JOURNAL OF APPLIED TOXICOLOGYJ. Appl. Toxicol. 21, S43–S46 (2001)DOI:10.1002/jat.804

Progress on the Road to New Nerve AgentTreatments†

Clarence A. Broomfield* and Stephen D. KirbyUS Army Medical Research Institute of Chemical Defense (MRICD), Biochemical Pharmacology Branch, PharmacologyDivision, 3100 Ricketts Point Rd, Aberdeen Proving Ground, MD 21010, USA

Key words: nerve agents; scavengers; history; butyrylcholinesterase; mutagenesis; OPAHA; carbamates; carboxylesterase.

In the 50 years since nerve agents were developed a great deal has been learned about their acute toxicity,treatment and prophylactic strategies. However, the currently fielded treatments are not significantly differentfrom those available at the end of World War II. Reasons for this lack of progress and strategies to circumventthose intrinsic problems that have impeded progress are discussed, with emphasis on the development ofscavengers to be introduced as prophylactics that will significantly reduce the effective dose and thus protectagainst multiple times the normal LD50. Published in 2001 by John Wiley & Sons, Ltd.

A little over 50 years have passed since nerve agentswere developed during World War II. In the interven-ing period a great deal of time and effort has beenexpended in studies of mechanisms, symptomatic treat-ments, acetylcholinesterase (AcChE) reactivators and pro-tective measures. Our current understanding of all aspectsof nerve agent actions, sequelae and prophylactic strate-gies is orders of magnitude greater than it was at the endof World War II and it is possible to protect the lives ofsoldiers against multiple LD50 values of those highly toxiccompounds and to ameliorate some of their symptomsand sequelae as well. Nevertheless, the treatments thatare currently in the field for all of the world’s armies arenot significantly different from those available at the endof World War II and they fail in many important respects.The reason for this is that all pretreatment and treatmentstrategies are constrained by human physiology and thecharacteristics of the nerve agents themselves. No matterhow effective a protective inhibitor, such as a carbamate,may be to block acetylcholinesterase against reaction withnerve agents, after an exposure there will always be atime during which acetylcholine will build up pendingreactivation of the enzyme, causing a period of incapac-itation. No matter how effective the reactivator, it mustreach the inhibited enzyme and reverse the inhibition intime to protect life or it is useless. Stronger anticholiner-gics and superior anticonvulsants are being developed, butthey must be delivered in a timely fashion if they are tobe effective. When we reviewed these facts several yearsago it became apparent that it would be extremely diffi-cult to improve on the existing pharmacological protectionagainst nerve agents because the basic limiting factor isthe speed at which the treatment drugs are able to reach thecritical nerve endings compared with the speed of actionof the agents. Given this situation, the best way to provide

* Correspondence to: C. A. Broomfield, US Army Medical ResearchInstitute of chemical Defense, Biochemical Pharmacology Branch, Phar-macology Division, 3100 Ricketts Point Rd, Aberdeen Proving ground,MD 21010, USA.† This article is a U.S. Government work and is in the public domainin the U.S.A.

complete protection of personnel against the effects ofnerve agents, without incapacitation or behavioral deficits,is to equip them with a scavenger that will reside in thebloodstream and be able to intercept and destroy nerveagent molecules before they reach a critical target.

The concept of using scavengers to reduce the toxicdose is neither new nor inspired. As early as 1956, Mainshowed that he could protect rats against paraoxon byinjecting exogenous paraoxonase.1 It was well establishedbefore 1983 that rodents enjoy natural protection fromnerve agents due to an endogenous scavenger, carboxy-lesterase, that is present at high levels in their serum.2 – 6

