Protective antigen (PA) is a central virulence factor of Bacillus anthracis and a key component in anthrax vaccines. PA binds to target cell receptors, is cleaved by the furin protease, self-aggregates to heptamers, and finally internalizes as a complex with either lethal or edema factors. Under mild room temperature storage conditions, PA cytotoxicity decreased (t1 ⁄ 2 ≈ 7 days) concomitant with the generation of new acidic isoforms, probably through deamidation of Asn residues. Ranking all 68 Asn residues in PA based on their predicted deamidation rates revealed five residues with half-lives of <60 days, and these residues were further analyzed: The Gram-positive spore-forming bacterium Bacillus anthracis, the causative agent of anthrax, produces a bipartite A/B-type toxin. The B subunit is the 83-kDa protective antigen (PA) 3 receptor-binding moiety (named for its use as a vaccine), and the two catalytic A subunit moieties are edema factor (EF; 89 kDa) and lethal factor (LF; 90 kDa) (1). EF is a Ca 2ϩ -and calmodulin-dependent adenylate cyclase (2). LF is a Zn 2ϩ protease that cleaves and inactivates mitogen-activated protein kinase kinase-1 and -2 (3, 4). Following PA binding to cell receptors (5-8), it is cleaved by a furin family protease (9) to a 20-kDa N-terminal fragment with no known further function and to a 63-kDa fragment that forms ring-shaped heptamers on the cell surface (10). The heptamers then bind up to three molecules of EF or LF (11,12). Following binding, the heptamer-LF/EF complex undergoes endocytosis, and LF or EF is released to the cytosol (13-15). The crystal structure (16) and functional studies (11,12,(17)(18)(19)(20) have demonstrated that the PA polypeptide is folded into four distinct domains with well defined functions. Domain I (residues 1-258) prevents premature PA polymerization and harbors the furin cleavage site, which is located in an unstructured flexible loop; domain II (residues 259 -487) is involved in heptamerization and is in the membrane insertion loop; domain III (residues 488 -595) is also involved in heptamerization (18) PA has been reported previously to be a thermally unstable protein, losing its in vitro cytotoxic activity upon storage at 37°C within 48 h (32) or within few minutes above 40°C as a result of aggregation (33). Protein loss of function under mild storage conditions is generally attributed to a variety of nonenzymatic modifications such as deamidation, isomerization, oxidation, and alternative disulfide pairings (34, 35). Spontaneous deamidation, which occurs mainly at Asn side chains and at a much slower rate at Gln residues (36), is a major and well documented degradation pathway in proteins. Deamidation rates depend on pH, temperature, primary sequence ("nearest neighbor" effect), and protein conformation (37-40). Deamidation proceeds by nucleophilic attack on the side chain carbonyl carbon of Asn by the nitrogen of the adjacent peptide bond, resulting in the formation of an unstable five-member succinimide ring, which is hydrolyzed to produce m...

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This report presents a non-lethal method for estimating a range of therapeutic doses of bisquaternary oximes that serve as antidotes against organophosphorus poisoning. We have estimated therapeutic oxime doses that are equivalent in their relative toxicity rather than selecting arbitrary fractions of their LD(50). Thus, toxic signs of the oximes HI-6, HLö-7, Toxogonin, AB-8 and AB-13 were monitored quantitatively in baboon monkeys and beagle dogs. Using Toxogonin as a reference oxime, a calculated unit of equivalent dose (CED) was defined as the oxime dose equal to the ratio between its minimal toxic dose (MTD) and the therapeutic ratio (TR) of Toxogonin i.e. CED = MTD/TR. Assuming that the tails of dose-response curves of toxicity for bisquaternary oximes are shallow and similar to one another, one could substitute the ED(10) for the MTD. The ED(10) values for bisquaternary oximes were estimated using the log-log model following experimental observations and quantitative scoring of toxic signs in dogs and monkeys. The MTD values then were calculated using the ED(10) values and the experimental therapeutic dose of the reference oxime Toxogonin. The following CED values were obtained for AB-8, AB-13, Toxogonin, HI-6 and HLö-7 in dogs (d) and monkeys (m): 98.7, 74.2, 30.0, 14.5 and 12.1 (d) and 281.9, 232.1, 41.7, 192.9 and 92.9 (m) micromol kg(-1), respectively. The antidotal efficacy of these oximes against poisoning by the nerve agent tabun was determined in dogs and monkeys. These dose-dependent efficacy data were obtained at 0.3 x CED, 1 x CED and 3 x CED of oximes in combination with atropine. These data provide comparative therapeutic values using oxime doses based on their relative toxicity. The highest antidotal efficacy against tabun in dogs was obtained for toxogonin, whereas HLö-7 and AB-13 were most efficacious in monkeys.

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