Amiodarone and Bepridil Inhibit Anthrax Toxin Entry into Host Cells

Abstract

Anthrax lethal toxin is one of the fundamental components believed to be responsible for the virulence of Bacillus anthracis. In order to find novel compounds with anti-lethal toxin properties, we used a cell-based assay to screen a collection of approximately 500 small molecules. Nineteen compounds that blocked lethal toxinmediated killing of RAW 264.7 macrophages were identified, and we report here on the characterization of the two most potent antitoxic compounds, amiodarone and bepridil. These drugs are used to treat cardiac arrhythmia or angina in humans at doses similar to those that provide protection against lethal toxin in vitro. Our results support a model whereby the antitoxic properties of both drugs result from their ability to block endosomal acidification, thereby blocking toxin entry. Amiodarone was tested in vivo and found to significantly increase survival of lethal toxin-challenged Fischer rats.Bacillus anthracis is a gram-positive bacterium that is the causative agent of anthrax. Two major virulence factors that contribute to disease are lethal toxin (LT) and edema toxin (ET), and these genes are located on an extrachromosomal plasmid, pXO1 (10). Both LT and ET are bipartite toxins that rely on the same binding moiety, protective antigen (PA), to mediate the delivery of their respective enzymatic subunits into the host cytosol. LT consists of PA and lethal factor (LF), a metalloprotease that cleaves the N terminus of mitogen-activated protein kinase kinases 1 to 4, 6, and 7 (15, 49). ET, on the other hand, consists of PA and edema factor (EF), a calciumand calmodulin-dependent adenylate cyclase that raises cyclic AMP (cAMP) levels once inside the host cell (30).Cellular entry of either EF or LF begins with PA binding to a cell surface receptor. Receptor-bound PA undergoes furin cleavage, a step that allows it to heptamerize and form a structure known as the prepore (26, 34). The prepore then associates with up to three catalytic subunits and undergoes receptor-mediated endocytosis (38). Acidic pH of the endocytic compartment triggers conformational changes in the PA heptamer, causing it to insert into the membrane of the endosome (34). Insertion results in formation of a pore through which LF and EF translocate, thus gaining access to the cytosol of the host cell (28, 29, 52).Importantly, both LT and ET are lethal when administered independently to laboratory animals, suggesting a role for both toxins in the pathogenic process (14,17,35). At the cellular level, however, the response to each toxin depends on cell type. LT treatment of macrophages derived from certain inbred mouse strains results in rapid cell lysis (6, 18), whereas a less dramatic cytotoxic effect has been seen in endothelial cells and dendritic cells, among others, where prolonged treatment over several days results in reduced viability (reviewed in reference 3). Yet, most cell types tested so far do not die in response to LT. Rather, more subtle effects have been characterized mostly in immune cells, such as neutrophils and ...

Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses

“…The requirement for low pH found in late endosomes is a characteristic shared by toxins, viruses, and natural receptor/li- gand pairs such as EGFR/EGF, which were blocked by EGA. Many of the known inhibitors of LT target this step by directly neutralizing endosomal pH (27,28). However, EGA does not block acidification of endosomes as determined by Lysotracker red staining (Fig.…”

“…MEK2 cleavage assay was performed as described previously with minimal modification (28). Briefly, six-well plates were seeded with 2 × 10 6 RAW 264.7 cells per well and allowed to grow overnight.…”

“…Neutralization of acidic compartments by lysosomotropic agents like NH 4 Cl is a wellestablished technique for blocking anthrax toxin entry (27,28). The observed block to pore formation raised the possibility that EGA functions by neutralizing low-pH-dependent steps in the endocytic pathway.…”

Rescue of Progranulin Deficiency Associated with Frontotemporal Lobar Degeneration by Alkalizing Reagents and Inhibition of Vacuolar ATPase

“…Such drugs may be tolerated without major adverse side effects, as shown for CQ, a frequently used malaria drug (Solomon and Lee, 2009). Drug concentrations used in this study are therapeutically achievable in plasma of patients during treatment (Sanchez et al, 2007). For treatment of FTLD-TDP, it is of obvious interest that the potential drugs are able to cross the blood-brain barrier.…”

Anthrax Toxins

“…Many compounds protect through inhibition of proper PA63 oligomer formation or by blocking translocation of LF to cytosol, either through inhibition of receptor -mediated endocytosis or acidifi cation of endosomes (Artenstein et al, 2004 ;Menard et al, 1996 ;Moayeri et al, 2006 ;Sanchez et al, 2007 ). Other inhibitors such as phosphatase inhibitors identifi ed in two separate studies have been implicated in interfering with LF cleavage of Mek substrates through unknown mechanisms (Kau et al, 2002 ;Panchal et al, 2007 ).…”