Protection against Anthrax Toxemia by Hexa- d -Arginine In Vitro and In Vivo

Abstract

The anthrax toxin protective antigen precursor is activated by proteolytic cleavage by furin or a furin-like protease. We present here data demonstrating that the small stable furin inhibitor hexa-D-arginine amide delays anthrax toxin-induced toxemia both in cells and in live animals, suggesting that furin inhibition may represent a reasonable avenue for therapeutic intervention in anthrax.Anthrax toxin consists of three polypeptides, the protective antigen (PA), lethal factor (LF), and edema factor (EF) (reviewed in references 1, 2, 3, and 13). The PA is secreted by the anthrax bacterium as a precursor molecule which must be proteolytically activated by furin (8,11,18,26) and/or furinlike proteases, such as PACE4 (9). Cleavage occurs at a surface loop and releases PA 20 , the N-terminal fragment of PA. Loss of this N-terminal fragment results in self-association of PA 63 , the C-terminal fragment of PA, forming membrane-inserting heptamers capable of binding LF and/or EF (20). These complexes are taken up into cells and enter the lumen of the acidic endosomal compartment, from which LF emerges to initiate enzymatic reactions that ultimately result in cell death (1,12,15).Through the use of combinatorial chemistry techniques, workers in our laboratory have developed the compound hexa-D-arginine (D6R) as a potential therapeutically useful furin inhibitor (4). Studies that used Pseudomonas aeruginosa exotoxin A (PEA), a bacterial toxin which also requires furin processing, have shown that D6R effectively blocks PEA-induced cell death in vitro and in vivo (21). In this study, we have tested the efficacy of D6R against anthrax exotoxins (PA plus LF) in cells and in two animal models.D6R inhibits the cytotoxicity of anthrax toxin in a murine alveolar macrophage cell line. To evaluate the ability of D6R to protect the murine alveolar macrophage cell line RAW 264.7 (ATCC TIB-71) from anthrax toxin-induced cytotoxicity, we first established the dose of toxin representing the 50% effective concentration (EC 50 ) (Fig. 1A), and we then treated cells with different concentrations of D6R in combination with this dose. A concentration of 25 ng of PA per ml in the presence of 12 ng of LF per ml was required for lysis of half of the murine alveolar macrophages after 3 h of incubation with toxins (at EC 50 ). The inclusion of 1 M D6R protected 16% of the cells from death at 6 h, while 100 M D6R increased this survival rate to 36% compared with that of cells treated with anthrax toxin alone (0% survival rate) and with that of the untreated control group (100% survival rate, P Ͻ 0.0001) (Fig. 1B). To determine whether multiple administration of D6R would exhibit improved protective effects against anthrax toxin-induced cytotoxicity, we treated cells with D6R prior to administration of toxin and also tested multiple applications of D6R (10 M) every hour after application of toxin, but no improved efficacy was detected (data not shown).To confirm the protective effect of D6R on the cleavage of PA 83 , we analyzed RAW 264.7 cell cl...

Cleavage of the papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for infection

“…Furin inhibitors that prevent the activation of bacterial toxins, such as anthrax toxin (40), are under development. Results from mice protected against anthrax toxemia by systemic treatment with furin inhibitors suggest that topical application might have acceptable side effects (41).…”

Inhibition of Furin by Polyarginine-containing Peptides

“…Protection of Anthrax Toxemia by D9R Amide in RAW264.7 Cells-Previous studies showed that the D6R amide prevents anthrax toxemia in RAW cells by inhibiting PA cleavage (9). Because D9R amide exhibited a better K i for furin in enzymatic assays, we tested whether this nonamer is also more potent in blocking anthrax toxemia (Fig.…”

“…L6R (hexa-L-arginine), 1 for example, exhibits the low inhibition constant (K i ) of 114 nM (7), and the D-forms of these polyarginines were also shown to be inhibitory. Moreover, D6R amide has been shown to block the activation of Pseudomonas aeruginosa exotoxin A (8) and to protect against anthrax toxemia both in vivo and in vitro (9).…”

Lethal and Edema Toxins in the Pathogenesis of Bacillus anthracis Septic Shock

“…Mutant forms of the PA molecule (dominant-negative inhibitors) with abnormal 2␤2-2 ␤3 loops that form dysfunctional heptamers with wild-type PA prevented the uptake and transport of LF or EF and were effective with LeTx challenge in cell culture and Fisher 344 rats (80,81). Hexa-d-arginine, a furin inhibitor that blocks PA cleavage and heptamer formation, was protective with LeTx challenge in murine alveolar macrophages and Fisher 344 rats (82). Finally, soluble PA receptor protected against LeTx in in vitro studies (13).…”