Book of Abstracts: Albany 2007
June 19-23 2007
Novel Therapy For Anthrax
In view of the recent terrorist attacks, the civilian populations and military forces are under an increased threat from the exposure of Bacillus anthracis, the causative agent of deadly inhalation anthrax (1). However, combating B. anthracis in a bio-threat scenario requires a therapy that is readily available, rapidly effective, and stable (and yet non-toxic) under physiological conditions. Unfortunately, very few current drugs and vaccines meet these criteria. What we need is a therapeutic agent that possesses the following attributes: (i) it targets the major virulence factors of B. anthracis at a very early stage, (ii) it binds strongly to the functional site(s) of the virulence factors, and (iii) this binding is not thwarted by the mutations on the virulence factors through which pathogens develop resistance against drugs and vaccines. Toward this end, we have chosen the three toxins from B. anthracis, namely ? protective antigen (PA), lethal factor (LF), and edema factor (EF), as putative therapeutic targets. As shown in the Figure, the binding of PA to the host cell receptor constitutes the very first step of the multi-step intoxication process. Upon cytosolic entry, both LF and EF cause toxic effects (2). Here, we describe the structure-based design and in vitro and in vivo testing of a chimeric protein that blocks the binding of PA to host cells. The chimeric protein consists of three domains. The first one is the von Willebrand factor A (VWA) domain, which is a PA-specific ligand; as shown in the Figure, the VWA domain is derived from the extra-cellular portion of the PA receptors, CMG2 and TEM8. The second one is the Fc domain, which, under native conditions, dimerizes via the formation of disulfide bridges. The third and final one is the linker domain that allows native folding of VWA and Fc without any steric interference. The chimeric VWA-Fc protein is, therefore, a bivalent PA ligand. In addition, the attachment of VWA to the Fc frame facilitates correct folding
The possible steps in anthrax intoxication (bottom) and the development of a therapeutic protein (top) that blocks the intoxication. Capillary morphogenesis protein 2 (CMG2) and tumor endothelial marker 8 (TEM8) have, so far, been identified as the putative PA receptors (2). Upon binding, PA83 gets cleaved by host furin protease. The cleaved PA63 forms a heptamer and attaches to LF or EF. The (PA63)7-LF/EF complex is translocated from the plasma membrane to the endosome from where LF or EF is released to the cytosol leading to host toxicity and morbidity (2). Based upon our experience in developing chimeric proteins for therapeutic and serological use (3-5), we proceeded to develop a chimeric protein to block the binding of PA83 to the receptor. Both schematic and atomic models of the VWA-Fc/PA complex are shown (color coding: VWA, yellow; Fc, green; linker, magenta; PA, red). The atomic model on the top is the energy-minimized average structure of the last 500-ps snapshots from the 1 ns MD trajectory of the complex of VWA-Fc and PA. The initial model was derived from the crystal structure of the complex of VWA-and PA (6).
The designed VWA-Fc chimera is expressed in mammalian cells. The purified chimera shows a high-affinity for PA (dissociation constant, KD=0.2 nM) by surface plasmon resonance (BIAcore) measurements. Also at nanomolar concentrations, the VWA-Fc chimera inhibits the lysis of mouse macrophages by the bipartite PA/LF toxin. Finally, to test the in vivo efficacy, a fairly low dose of VWA-Fc chimera is administered intravenously to Fisher (C13) rats at the same time of the PA/LF exposure or one hour prior to the exposure. The VWA-Fc rescues the rats from lethality during four days of study period without any toxic side effect. Therefore, the results of our study indicate that the VWA-Fc chimera can provide an effective therapy against anthrax in case of a terrorist attack, i.e., the therapy can be administered in the emergency room to the civilians exposed to B. anthracis as well as to the soldiers before they are sent to the battlefields where they may be exposed to this bio-threat agent.
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