Bacillus anthracis causes anthrax disease by growing to high numbers in the blood and secreting the anthrax exotoxins, consisting of three components: protective antigen (PA), lethal factor (LF), and edema factor (EF). Anthrax infections are initiated when B. anthracis spores enter a potential host organism by ingestion, inhalation, or skin abrasion. The spores then germinate and replicate as toxin-producing vegetative bacteria, which through secreting anthrax toxins to overcome the host innate immune responses to establish successful systemic infections and eventually kill host through the actions of the toxins. Among the three components of anthrax toxins, PA is the cellular binding moiety, which binds to its cellular receptors, Tumor Endothelium Marker-8 (TEM8) and Capillary Morphogenesis Protein-2 (CMG2). PA is then proteolytically activated by the ubiquitously expressed cell surface furin protease, resulting in the formation of active PA oligomer, which in turn binds and translocates the two enzymatic moieties LF and EF into the cytosol of cells. LF, which forms lethal toxin (LT) with PA, cleaves several mitogen-activated protein kinase kinases (MEKs) and rodent inflammasome sensor Nlrp1. EF, which forms edema toxin (ET) with PA, is an adenylate cyclase that generates abnormally high concentrations of cAMP. Both LT and ET are lethal to animals. TEM8 and CMG2 are the two well-characterized anthrax toxin receptors, each containing a von Willebrand factor A (vWA) domain responsible for PA binding. Recently, a cell-based analysis was used to implicate another vWA domain-containing protein, integrin 1 as a third anthrax toxin receptor. To explore whether proteins other than TEM8 and CMG2 function as anthrax toxin receptors in vivo, in the year of 2013, we further evaluated the sensitivity of the mice lacking TEM8 and/or CMG2 to anthrax toxins. Specifically, we used as an effector protein the fusion protein FP59, a fusion between the PA-binding domain of LF and the catalytic domain of Pseudomonas aeruginosa exotoxin A. FP59 is at least 50-fold more potent than LF in the presence of PA, with 2 ug PA + 2 ug FP59 being sufficient to kill a mouse. While TEM8-/- and wild type control mice succumbed to a 5 ug PA + 5 ug FP59 challenge, CMG2-/- mice were completely resistant to this dose, confirming that CMG2 is the major anthrax toxin receptor in vivo. To detect whether any toxic effects are mediated by TEM8 or other putative receptors such as integrin 1, CMG2-/-/TEM8-/- mice were challenged with as many as five doses of 50 ug PA + 50 ug FP59. Strikingly, the CMG2-/-/TEM8-/- mice were completely resistant to the 5-dose challenge. These results strongly suggest that TEM8 is the only minor anthrax toxin receptor in vivo and that other proteins implicated as receptors do not play this role. Developments of anti-toxin agents for anthrax prevention and therapy are urgently needed. In the year of 2013, by collaborating with Drs. Andrew Orry (MolSoft LLC) and Michael Peredelchuk (University of Texas Brownsville), we performed a virtual ligand screen of a library of 10000 members to identify compounds predicted to bind to PA and prevent its oligomerization. Four of these compounds slowed PA oligomaerization, and two of those protected cells from intoxication at concentrations of 1-10 M. Exploration of the protective mechanism showed decreased SDS-resistant PA oligomer on cells and, surprisingly, decreased amounts of activated PA. In vitro assays showed that one of the inhibitors blocked furin-mediated cleavage of PA, apparently through its binding to the PA substrate. Thus, we have identified inhibitors that can independently block both PA's cleavage by furin and its subsequent oligomerization. Lead optimization on these two backbones may yield compounds with high activity and specificity for the anthrax toxins. We previously developed small molecule inhibitors of LF proteolytic activity (LFIs) and demonstrated their in vivo efficacy in a rat lethal toxin challenge model. In the year of 2013 we further showed that these LFIs protect against lethality caused by anthrax infection in mice when combined with sub-protective doses of either antibiotics or neutralizing monoclonal antibodies that target edema factor. Significantly, these inhibitors provided protection against lethal