Saturday, March 17, 2012

2 September 2010 – The Men Who Genetically Engineer Goats


It might just be my imagination, but for some reason, butyrylcholinesterase seems to be popping up in the news a lot these days.

Butyrylcholinesterase - or, as it’s more commonly known, BChE, or serum cholinesterase - is a complex enzyme that occurs naturally in human blood, and is structurally rather similar to acetylcholinesterase, or AChE.  That latter enzyme probably sounds more familiar to you.  This is because it is the enzyme that is bound (i.e., deactivated) by exposure to organophosphorous compounds, such as the G and V nerve agents (note 1).  Simply put, the transmission of electrical messages between axons and dendrites of nerve cells is effected by acetylcholine, which is subsequently dismantled into its acetyl and choline groups by AChE.  Inhibition of AChE leads to what amounts to continuous firing of nerves, resulting in symptoms ranging from pupillary dilation, excessive salivation and production of mucous, and shortness of breath, to convulsions, cardiac arrest and death, usually through suffocation due to paralysis of the diaphragm.

Toxicity is only one part of the problem of dealing with nerve agent poisoning, or with organophosphorous (OP) poisoning in general.  This has been a common difficulty for workers in the agricultural industry, and became especially prevalent in the years following the EPA’s ill-advised ban on DDT in the 1970s.  DDT - which has never been associated with any human fatalities or chronic illnesses - was replaced with a variety of pesticides, including the OP insecticide Malathion, which in the first few years after its introduction caused hundreds of cases of OP poisoning.  In Pakistan in 1976, for example, during malathion spraying for mosquitoes to prevent a malaria epidemic (a function previously performed by DDT), 2500 of 7800 spraying personnel became ill, and 5 died.  Such poisoning cases, whether in a civilian or a military setting, are treated with a cocktail of drugs, the commonest of which are stimulants to keep the body functioning despite AChE deactivation (the commonest one being atropine); oxime reactivators (such as 2-PAM chloride, or the HI-6 developed in Canada) which are designed to un-bind the OP agent from the receptor sites on the AChE molecule; and anticonvulsants, like diazepam, to control the muscle spasms induced by AChE inactivation.  The only pre-treatment for potential nerve agent poisoning, however, has long been pyridostigmine bromide (PB).  PB, a drug also used to treat myasthenia gravis (an autoimmune neuromuscular disorder), does not protect against OP poisoning, but it facilitates post-exposure treatment, basically by occupying AChE receptor sites (effectively preventing the OP compound from doing so), and then giving them up readily once an oxime reactivator is introduced.

PB is not good for you (it’s been accused of being responsible for, amongst other things, “Gulf War Syndrome”), but it’s a necessary evil because of the problem of “aging”. Think of an OP molecule as a two-part mechanism.  One part is the “deactivator”; the other is the “handle”.  The “deactivator” binds to the AChE receptor site, deactivating the enzyme; while the “handle” is what an oxime reactivator grabs on to in order to extract the OP compound from the enzyme.  With all OP compounds, the “handles” sooner or later break off.  “Aging” is a measure of how long it takes between an OP challenge, i.e. when a nerve agent enters the body and begins to deactivate AChE, and when the “handle” breaks off the agent molecule, leaving part of its structure stuck to the enzyme receptor site (see Figure 1).     Once the “handle” is gone, it’s virtually impossible for an oxime reactivator like 2-PAM or HI-6 to extract the “deactivator” and allow the enzyme to function again.  Some OP compounds, like VX, age relatively slowly.  Others, like Soman, age very, very quickly.  In cases of Soman intoxication, the half-life for aging (the amount of time it takes for 50% of the agent molecules bound to enzyme molecules to lose their “leaving group”, the alcohol radical) is about 2 minutes.  Basically, if someone is exposed to Soman - an agent which was produced and stockpiled by the USSR, in both neat and thickened varieties, in quantities measuring in the thousands of tonnes - the oxime reactivator has to be administered within only a few minutes, or the individual is unlikely to recover (see here for a description of the problem of Soman 'aging').

