positions shown to be important for binding the HIV envelope, and recombining favorable substitutions to greatly improve the potency of <b>2C</b>. The most potent candidate peptides gain neutralization breadth and inhibit CXCR4 and CXCR4/CCR5-using viruses, rather than CCR5-tropic strains only. We found that gains in potency in the absence of toxicity were highly dependent on amino acid position and residue type. Using virion capture assays we show that <b>2C</b> and the new peptides inhibit capture of CD4-bound HIV-1 particles by antibodies whose epitopes are located in or around variable loop 3 (V3) on gp120. Analysis of antibody binding data indicates that interactions between CCR5 ECL2-derived peptides and gp120 are localized around the base and stem of V3 more than the tip. In the absence of a high-resolution structure of gp120 bound to coreceptor CCR5, these findings may facilitate structural studies of CCR5 surrogates, design of peptidomimetics with increased potency, or use as functional probes for further study of HIV-1 gp120–coreceptor interactions

Characterization and Carbohydrate Specificity of Pradimicin S

The pradimicin family of antibiotics is attracting attention due to its anti-infective properties and as a model for understanding the requirements for carbohydrate recognition by small molecules. Members of the pradimicin family are unique among natural products in their ability to bind sugars in a Ca²⁺-dependent manner, but the oligomerization to insoluble aggregates that occurs upon Ca²⁺ binding has prevented detailed characterization of their carbohydrate specificity and biologically relevant form. Here we take advantage of the water solubility of pradimicin S (PRM-S), a sulfated glucose-containing analogue of pradimicin A (PRM-A), to show by NMR spectroscopy and analytical ultracentrifugation that at biologically relevant concentrations, PRM-S binds Ca²⁺ to form a tetrameric species that selectively binds and engulfs the trisaccharide Manα1–3­(Manα1–6)­Man over mannose or mannobiose. In functional HIV-1 entry assays, IC₅₀ values of 2–4 μM for PRM-S corrrelate with the concentrations at which oligomerization occurs as well as the affinities with which PRM-S binds the HIV surface envelope glycoprotein gp120. Together these data reveal the biologically active form of PRM-S, provide an explanation for previous speculations that PRM-A may contain a second mannose binding site, and expand our understanding of the characteristics that can engender a small molecule with the ability to function as a carbohydrate receptor

Comparison of equilibrium dissociation constants for the binding of ScFvs to 5-helix determined by ITC and HIV-1 neutralization potency in an Env-pseudotyped neutralization assay.

a<p>ITC was carried out at 28°C in 10 mM Tris-HCl, pH 7.6, 150 mM NaCl.</p>b<p>The neutralization IC₅₀ values for Fab8066, taken from ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Gustchina2" target="_blank">[22]</a>, are provided for comparison. The <i>K</i>_D for binding to 5-helix, also determined by ITC under the same conditions used for the ScFvs here, is from ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Gustchina5" target="_blank">[35]</a>.</p>c<p>Sc62 is derived from the parental Fab8062 and has four mutations in the CDR-H2 loop relative to Fab8066/Sc66: I53L, T56F, T57A and N58V.</p

Native-PAGE band-shift and SEC-MALS analyses of core^S-antibody complexes.

<p>Interaction of (<i>A</i>) Fab8066, (<i>B</i>) Sc66 and (<i>C</i>) Sc62 with the six-helix bundle core^S antigen (Ag). Left panels: 10 µM core^S trimer mixed with Fab or ScFv (shown above the lanes) in molar ratios of 1∶1, 1∶2 and 1∶3 were subjected to 20% homogeneous native-PAGE. Core^S and antibody are color coded orange and blue, respectively. Calculated molecular weights of Fab8066, Sc66, Sc62, core^S and their complexes are indicated in kDa. Right panels: Protein mixtures (total of 200 µg) at a trimer (core^S) to antibody ratio of 1∶1 were subjected to SEC-MALS. Experimental average masses and compositions are indicated. Also shown in panel A (right) are the SEC-MALS traces for core^S alone (black), corresponding to a trimer (of calculated mass 3×8284 g/mol), and a 1∶1 mixture of core^S and Fab8062 (dashed gray) which shows no evidence of complex formation. The black arrows in panels B and C (right) indicate the retention volume of Sc66 (see Figure S2C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683.s001" target="_blank">File S1</a>). In panel C (right), the major peak corresponds to uncomplexed ScFv and core^S which co-elute, while the minor peak represents a barely detectable amount of 1∶1 Sc62:core^S complex. Observed masses (g/mol) for free Fab8066, Fab8062 and Sc66 are 47060±559, 48560±629 and 27630±774, respectively.</p

Determining the upper limit of affinity for the binding of Fab8066 to core^S by native-PAGE and an approximate <i>K</i>_D by SEC-MALS.

