Polyvalent inhibitors of anthrax toxin that target host receptors

Abstract

Resistance of pathogens to antimicrobial therapeutics has become a widespread problem. Resistance can emerge naturally, but it can also be engineered intentionally, which is an important consideration in designing therapeutics for bioterrorism agents. Blocking host receptors used by pathogens represents a powerful strategy to overcome this problem, because extensive alterations to the pathogen may be required to enable it to switch to a new receptor that can still support pathogenesis. Here, we demonstrate a facile method for producing potent receptor-directed antitoxins. We used phage display to identify a peptide that binds both anthrax-toxin receptors and attached this peptide to a synthetic scaffold. Polyvalency increased the potency of these peptides by >50,000-fold in vitro and enabled the neutralization of anthrax toxin in vivo. This work demonstrates a receptor-directed anthrax-toxin inhibitor and represents a promising strategy to combat a variety of viral and bacterial diseases.

Fig. 1.

Identification of peptides that bind the I domains of anthrax-toxin cellular receptors ANTXR1 and ANTXR2. (A and B) Phage from the unselected 12-mer library or pooled phage obtained after three rounds of panning against immobilized ANTXR1 (A) or immobilized ANTXR2 (B) were subjected to ELISA. In each case, the phage pool isolated after three rounds bound specifically to the I domain (black bars) and was inhibited by PA (gray bars). Immobilized BSA was used as a control (white bars).

Fig. 2.

Characterization of AWPLSQLDHSYN-functionalized polyvalent inhibitors in vitro. (A) Inhibition of anthrax toxin-induced RAW264.7 cytotoxicity by polyvalent liposomes presenting AWPLSQLDHSYN (■) or control thioglycerol-functionalized liposomes (●). (B) Phage presenting the sequence AWPLSQLDHSYN, which were isolated by panning against ANTXR1, bind specifically to both ANTXR1 and ANTXR2 (black bars) but do not bind to BSA (white bars). Binding of phage to the I domains was inhibited by PA (gray bars). (C) Binding of fluorescein-containing liposomes presenting the sequence AWPLSQLDHSYN (black bars), the sequence HTSTYWWLDGAP (white bars), and thioglycerol (gray bars) to ANTXR2, [PA₆₃]₇, PA, and BSA. (D) Binding of fluorescein-containing liposomes presenting AWPLSQLDHSYN (black bars) and thioglycerol-functionalized control liposomes (gray bars) to CHO-R1-ANTXR1, CHO-R1-ANTXR2, and receptor-deficient CHO-R1.1 cells.

Fig. 3.

The AWPLSQLDHSYN-functionalized liposomes prevent LF_N binding to oligomeric PA_63. (A) CHO-R1.1 cells (lane 1) or CHO-R1-ANTXR1 cells (lanes 2–5) were treated for 2 h with PA alone (lanes 1 and 2); PA and AWPLSQLDHSYN-functionalized liposomes (lane 3), PA and HTSTYWWLDGAP-functionalized liposomes (lane 4), or PA and thioglycerol-functionalized liposomes (lane 5). Cells were washed with PBS and lysed, and the resulting lysates were subjected to Western blotting using anti-PA antibody. (B) The association of [³⁵S]LF_N with cells in the presence of PA and liposomes functionalized with either AWPLSQLDHSYN (white bars), HTSTYWWLDGAP (black bars), or thioglycerol (gray bars) was measured by scintillation counting.