Ideally, given such a short window for effective post-exposure treatment, you’d want an effective prophylaxis (pre-treatment) just in case someone was likely to be exposed to an OP agent, especially a fast-aging one like Soman.  This is why PB, despite its many drawbacks, was approved for pre-treatment use.  And it’s also where the men who genetically engineer goats come into the picture.

A few years ago, a Canadian company, Nexia Technologies of Montreal, began producing a novel product called Biosteel™.  In a nutshell, Biosteel is spider silk, and it was used for, among other things, producing bullet-proof vests.  The interesting part is where it comes from: it is extracted from milk produced by goats injected with recombinant spider DNA designed to express the silk protein.  Peter Parker doesn’t look so science-fictiony now, does he?  Since protein production is simply a matter of ensuring that the right genes are introduced and operational, Nexia received a $2M contract from DRDC Suffield to try the same thing with the human gene for producing BChE.  Why BChE?  Well, because as enzymes go, it’s a molecular version of the Terminator.  Although it only occurs naturally in very small quantities in human blood, it has the remarkable property of latching onto and tearing apart just about any toxin that’s likely to find its way into you.  Experiments with animals have shown that BChE, in sufficient concentration, is capable of destroying - with virtually no ill effects to the subject - many different poisonous chemicals.  And not only OP compounds and nerve agents, either; it’s been used to treat massive overdoses of cocaine and heroin.  In experiments, animals buffered with injections of BChE and subsequently intoxicated with multiple lethal doses of VX or Sarin have recovered without so much as manifesting any of the symptoms of nerve agent poisoning.

Sounds perfect, right?  Well, there was still a small problem.  First, breeding the goats cost about a million bucks each.  Second, they only produced small (milligram) quantities of BChE.  And third, our bodies tend to adapt to unusual chemical situations.  It seems that if you make a habit of injecting abnormally huge amounts of a human enzyme into your body, your metabolic chemistry becomes very adept at cleaning it out.  In other words, the more you use BChE as a pre-treatment, the less effective it will become over time.  As a result, BChE seems to be getting more press these days as a highly effective post-exposure treatment, both for exposure to OP compounds, and for overdoses of illegal narcotics.

As for Nexia…well, sadly, GOC contracts were not enough to keep the company going, and it was bought out a few years back by the Maryland-based PharmAthene, a major US pharmaceutical company.  Human BChE extracted from the milk of genetically engineered goats is now being trialled under the trade name Protexia™.  In 2006, PharmAthene was granted a US$219M contract by DoD to develop Protexia both as a pre-exposure prohylaxis against, and a post-exposure therapy for, nerve agent poisoning.  The initial contract called for the production and stockpiling of 90,000 doses.  In sufficient concentration, Protexia might be able to protect a soldier (or anyone else) from just about any sort of chemical toxin.  To paraphrase Jeff Bridges, the US could very well be on the way to becoming the first superpower to create super powers.

But first…somebody’s going to have to milk a LOT of goats.



1) The classical G-Agents are Tabun (GA), Sarin (GB) and Soman (GD).  There are other variants, including Cyclo-sarin (stockpiled by Saddam Hussein, so-called because of the use of cyclohexanol instead of ethanol as an alkylating agent), and the intermediate volatility agent, GV.  The V-Agents are VX, its Russian variant Vx, a Chinese variant, Amiton (VG), and a variety of other analogues with slightly different physical and toxicological properties.


Huang, Yue-Jin, et al., “Recombinant human butyrylcholinesterase from milk of transgenic animals to protect against organophosphate poisoning” []

Huang, Yue-Jin, et al., “Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin” []

Ted A. Loomis and Dennis Johnson, “Aging and reversal of soman-induced effects on neuromuscular function with oximes in the presence of dimethyl sulfoxide”, Toxicology and Applied Pharmacology, Volume 8, Issue 3, May 1966, Pages 533-539

F. Nachon, et. al., “Aging mechanism of butyrylcholinesterase inhibited by an N-methyl analogue of tabun: implications of the trigonal-bipyramidal transition state rearrangement for the phosphylation or reactivation of cholinesterases”. Chemico-biological Interactions, 2010;187(1-3):44-8

J.L. Sporty, et. al., “Immunomagnetic separation and quantification of butyrylcholinesterase nerve agent adducts in human serum”. Analytical Chemistry, 2010;82(15):6593-600