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 antitoxins.

Figure 1

Structure-based design of heptavalent anthrax toxin inhibitors. A) Structure of the LF-binding face of [PA₆₃]₇. Residues 184, 187, 197, and 200, which form part of the peptide-binding site are shown in purple. B) Structure of the core, β-cyclodextrin. C) Scheme illustrating the binding of a heptavalent inhibitor, synthesized by the attachment of seven inhibitory peptides to the β-cyclodextrin core via an appropriate polyethylene glycol linker, to [PA₆₃]₇

Figure 2

Mutations in PA₆₃ diminish binding of phage displaying HYTYWWLD. Phage displaying HYTYWWLD peptide were added to wells containing adsorbed mutant or wild-type (WT) [PA₆₃]₇ as indicated. Unbound phage were removed by washing and the bound phage were detected with an anti-M13 phage antibody conjugated to horseradish peroxidase.

Figure 3

Clusters representing conformations sampled by the HTSTYWWLDGAP peptide, obtained using replica exchange molecular dynamics simulations. The figure was generated using the program VMD.

Figure 4

(A) Residues of [PA₆₃]₇ that are contacted by the peptide in docked complexes 4-3 (blue) and 4-4 (red) are shown as space-filled atoms. Individual protein chains of the heptamer (each colored differently) are also shown as transparent background. Both 4-3 and 4-4 dock at the interface of two monomeric units and their contact sites include amino acid residues identified as important by mutagenesis. (B) Close-up view of the docked conformation 4-4 showing HTSTYWWLDGAP peptide (green) and the [PA₆₃]₇ contact residues (red) as space-filled atoms. Two of the chains in the heptameric unit that 4-4 is in contact with are also shown as transparent background.

Figure 5

Synthesis scheme of heptavalent anthrax toxin inhibitor. (a) TBDMSCl, pyridine, 0 °C - rt. (b) NaH, MeI, THF (c) NH₄F, MeOH, reflux (d) NaH, propargyl bromide, DMF, 0 °C - rt. (e) CuSO₄, sodium ascorbate, THF:H₂O:BuOH (0.5:1:1), 80 °C (f) Chloroacetic anhydride, triethylamine (g) Peptide, DMF, DBU, triethylamine.

Figure 6

Characterization of a well-defined heptavalent anthrax toxin inhibitor. A) Inhibition of anthrax toxin-induced cytotoxicity of RAW264.7 cells by heptavalent inhibitors presenting HTSTYWWLDGAP (open circles) or control thioglycerol-functionalized molecules (closed circles). B) Stability of heptavalent inhibitor in serum. Heptavalent inhibitor was incubated in 80% serum for indicated times and the IC₅₀ was determined in a RAW264.7 cell cytotoxicity assay.

Figure 7

Influence of linker length on the activity of heptavalent inhibitors. The indicated PEG linkers were used to join the HTSTYWWLDGAP peptide to β-cyclodextrin and the IC₅₀ values of the resulting inhibitors were measured in a RAW264.7 cell cytotoxicity assay. The error bars represent the standard deviation from four separate experiments. Asterisks indicate that inhibitory activity was not detected.