Medical Aspects of Chemical Warfare (2008) - The Black Vault

Medical Aspects of Chemical Warfarebiological residence time, rHu BChE was modified toinclude polyethylene glycol adducts. The polyethyleneglycolylated material had a pharmacokinetic profilesimilar to that of the pHu BChE, 55,59 suggesting that differencesin pharmacokinetics between plasma-derivedand recombinant enzymes can be addressed using invitroposttranslational modifications. Efficacy studiesusing rHu BChE from transgenic goat milk in guineapigs against soman and VX have yielded results similarto those previously described that used pHu BChE. 59These results suggest that effective recombinant stoichiometricbioscavengers can be developed, potentiallyproviding a source for sufficient material for militarymembers and civilians (such as first responders, emergencymedical personnel, and agricultural workers)that may be occupationally exposed to OP pesticides.CATALYTIC BIOSCAVENGERSAlthough stoichiometric scavengers are able to affordgood protection as long as the enzyme level inthe body is higher than the amount of OP, they havea relatively high molecular weight; a comparativelylarge quantity is required to neutralize a small amountof nerve agent. A catalytic scavenger, even having thesame high molecular weight, could be administeredin smaller quantities and would potentially producethe same or greater extent of protection. It would alsobe advantageous because it would not be consumedin the process of detoxifying the nerve agent, makingit available to protect against multiple OP exposures.Enzymes with intrinsic, catalytic, anti-OP activitiescome from a variety of sources, such as the OP hydrolasefrom Pseudomonas diminuta, 60 the OP anhydrasefrom Alteromonas haloplanktis, 61 and human paraoxonase1 (Hu PON1). 62–66 Recombinant OP hydrolasefrom Pseudomonas diminuta was shown to protect miceagainst behavioral side effects and lethality caused bysoman. 67 Similarly, pretreatment with only OP hydrolasepurified from Pseudomonas species was shown toprotect mice from lethality due to paraoxon, diethylfluorophosphate,and tabun. 68,69 Most of these enzymespossess short circulation times in vivo, and none hasthe ability to hydrolyze all known toxic OPs, nor doany have the high turnover required to dispose of theOPs from blood in one circulation time. In addition,these bacterial enzymes are likely to initiate potentimmune responses in humans; therefore, they are notsuitable for repeated use. Bacterial enzymes couldconceivably be useful for skin protection as active componentsof topical skin protectants or covalently boundto the cornified layer of epidermis. 70 OPs can also bedetoxified through enzymatic oxidation of their alkylchains. In particular, breakdown of VX by horseradishperoxidase 71 or by Caldariomyces fumago chloroperoxidase72 could be used in a polyfunctional active topicalskin protectant and for skin decontamination.Conversely, Hu PON1 can possibly afford protectionwithout the potential complication of inducingan immune response. However, Hu PON1 does notpossess the desired catalytic activity at a rate that is fastenough for use as a nerve agent pretreatment. Becauseagent must be cleared from the bloodstream withinone circulation time (1 to 2 minutes) before it reachescritical targets, 15 a functional catalytic scavenger musthave both a lower K m(a measure of the strength ofbinding of a substrate to the enzyme) and a highturnover number (k cat). Research efforts were directedtoward creating such an enzyme by specific mutationof enzymes such as Hu BChE and Hu PON1. Hu BChEmutation designs were based on the fact that OP inhibitorsare hemisubstrates for this enzyme. The acylationreaction is similar to that of normal substrates, but thesubsequent reaction, equivalent to deacylation of theactive site serine, cannot be affected because the aminoacid group responsible for dephosphylation is not inthe appropriate position. 73,74The perceived solution to this problem was to inserta second catalytic center into the active site specificallyto carry out the dephosphylation step of the reaction. 74Applying this rationale, wild-type Hu BChE was mutatedin the oxyanion hole to create a mutated enzyme,G117H, with the ability to catalyze the hydrolysis ofsarin, diisopropylfluorophosphate (DFP), paraoxon,VX, and other nonaging nerve agents. 74,75 Aging andreactivation are parallel first-order reactions in phosphylatedenzymes. In the reactivation reaction, thephosphoryl group is removed from the active siteserine residue (Ser198), restoring activity, whereas inthe aging reaction one of the alkyl groups is removedfrom the phosphoryl group, rendering the inhibitedenzyme nonreactivatable. To catalyze the hydrolysis ofrapidly aging nerve agents such as soman, it is necessaryto slow the rate of the aging reaction so that reactivationis faster. This was accomplished by replacingthe carboxyl group Glu197 adjacent to the active siteserine with an amide group. 76 Although these effortswere successful, the mutants have catalytic activitiesthat are still too slow for practical use.Hu PON1 is currently being subjected to mutationin efforts to generate faster catalytic antinerve agentenzymes. Because OPs are “accidental” substrates forparaoxonase, 62,64 it is likely that activity improvementcan be realized through protein engineering. Two ofthe major difficulties in designing appropriate site-250