Structure-Based Design of a Heptavalent Anthrax Toxin Inhibitor

Abstract

The design of polyvalent molecules, consisting of multiple copies of a biospecific ligand attached to a suitable scaffold, represents a promising approach to inhibit pathogens and oligomeric microbial toxins. Despite the increasing interest in structure-based drug design, few polyvalent inhibitors based on this approach have shown efficacy in vivo. Here we demonstrate the structure-based design of potent biospecific heptavalent inhibitors of anthrax lethal toxin. Specifically, we illustrate the ability to design potent polyvalent ligands by matching the pattern of binding sites on the biological target. We used a combination of experimental studies based on mutagenesis and computational docking studies to identify the binding site for an inhibitory peptide on the heptameric subunit of anthrax toxin. We developed an approach based on copper-catalyzed azide-alkyne cycloaddition (click-chemistry) to facilitate the attachment of seven copies of the inhibitory peptide to a β-cyclodextrin core via a polyethylene glycol linker of an appropriate length. The resulting heptavalent inhibitors neutralized anthrax lethal toxin both in vitro and in vivo and showed appreciable stability in serum. Given the inherent biocompatibility of cyclodextrin and polyethylene glycol, these potent well-defined heptavalent inhibitors show considerable promise as anthrax anti-toxins.

Design of Monodisperse and Well‐Defined Polypeptide‐Based Polyvalent Inhibitors of Anthrax Toxin

“…[10] Polyvalent inhibitors have been created by attaching multiple copies of this peptide to a variety of scaffolds such as polymers, [12] liposomes, [13] and β-cyclodextrin. [14] Structure-based design is a particularly effective strategy to design polyvalent inhibitors of anthrax lethal toxin and other targets. [11][12][13][14][15][16] Ligand spacing and valency are key parameters that influence the efficacy of polyvalent molecules (Scheme 1A).…”

“…[14] Structure-based design is a particularly effective strategy to design polyvalent inhibitors of anthrax lethal toxin and other targets. [11][12][13][14][15][16] Ligand spacing and valency are key parameters that influence the efficacy of polyvalent molecules (Scheme 1A). [12][13][14]17] The ligand spacing is simply the distance separating adjacent ligands on a polyvalent molecule.…”

“…[11][12][13][14][15][16] Ligand spacing and valency are key parameters that influence the efficacy of polyvalent molecules (Scheme 1A). [12][13][14]17] The ligand spacing is simply the distance separating adjacent ligands on a polyvalent molecule. [5,13,14] Matching the ligand spacing on the polyvalent molecule to the distance between ligand binding sites on the target molecule has been shown to be an effective strategy to design potent polyvalent ligands.…”

“…[12][13][14]17] The ligand spacing is simply the distance separating adjacent ligands on a polyvalent molecule. [5,13,14] Matching the ligand spacing on the polyvalent molecule to the distance between ligand binding sites on the target molecule has been shown to be an effective strategy to design potent polyvalent ligands. [16,18] For a linear polymer or polypeptide, ligand spacing can be manipulated by choosing a linker of appropriate length to separate adjacent ligands on a polyvalent molecule.…”

“…[12] Matching the valency of the polyvalent molecule to the number of binding sites on the target molecule may also be an effective strategy. [5,12,14] For polyvalent conjugates, the number of ligand molecules attached to the scaffold can usually be adjusted by varying the relative molar ratio of ligands to scaffold during a conjugation reaction. Bioconjugation coupling strategies such as amine-activated ester coupling or sulfhydryl-maleimide coupling have been the preferred strategies used to attach multiple copies of ligand molecules to scaffolds.…”