Enzymatic Detoxification Of Chemical Warfare Agents

We succeeded in completing our objectives for this research project. These accomplishments include the following: (a) synthesis and development of a flexible organophosphate substrate analog library for each of the stereoisomers of GB, GD, and VX. These compound have been utilized in the high throughput screening of mutant protein libraries; (b) site-directed mutagenesis of each amino acid within the active site of the bacterial phosphotriesterase; (c) development of mutagenic protocols for the efficient construction of protein libraries with altered catalytic properties; (d) development of high throughput spectrophotometric screens for characterization of individual mutant proteins derived from the enzyme libraries.

Based on results previously restricted for military use and inaccessible to the public, this practice-oriented handbook introduces the use of enzymes for fast and efficient decontamination of B/C weapons in various scenarios, including terrorist attacks. It draws on the internationally recognized technological leadership of the German armed forces, whose anti-B/C technology is among the most advanced worldwide. The text is rounded off with a look at future perspectives.

The primary objective of this research program is the development of a versatile enzyme-based system that is fully optimized for the decontamination, destruction, and detection of know chemical warfare agents. The catalytic properties of the wild-type enzyme phosphotriesterase are being enhanced and broadened for the hydrolic detoxification of the 0- and V-type nerve agents and their associated analogs. This goal is being achieved by the reorganization of the active site of the wild type phosphotriesterase through combinatorial and direct-mutagensis to create libraries of mutant enzymes with altered catalytic properties. High throughput screening methods have been developed to permit the isolation of those mutant proteins with improved catalytic properties toward the hydrolysis of all biologically toxic stereoisomers of the 0-and V-type agents. The individual amino acid residues within the active site that dictate the substrate and catalytic efficiency of this enzyme have been identified and characterized. Two to 10- fold improvements with single site- mutant proteins have been obtained in the turnover numbers for a subset of soman and sarin analogs. Fully optimized mutants will be created and identified by enhanced combinatorial mutagensis methods.

There are some 15,000 known organophosphorus chemicals. Some of these OP's, including VX and paraoxon, demonstrate an acute neurotoxicity due to the inhibition of cholinergic enzymes. Organophosphorus chemical warfare agents and pesticide neurotoxins are subject to hydrolysis by OP degrading enzymes. To be useful as a bioremediation or anti-chemical warfare agent, the enzyme must be tailored for, and integrated into, a practical application platform. Several studies have established enzyme-based countermeasures, describing such diverse applications as decontaminating foams for surface remediation, encapsulating enzyme with liposome for in vivo therapy, enzyme attachments to surfaces for biosensors and development of a corn expression system for large-scale enzyme production. The goal of the research described here is to select, investigate and improve the operational potential of organophosphate-degrading enzymes including Organophosphorus Hydrolase (OPH, 3.1.8.1) and Organophosphorus Acid Anhydrolase (OPAA, 3.1.8.2). Using saturation kinetics, the catalytic efficiencies of these two major detoxification enzymes were characterized with substrates representing each class of OP neurotoxin, phosphotriester, phosphothioate and phosphofluoridate. OPH presents superior kinetic parameters with each OP class tested. Variants of OPH were created to increase the operational effectiveness of OP hydrolytic enzymes against phosphorothioates. An H254S/H257L mutation in the active site resulted in an improvement in the kinetics (kcat/KM) for the phosphorothioate, demeton-S. To screen potential vascular protection therapies, an in vitro protocol was developed to predict enzymatic effectiveness for protection of acetylcholinesterase from acute OP-inhibition. The protection abilities of the enzymes were directly related to their second order rate constants as inhibitory levels of OP are below the KM of the enzymes. Consideration of contaminant nature concentration and enzyme kinetic parameters, kcat and KM, is critical to understanding decontamination and effective use of enzyme technology. These technologies continue to develop and provide promising new decontamination tools for OP compounds.

Enzymes in Action is a timely survey of a modern development in organic chemistry. It is clear that bioreagents demand that organic chemists think in a different way. If they do so, they will open up new avenues of exciting, new chemistry that will permit problems to be solved in an elegant way. The first section covers the concepts necessary to understand enzymes in molecular operations. The second section covers heteroatom enzyme chemistry, with considerable attention being given to the use of enzymes in the detoxification of chemical warfare agents and their application in environmental problems. The final section highlights the strategic use of enzymes in organic chemistry. It is clear that the term `green chemistry' is appropriate, since enzyme mediated processes occur under mild, environmentally benign conditions, and enzymes enable chemists to perform new chemical operations that would otherwise be difficult to achieve at all.

Handbook of Toxicology of Chemical Warfare Agents, Third Edition, covers every aspect of deadly toxic chemicals used in conflicts, warfare and terrorism. Including findings from experimental as well as clinical studies, this essential reference offers in-depth coverage of individual toxicants, target organ toxicity, major incidents, toxic effects in humans, animals and wildlife, biosensors and biomarkers, on-site and laboratory analytical methods, decontamination and detoxification procedures, and countermeasures. Expanding on the second edition, Handbook of Toxicology of Chemical Warfare Agents has been completely updated, presenting the most recent advances in field. Brand new chapters include a new chapter on emergency preparedness, coverage of the chemical warfare agents used in Syria, the use of the Novichok agent in the UK, and more. Unites world-leading experts to bring you cutting-edge, agent-specific information on Chemical Warfare Agents (CWA) and their adverse effects on human and animal health, and the environment Provides you with all the information you need on CWA modes of action, detection, prevention, therapeutic treatment and countermeasures New to this edition: a full update to reflect the most recent advances in the field and new chapters on emergency preparedness, the chemical warfare agents used in Syria, and the use of the Novichok agent in the UK

Many books cover the emergency response to chemical terrorism. But what happens after the initial crisis? Chlorine, phosgene, and mustard were used in World War I. Only years after the war were the long-term effects of these gases realized. In the 60s, 70s, and 80s, these and other agents were used in localized wars. Chemical Warfare Agents: Tox

Organophosphorus (OP) nerve agents are some of the most potent toxic agents conceived by mankind, are specific inhibitors of cholinesterase (ChE), and therefore are a serious threat to military and civilian personnel. OP nerve agents can exist as a liquid and contaminate skin, penetrate, resulting in deleterious systemic effects including a cholinergic crisis. Another serious problem that may be encountered while caring for personnel contaminated with organophosphorus chemical warfare nerve agents is the possibility that there will be cross-contamination to the medical personnel. In addition, during combat or terrorist acts, individuals might be exposed to chemical toxins before they don their protective gear. We have been developing enzyme immobilized polyurethane foams that can effectively decontaminate the skin and other such exposed surfaces of the organophosphorus toxins in a wide variety of environmental conditions. With additives, the sponges provided increased protection for guinea pigs exposed to neat soman and VX compared to currently fielded kits, and reduces methylene blue uptake in mustard-exposed animals. Sponges have several criteria to meet to be effective personal decontaminants and detoxifiers of chemical warfare agents. The sponges must be lightweight for individual use, yet be shelf-stable under environmental conditions found in the field. In this manner, the sponges would provide immediate use for either the individual or a buddy in a field.