infection when administered as a monotherapy. As little as two doses (10 mg/kg) administered at 2 h and 8 h after spore infection was sufficient to provide a significant survival benefit in infected mice. Administration of LFIs early in the infection was found to inhibit dissemination of vegetative bacteria to the organs in the first 32 h following infection. In addition, neutralizing antibodies against edema factor also inhibited bacterial dissemination with similar efficacy. Together, our findings confirm the important roles that both anthrax toxins play in establishing anthrax infection, and demonstrate the potential for small molecule therapeutics targeting these proteins. One area of work in this project seeks to use the toxic action of modified anthrax toxins to target cancer. In the year of 2013, by collaborating with Dr. Ralph Abi-Habib (Lebanese American University, Beirut, Lebanon), we attempt to target the mitogen-activated protein kinase (MAPK) pathway in acute myeloid leukemia (AML) cells using a recombinant anthrax LT. We tested potency of LT on a panel of 11 human AML cell lines. Seven cell lines showed cytotoxic responses to LT. Cytotoxicity of LT was mimicked by the specific MEK1/2 inhibitor U0126, indicating that LT-induced cell death is mediated through the MEK1/2-(ERK1/2 branch of the MAPK pathway. The four LT-resistant cell lines were sensitive to the phosphatidylinositol 3-kinase inhibitor LY294002. Co-treatment of AML cells with both LT and LY294002 did not lead to increased sensitivity, showing a lack of additive/synergistic effects when both pathways are inhibited. Flow cytometry analysis of MAPK pathway activation revealed the presence of phospho-ERK1/2 only in LT-sensitive cells. Staining for Annexin V/propidium iodide and active caspases showed an increase in double-positive cells and the absence of caspase activation following treatment, indicating that LT-induced cell death is caspase-independent and nonapoptotic. We have shown that a majority of AML cell lines are sensitive to the LF-mediated inhibition of the MAPK pathway. Furthermore, we have demonstrated that LT-induced cytotoxicity in AML cells is nonapoptotic and dependent on phospho-ERK1/2 levels.

Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
2013
Total Cost
$847,612
Indirect Cost
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Liu, Shihui; Liu, Jie; Ma, Qian et al. (2016) Solid tumor therapy by selectively targeting stromal endothelial cells. Proc Natl Acad Sci U S A 113:E4079-87
Vrentas, Catherine E; Moayeri, Mahtab; Keefer, Andrea B et al. (2016) A Diverse Set of Single-domain Antibodies (VHHs) against the Anthrax Toxin Lethal and Edema Factors Provides a Basis for Construction of a Bispecific Agent That Protects against Anthrax Infection. J Biol Chem 291:21596-21606
Moayeri, Mahtab; Tremblay, Jacqueline M; Debatis, Michelle et al. (2016) Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice. Clin Vaccine Immunol 23:213-8
Chen, Zhaochun; Schneerson, Rachel; Lovchik, Julie A et al. (2015) Bacillus anthracis Capsular Conjugates Elicit Chimpanzee Polyclonal Antibodies That Protect Mice from Pulmonary Anthrax. Clin Vaccine Immunol 22:902-8
Wein, Alexander N; Peters, Diane E; Valivullah, Zaheer et al. (2015) An anthrax toxin variant with an improved activity in tumor targeting. Sci Rep 5:16267
Martin, Erik W; Buzza, Marguerite S; Driesbaugh, Kathryn H et al. (2015) Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden. Oncotarget 6:33534-53
Bekdash, Amira; Darwish, Manal; Timsah, Zahra et al. (2015) Phospho-MEK1/2 and uPAR Expression Determine Sensitivity of AML Blasts to a Urokinase-Activated Anthrax Lethal Toxin (PrAgU2/LF). Transl Oncol 8:347-57
Bachran, C; Hasikova, R; Leysath, C E et al. (2014) Cytolethal distending toxin B as a cell-killing component of tumor-targeted anthrax toxin fusion proteins. Cell Death Dis 5:e1003
Peters, Diane E; Hoover, Benjamin; Cloud, Loretta Grey et al. (2014) Comparative toxicity and efficacy of engineered anthrax lethal toxin variants with broad anti-tumor activities. Toxicol Appl Pharmacol 279:220-9
Maier, Nolan K; Crown, Devorah; Liu, Jie et al. (2014) Arsenic trioxide and other arsenical compounds inhibit the NLRP1, NLRP3, and NAIP5/NLRC4 inflammasomes. J Immunol 192:763-70

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