The first real effort at MRICD towards investigating thedevelopment of a protective scavenger was a projectto purify an organophosphorus acid anhydride hydrolase(OPAH, initially called ‘DFPase’; to eliminate the confu-sion caused by a plethora of names given to this class ofenzymes, this nomenclature was adopted at a Workshopin Woods Hole, MA, June 1987) from hog kidney, a richsource of that activity. Once the protein was obtained inrelatively pure form, however, it was discovered that ithas a strong preference for the P(+) or inactive forms ofsoman and thus was ineffective at detoxifying the agent.A few years later, Dr Ludwig Sternberger conceived theidea of actively immunizing against soman, and severalyears were invested in the development of haptens andimmunogens to protect against that most refractory com-pound. Again, mice were used as test animals, and theamount of protection achieved was not large enough tojustify full-scale development. After reviewing the data inlight of present knowledge, one is led to wonder whetherthe protection would have been high enough to pursuehad the mice been treated with CBDP (a specific inhibitorof carboxylesterase) prior to challenge with soman, as iscommon practice now. Following the demise of the immu-nization program we were able to show that the OPAH inhuman blood, the ‘paraoxonase’ used by Main to pro-tect rats against paraoxon, is able also to catalyze thehydrolysis of soman and sarin, and that there is a widevariation among individuals in the activity of that enzyme.Because at that time we did not recognize its identity with

Published in 2001 by John Wiley & Sons, Ltd.Received 16 February 1999

Accepted 10 June 1999

S44 C. A. BROOMFIELD AND S. D. KIRBY

Main’s paraoxonase, we called it ‘somanase’.7 Subsequentinvestigation revealed that ‘somanase’ was identical tothe previously described ‘paraoxonase’. Our initial dataindicated that the enzyme might be induced by low-levelexposures to nerve agents, but studies in animals showedthat it did not appear to be inducible. This activity, usuallyreferred to as ‘PON’,8 has been shown now to consist ofthree separate but related enzymes and the most abundanthas been isolated, purified and sequenced and its gene hasbeen cloned and expressed in eukaryotic cells.9

During the next few years, the effort on scavengerswas greatly reduced, with work limited to attempts toisolate and characterize OPAH enzymes from rat liver10

and NG108-15 cells,11 but we were able to show duringthis period that certain of the enzymes of this type wouldreact with and detoxify the P(−) isomers of soman.12

Then, in the late 1980s, technology had advanced to thepoint where new approaches could be considered. TheMRICD commander and his successors were interested inapplying state-of-the-art solutions to the problem of nerveagent intoxication. The cholinesterases now were availablein reasonably pure form and they were used to showthat stoichiometric scavengers (one molecule of scavengerreacts irreversibly with one molecule of toxin) were ableto protect animals against the effects of nerve agentswithout measurable side-effects.13,14 They also requirea large amount of material to destroy a small amountof toxin. Using a bacterial OPAH from Pseudomonasdiminuta we were able to show that catalytic scavengers(enzymes that catalyze the hydrolysis of the toxins) alsoare effective.15 Unfortunately, the enzymes that occurin nature are generally inefficient; they have high Km

values and low turnover numbers. In general they are alsoimmunogenic when administered to humans.

Based on these results, we were convinced that ourbest chance of providing a truly effective pretreatmentthat could protect soldiers against multiple lethal dosesof nerve agents, without undesirable side-effects or tem-porary incapacitation, lay in the development of catalyticscavengers of human origin, having low Km values andhigh turnover numbers. Because enzymes of that descrip-tion are not available in nature, as far as we are aware,we made the decision to attempt to create enzymes withthe required characteristics using emerging technology.We initiated at MRICD a dual approach to developscavengers with the characteristics needed for practicalapplication. One approach was to attempt to develop cat-alytic monoclonal antibodies with the desired properties,using organophosphorus transition-state analogs to elicitthe specificity needed. This work produced one clone thatapparently displayed the desired specificity16 but it wassubsequently lost and could not be reproduced; eventu-ally the effort was abandoned. It is interesting to note thatan investigator in Finland recently published results on amonoclonal catalytic antibody that has characteristics verysimilar to those of the antibody produced at MRICD.17

