Novel protein-based solutions for organophosphorus nerve agent detection and elimination Public Deposited

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  • March 22, 2019
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  • Hemmert, Andrew C.
    • Affiliation: School of Medicine, Department of Biochemistry and Biophysics
Abstract
  • Organophosphorus (OP) nerve agents are some of the deadliest chemicals ever synthesized by man. These toxins, which include sarin, soman, cyclosarin, tabun, and VX, inhibit the neurotransmitter-regulating enzyme, acetylcholinesterase (AChE). This leads to continual acetylcholine muscarinic and nicotinic receptor stimulus and may eventually result in death due to prolonged muscles contraction and diaphragm incapacitation. Current treatments for OP poisoning include injections of atropine, to dampen acetylcholine stimulation, a strong-nucleophile oxime, such a 2-pralidoxime, to reactivate inhibited AChE, and diazepam for seizures. These treatments are limited, however, because they do not protect against poisoning, cannot be administered prior to exposure, and don't address the long-term side effects associated with nerve agent poisoning. Additionally, there is no broad-spectrum oxime effective against all nerve agents. A better therapeutic would be a prophylactic molecule capable of catalytically degrading the OP prior to AChE inhibition. Protein-based therapeutics are an emerging remedy for OP toxicity. It has been shown that pre-administration of excess AChE in mice can protein against 8-10 normally lethal doses of soman. Current enzyme therapeutics can be categorized as either stoichiometric or catalytic. Stoichiometric OP binders, such as AChE or the homologous butyrylcholinesterase (BChE), benefit from nanomolar (nM) dissociation constants, but suffer in their ability to recover after OP exposure, thereby requiring large enzyme doses for effective treatment. Catalytic protein therapeutics, including serum paraoxonase (PON1) or the bacterial organophosphate hydrolase (OPH) exhibit rapid rates of in vitro nerve agent hydrolysis, but are limited by high dissociation constants, making them ineffective in vivo. Human carboxylesterase 1 (hCE1) is a liver serine hydrolase in the same [alpha]/[beta] super family as AChE and BChE, which may have more favorable attributes as an OP bioscavenger. Indeed rodents express a serum carboxylesterase that affords them high levels of protection against OPs. Using structural and biochemical studies, we determined the stereopreference, rates of spontaneous reactivation, and availability of rapid oxime-assisted reactivation of hCE1 with nerve agents. Next, using structurally guided protein design, we engineered a form of the hCE1 that combines the benefits of both bioscavenger classes, exhibiting nM dissociation constants and enhanced rates of hydrolysis, up to 48,700-fold, against the nerve agents sarin, soman, and cyclosarin. Finally, novel mutants of hCE1 were developed that exhibit-increased rates of reactivation against specific agents, and can be utilized to detect and identify chemical agents.
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  • ... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics.
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  • Redinbo, Matthew R.
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