<p>(<i>A</i>) Decreasing concentrations (10 to 1.25 µM) of core^S trimer mixed with a 2-fold molar excess of Fab8066 and subjected to native-PAGE with Coomassie staining (upper panel), and decreasing concentrations (1 to 0.25 µM) of core^S trimer mixed with a constant 1 µM concentration of Fab8066, visualized by silver staining. Core^S and Fab8066 are color coded orange and blue, respectively. (<i>B</i>) Injection of 3 µg core^S mixed with 6 µg Fab8066 on a BioSep-SEC-S 2000 column (0.46×30 cm) at a flow-rate of 0.35 ml/min equilibrated in 10 mM Tris-HCl, pH 7.6, 150 mM NaCl (buffer A). The elution profile (black) is shown superimposed on deconvoluted peaks for the major (∼85%) core^S-Fab8066 complex (red) and free Fab8066 (blue). The measured mass of the complex is shown beside the peak. A <i>K</i>_D of ∼70 nM was estimated on the basis of the calculated concentration of the complex and free Fab. Deconvolution of the SEC-MALS profile was carried out using the program PeakFit (Seasolve Software, Inc. Framingham, MA).</p

Native-PAGE, SEC-MALS and CD analysis of 6-helix-antibody complexes.

<p>(<i>A</i>) Native-PAGE in the presence of increasing molar ratios of Sc66 to 6-helix. 6-helix to Sc66 ratios are shown above the respective lanes and the observed stoichiometry of the complexes (purple, 6-helix; blue, Sc66) and expected molecular weights (kDa) are indicated. (<i>B</i>) SEC-MALS for 6-helix alone (orange) and a 1∶1 mixture of 6-helix with Sc66 (black). Average compositions and masses are indicated next to the peaks. (<i>C</i>) CD spectra of 6-helix alone (black) and a 1∶2 mixture of (6 helix+Sc66) minus Sc66 alone (orange). The CD data indicate that there is no change in helicity of 6-helix upon complexation with Sc66.</p

Mass, SDS-PAGE and CD analyses of core^SP-antibody complexes.

<p>(<i>A</i>) SEC-MALS of core^SP (black), Fab8066 (blue) and Sc66 (orange) (top) and of the core^SP-Fab8066 (middle) and core^SP-Sc66 (bottom) complexes (mixed at a molar ratio of 1∶1 six-helix bundle to antibody). Average masses and compositions are indicated next to the peaks. (<i>B</i>) SDS-PAGE of peak fractions (numbering of lanes corresponds to elution volume) collected from SEC-MALS confirm the composition of the peaks indicated on the SEC-MALS traces in panel A. Top, core^SP alone; middle, core^SP+Fab8066 (H and L denote heavy and light chains, respectively); bottom, core^SP+Sc66. (<i>C</i>) CD of core^SP alone (black); a 1∶1 mixture of (core^SP+Fab8066) minus Fab8066 alone (blue); a 1∶1 mixture of (core^SP+Sc66) minus Sc66 alone (orange); and the C34 peptide (green, corresponding to the C-HR) alone. The CD data indicate that there is no change in helicity of core^SP when complexed to either Fab8066 or Sc66.</p

Interaction of Fab8066 with 5-helix and design of the corresponding ScFv Sc66.

<p>(<i>A</i>) Overall interaction of Fab8066 with 5-helix, (<i>B</i>) detailed view of the interaction of the CDR-H2 loop of Fab8066 (yellow) with the two exposed N-HR helices (green) of 5-helix, and (<i>C</i>) interaction of the CDR-H1 and CDR-H2 loops of Fab8066 (yellow) with two N-HR helices (white) and one C-HR helix (orange) of 5-helix. The addition of a third C-HR helix (transparent orange) to 5-helix to complete the six-helix bundle would result in steric clash with the CDR-H1 and CDR-H2 loops. Color coding in panels A and B is as follows: N-HR and C-HR helices of 5-helix are shown in green and orange, respectively; the CDR heavy and light chain loops of Fab8066 are shown in yellow and white, respectively; the remainder of the heavy and light variable domains are shown in dark red and blue, respectively; the light and heavy constant domains of Fab8066 are shown in pink and light blue, respectively. In panel C, the three N-HR helices are shown in white and residues mapped by alanine scanning mutagenesis of a six-helix bundle construct as the epitope for binding Fab8066 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Gustchina1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Louis3" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Gustchina2" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Miller1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Luftig1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Nelson1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Choudhry1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Zhang1" target="_blank">[27]</a>, are indicated on one of the N-HR helices (helix <i>Na</i>). Also shown in panel A is the design employed to construct the corresponding ScFv by linking the C-terminus of the light chain variable domain (blue) to the N-terminus of the heavy-chain variable domain (dark red) via a 15-amino acid linker (3×GGGGS). The coordinates are taken from PDB IDs 3MA9 (Fab8066/5-helix complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Gustchina5" target="_blank">[35]</a>) and 1SZT (core^S trimer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104683#pone.0104683-Tan1" target="_blank">[16]</a>).</p