The second approach was an effort to construct a moreefficient OPAH by protein engineering techniques. Ourinitial attempt involved site-directed mutagenesis of thehuman butyrylcholinesterase (BuChE) gene. The ratio-nale for this scheme was based on a hypothesis byJarv18 that organophosphates are hemisubstrates for thecholinesterases because they form chiral phosphylatedenzymes. The histidine-bound water molecule that nor-mally displaces the acyl group from the active site serine

in the course of normal hydrolysis is prevented stericallyfrom attacking the appropriate face of the tetrahedral phos-phorus atom, thus preventing reactivation of the enzyme.If this hypothesis is correct, we reasoned that it might bepossible to introduce a second nucleophilic center into theactive site in such a position that it could carry an acti-vated water molecule to the face of the phosphorus moietyopposite the phosphorus-serine bond and thereby reacti-vate the enzyme. To attempt this became possible whenthe crystal structure of AcChE was solved by Sussmanet al.19 For a number of reasons we felt that BuChE wouldbe more appropriate to test this hypothesis than AcChE:BuChE has a less restricted active site and is somewhatless stereoselective than AcChE; in addition, BuChE is anintrinsic plasma protein and therefore a successful mutantwould be expected to enjoy a longer biological half-life inthe plasma. Furthermore, although several natural mutantsof BuChE exist in nature, there is no evidence that theyare antigenic (i.e. persons transfused with blood contain-ing mutant forms do not develop antibodies) so the bodyappears to be more tolerant of mutations in BuChE thanin some other proteins. Therefore, we began by makinga computer model of human BuChE based on the crystalstructure of Torpedo AcChE, replacing those residues thatare different and then minimizing the energy of the result-ing structure.19 Having done that, we then measured thedistance between the α-carbon of serine 198 (the ‘activesite’ serine) and the α-carbon of histidine 438 (9.62 A onour model) and looked for residues on the opposite sideof the gorge with similar spacing and with the side-chainoriented in the general direction of serine 198.

We identified, among others, three glycine residues(numbers 115, 117 and 121) with reasonable spacings andthe appropriate orientation and decided to make mutantswith each of these residues substituted by histidine asa first attempt at our goal. Using phosphorothioate site-directed mutagenesis, these mutants were made, expressedand screened for the ability to catalyze the hydrolysisof butyrylthiocholine (BuSCh), benzoylcholine (BzCh),echothiophate, soman, sarin and diisopropyl fluorophos-phate (DFP) and for inhibition by echothiophate, soman,sarin, DFP and tetraisopropyl pyrophosphoramide (iso-OMPA).

Both G121H and G115H appeared to have no activityat all; the hydrolysis of neither benzoylcholine (BzCh) norbutyrylthiocholine (BuSCh) is catalyzed and there is noevidence of hydrolysis of any of the organophosphoruscompounds tested. On the other hand, G117H showedhydrolytic rates of AcSCh, BuSCh and BzCh comparablewith those of the wild type enzyme and it appeared tohave essentially normal or only slightly altered reactionkinetics with all of the substrates tested (Table 1). Furtherinvestigation showed that the rates of reaction with allof the organophosphorus inhibitors were greatly reduced(Table 2). Following inhibition by sarin, VX or DFP,spontaneous reactivation was much more rapid than in thewild type enzyme. This is the type of organophosphorushydrolase activity that we expected to see but it was muchlower than we hoped to find.

In an effort to explore the reaction mechanism ofthis mutant with the organophosphorus inhibitors, arelated mutant G117K, in which glycine at position 117is replaced by lysine instead of histidine, was madeand expressed. Lysine carries a positive charge just ashistidine does. Our hypothetical mechanism requires the

Published in 2001 by John Wiley & Sons, Ltd. J. Appl. Toxicol. 21, S43–S46 (2001)

NEW NERVE AGENT TREATMENTS† S45

Table 1—Comparison of substrate kinetic constants

Enzyme

type

p-Nitrophenyl

acetate

Km(mM)

BuSCh

Km(mM)

BuSCh

Kss(mM)

