Anthrax toxin protective antigen protein (PA, 83 kDa) binds to receptors on the surface of mammalian cells, is cleaved by the cell surface protease furin, and then captures either of the two other toxin proteins, lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa). The PA-LF and PA-EF complexes enter cells by endocytosis and LF and EF translocate to the cytosol. EF is a calcium- and calmodulin-dependent adenylyl cyclase that causes large and unregulated increases in intracellular cAMP concentrations. LF is a metalloprotease that cleaves several mitogen-activated protein kinase kinases (MEKs). Entry of anthrax toxin into cells depends on two related cell surface receptors, tumor endothelium marker 8 (TEM8) and capillary morphogenesis gene product 2(CMG2). TEM8 was initially identified as a protein upregulated in colon cancers. CMG2 has substantial sequence similarity to this candidate tumor marker. The tissue distribution and the relative importance of the two toxin receptors in toxin action are not well understood. During 2011 we extended analysis of toxin receptor in a collaboration with Keith Wycoff of Planet Biotechnology, Inc. While antibodies to PA have proven efficacious, an alternative strategy is to create receptor decoys. The CMG2 extracellular domain has high affinity to PA, but it would not be expected to have a long residence time in plasma. To improve its pharmacological properties, this domain was fused to the Fc domain of human IgG. Furthermore, to assure that adequate amounts could be produced, the fusion protein was expressed in plants, specifically tobacco plants. In the best clone, nearly 0.1% of the plant's wet weight was the Fc-CMG2 fusion protein, meaning that almost unlimited amounts of protein could be produced. Administration of the purified protein at 2 mg/kg body weight protected rabbits against a lethal challenge with fully virulent anthrax spores. The plant-produced protein was glycosylated, but the presence of this modification did not alter serum half-life, which was approximately 5 days. These data show that this receptor decoy protein has considerable potential for production as a therapeutic agent for anthrax. In other collaborative research done during 2011, we worked to improve small molecule inhibitors directed to the protease activity of LF. Our colleagues at Panthera, Inc., used an LF inhibitor described by Merck as a point of departure in design of more potent compounds. A large number of compounds were synthesized and tested for in vitro protease inhibition. From among the most potent of these, compounds with properties suggesting they could be useful in vivo as drugs were tested at the LBD for protective efficacy in the rat lethal toxin (PA + LF) challenge model. In this model, rats injected intravenously with lethal toxin experience a rapid and highly reproducible shock response that would lead to death in 60-100 minutes. This system provides a quantitative method for assessing and comparing the in vivo efficacies of compounds. One of these compounds, which is active in vitro at subnanomolar concentrations, provided 100% protection in the rat lethal toxin model. In a subsequent follow-up study, additional compounds were synthesized to examine the roles of substituents at several positions on the core structure. This led to identification of an improved group of compounds having Ki values of 0.1-2.0 nM. The best of these compounds protected rats from lethal toxin challenge at drug doses of only 2.5 mg/kg. These are among the most potent LF protease inhibitors yet described.
Showing the most recent 10 out of 31 publications
