Identification of a Peptide-Based Neutralizer That Potently Inhibits Both Shiga Toxins 1 and 2 by Targeting Specific Receptor-Binding Regions

Abstract

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagicEscherichia colithat occasionally causes fatal systemic complications. We recently developed a tetravalent peptide (PPP-tet) that neutralizes the cytotoxicity of Stx2 using a multivalent peptide library approach. In this study, we used this technique to identify a series of tetravalent peptides that bound to Stx1, another major Stx family member, with high affinity by targeting one receptor-binding site of the B subunit. One peptide, MMA-tet, markedly inhibited Stx1 and Stx2 cytotoxicity with greater potency than PPP-tet. After forming a complex with Stx1 through its specific receptor-binding region, MMA-tet did not affect vesicular transport of the toxin to the endoplasmic reticulum but substantially rescued inhibition of the protein synthesis induced by Stx1. Oral application of MMA-tet protected mice from a fatal dose of anE. coliO157:H7 strain producing both toxins. MMA-tet may be a promising therapeutic agent against the infection.

Affinity-Based Screening of Tetravalent Peptides Identifies Subtype-Selective Neutralizers of Shiga Toxin 2d, a Highly Virulent Subtype, by Targeting a Unique Amino Acid Involved in Its Receptor Recognition

“…Tetravalent peptides were synthesized with N-␣-9-fluorenylmethoxy carbonyl (Fmoc)-protected amino acids and standard (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate/1-hydroxybenzotriazole hydrate coupling chemistry as described previously (34). A Met-Ala sequence was included at the amino terminus of the tetravalent peptide so that its structure would be identical to that of MMA-tet, which was developed on the basis of the results of multivalent peptide library screening and found to effectively inhibit both Stx1a and Stx2a (36). The terminal amino groups of the tetravalent peptides were biotinylated with biotin (Sigma-Aldrich, USA) and 1-(bis[dimethylamino]methylene)-1H-benzotriazolium 3-oxide hexafluorophosphate (Peptide Institute Inc., Japan) in the last cycle of the peptide synthesis.…”

“…By targeting Stx2a receptor-binding site 3 or Stx1a site 1, we identified various tetravalent peptides demonstrating remarkable therapeutic potency in both a mouse model of EHEC infection (34,36) and a nonhuman primate model (19). Recently, we established a novel technique to determine a wide range of binding motifs for the B subunit by directly screening hundreds of divalent peptides synthesized on a cellulose membrane.…”

“…Accordingly, several compounds with clustered trisaccharides that can bind to the B subunit with high affinity have been developed and shown to effectively neutralize Stx both in vitro and in vivo (27)(28)(29)(30)(31)(32)(33). These compounds, however, cannot be customized to specific Stx subtypes, because all Stx subtypes recognize the trisaccharide as the natural binding unit.Previously, we developed a library of multivalent peptides exhibiting the clustering effect, from which we identified Stx-neutralizing tetravalent peptides by screening the library for highaffinity binding to the specific receptor-binding sites (33)(34)(35)(36). By targeting Stx2a receptor-binding site 3 or Stx1a site 1, we identified various tetravalent peptides demonstrating remarkable therapeutic potency in both a mouse model of EHEC infection (34, 36) and a nonhuman primate model (19).…”

“…Previously, we developed a library of multivalent peptides exhibiting the clustering effect, from which we identified Stx-neutralizing tetravalent peptides by screening the library for highaffinity binding to the specific receptor-binding sites (33)(34)(35)(36). By targeting Stx2a receptor-binding site 3 or Stx1a site 1, we identified various tetravalent peptides demonstrating remarkable therapeutic potency in both a mouse model of EHEC infection (34, 36) and a nonhuman primate model (19).…”

“…We targeted Asn16 and screened a series of tetravalent peptides synthesized on a cellulose membrane, the sequences of which contained the Bsubunit consensus binding motif (34,36,37). Using this approach, we identified two peptides that selectively neutralize Stx2d.…”