BuSCh b

value

Wild

type

6 ± 1 0.025 ± 0.006 0.9 ± 0.1 3.1 ± 0.4

G117H 13 ± 2 0.05 ± 0.01 0.8 ± 0.1 2.7 ± 0.3

G117K 13 ± 2 0.40 ± 0.06 44 ± 5 4.0 ± 0.2

E197Q ND 0.078 ± 0.005 23 ± 4 1.7 ± 0.1

G117H/

E197Q

ND 0.28 ± 0.02 120 ± 20 5.1 ± 0.5

Table 2—Comparison of organophosphorus inhibition reactionrates

Enzyme type Bimolecular inhibition rate constants (M−1

s−1)

VX GB GD P(−)C(−)

Wild type 30 400 ± 2800 22 400 ± 3700 >105

G117H 26.4 (20.4–35.2) 2.8 (2.4–3.3) 13 000 ± 2000

G117K 51.9 (28.7–98.0) 3400 (800–3500) >105

G117H/E197Q – – 610 ± 40

bifunctional nature of histidine, so it was anticipated thatthe lysine mutant would lack the ability to reactivaterapidly. Because the preliminary results indicated thatthe dephosphylation step is rate limiting, turnover ratescould be determined accurately by measuring the recoveryof BuChE activity after removal of excess inhibitor.Reactivation data are shown in Table 3. Mutant G117K isinhibited more rapidly and reactivates much more slowlythan G117H with each of the organophosphorus inhibitorstested. Comparison of kinetic data on substrate hydrolysis,inhibition by organophosphorus inhibitors and reactivationrates between these two mutants makes it clear that theobserved characteristics of G117H are not due to thepresence of a positive charge at the 117 position. Ofparticular interest are the reactivation rates of G117H.Assuming that reactivation equals turnover, these datarepresent rate enhancements for hydrolysis of GB andVX of ca. 100- and 2000-fold, respectively. This rate wasverified by direct measurement of the disappearance ofVX in a separate experiment. Upon inhibition of G117Hwith soman, no reactivation is observed and activity is notrestored upon treatment with 2PAM or N ,N ′-trimethylenebis-(pyridine-4-aldoxime) (TMB-4).

Ageing and reactivation are competing first-order reac-tions of the inhibited enzyme. Because the soman-inhi-bited enzyme could not be reactivated with oximes, it

Table 3—Comparison of organophosphorus reactivation rates(×10−5 min−1)

Enzyme type GB VX GD

Wild type <5 <5 NRa

E197Q <5 <5 NR

G117H 870 1200 NR

G117K <5 <5 NR

G117H/E197Q 6200 7800 600–12 800b

a NR = not reactivated.b The GD reactivation rate depends on the stereoisomer.

was assumed that the aging rate was much faster than thereactivation rate and that ageing occurred before turnovercould be achieved. The amino acid residue immediatelypreceding the active site serine in the cholinesterases isa conserved glutamic acid, and it has been implicatedin the ageing reaction.21 If ageing of soman-inhibitedBuChE could be slowed sufficiently, reactivation couldproceed, and it was expected that turnover of soman couldbe observed. Therefore, a double mutant, G117H/E197Q,was made and shown to catalyze the hydrolysis of soman,as expected.22

The question we are now left with is “Why are thesereactions so slow?” One explanation would be that theinsertion of this large side-chain in place of a glycineso affects the folding of the protein structure that it isno longer an effective enzyme. However, the fact thatthe natural butyrylcholine-hydrolyzing activity is affectedonly slightly indicates that the active site is generallyintact. Another explanation could be that our hypothesizedreaction mechanism is not correct. Although the expectedreactivation is seen and the G117K and G117H/E197Qmutants behave as expected, there are aspects of the reac-tion that do not fit the proposed mechanism. For exam-ple, the reactivation rates increase as pH is decreased,which is opposite to the expected result if reactivationis base-catalyzed as with deacylation.23 Several alterna-tive mechanisms have been proposed, including one byLockridge23 and two by Fortier,24 which are based onmolecular mechanics calculations. The Lockridge mecha-nism proposes that dephosphorylation is catalyzed by His438, just as with deacylation. The role of the His 117 inthis mechanism is to skew the oxyanion hole so that thephosphorus is open to attack by water and then to desta-bilize the P–O–Ser and P–O–R′ bonds. This mechanismaccurately predicts an increased rate of ageing as well asthe correct pH dependence for reactivation of the phos-phorylated enzyme. One of Fortier’s mechanisms invokesa phosphoryl histidine intermediate, which is then releasedby uncatalyzed hydrolysis by water or hydroxyl ion. Thiswould be a slow step that might explain the slow reactiva-tion rates. Unfortunately, none of these mechanisms fullyexplains the experimental data.

Our modeling studies indicated that the large histidineside-chain in the oxyanion hole might result in some dis-tortion of the trigonal bipyramidal transition state. Totest this possibility we studied inhibition of the 117Hmutants with carbamates and their subsequent reactivationrates.25 Because the carbamate reaction presumably pro-ceeds through the same tetrahedral transition-state struc-ture as normal substrates, their inhibition rates were notexpected to be affected by the histidine as much as thoseof the organophosphorus inhibitors. The results are shownin Table 4.

Table 4—Rates of carbamylation (ki ) and decarbamylation (k3)

Enzyme Physostigmine (phy) Pyridostigmine (pyr)type

ki

(M−1

min−1

)

k3 (min−1

) ki

(M−1

min−1

)

k3 (min−1

)

Wild type 1 050 000 0.006 10 000 0.0024

G117H 13 600 0.0018 250 0.0009

G117K 14 800 0.009 200 ND

G117H/

E197Q

14 0.0005 60 0.0008

Published in 2001 by John Wiley & Sons, Ltd. J. Appl. Toxicol. 21, S43–S46 (2001)

S46 C. A. BROOMFIELD AND S. D. KIRBY

Although these rates are slower than those of the wildtype enzyme, they are slowed to a much lesser extentthan are those with the organophosphorus compounds.From these results we conclude that the phosphorus tran-sition state is affected to a greater extent than that ofthe carbon compounds and might at least partly explainthe slow reactivation rates observed. Current efforts aredirected towards producing mutants that have histidine ina position to bind a water molecule that can attack theappropriate face of the phosphorus atom but do not affectthe transition-state structure. If such a structure can bemade, it could prove to be the universal OPAH neededfor use as a catalytic scavenger.

We are aware also that it might not be possible toincrease the efficiency of the BuChE mutants, given theconstraints of the secondary and tertiary structure of thatenzyme. Human carboxylesterase has many of the favor-able characteristics of BuChE but has a different speci-ficity (and therefore different binding contacts) and alarger active site cavity than BuChE. One of us (S.D.K.)already has begun to make modifications in the activesite region of that enzyme and has found that two single

mutants, G124H and G126H, catalyze the hydrolysis ofsarin; the efforts with human carboxylesterase are contin-uing. Work also is in progress on a human OPAH enzyme(PON1) that naturally has the specificity required for anerve agent scavenger and needs only some improvementin binding affinity (lower Km) and turnover rate. Strategiesfor introducing those modifications will be possible whena crystal structure for this class of enzymes is available.We are hopeful of seeing those critical data in the nearfuture.

Thus, we have reason to be very optimistic that thetime when we will have a human OPAH available fortesting as a practical nerve agent scavenger is not faroff. Advances in applications of gene therapy for thetreatment of certain diseases promise to present us with thepossibility of administering a highly effective scavengermolecule in the form of its gene. If proven to be safe,such a product would solve problems of stability, cost andbiological half-life required for military application andalso could provide a product for use in the civilian sectorto protect farm workers and others at risk of poisoningwith organophosphorus pesticides.

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Published in 2001 by John Wiley & Sons, Ltd. J. Appl. Toxicol. 21, S43–S46 (2001)