Perhaps a million years ago, a protohuman ate a poisonous berry. The berry was loaded with natural organophosphate molecules that probably protected it from insect pests. Unfortunately, those molecules could also wreak havoc on the nervous system of a larger animal, perhaps disabling or killing it. But the human ancestor’s blood contained an enzyme known as butyrylcholinesterase (BChE), which bound the toxic molecules, rendering them inert. Our forerunner survived.
Modern humans have unleashed upon themselves hazards far more terrifying than toxic plants: organophosphate-based nerve agents that can cause agonizing death within minutes of exposure. That’s what happened on Aug. 21, in a chemical weapons attack in Damascus, Syria, where 1,400 people were killed by the nerve gas sarin.
The attack heightens the urgency of protecting people from these weapons of mass destruction. Although antidotes including atropine and diazepam can arrest some symptoms, such as convulsions and asphyxiation, they do not prevent permanent neurological damage. And they can be used only after the fact. They cannot be deployed as a shield when a rogue nation threatens havoc.
However, that ancient enzyme BChE, which our systems still harbor in extremely small quantities, has long been eyed as a potential protectant. It, and other organophosphate-binding or -degrading enzymes, can be found in most creatures, from mammals to bacteria.
Advances in recombinant DNA technology and directed evolution have now made it possible to pursue the development of enzymes that might work as nerve-agent protectants and antidotes. Numerous research labs, mostly in the U.S., but also in Western European countries such as the U.K., are collaborating with U.S. Army and National Institutes of Health programs on such enzyme-based shield strategies.
Army-funded projects tend to focus on prophylactics for military personnel or first responders, while NIH’s Countermeasures Against Chemical Threats (CounterACT) program is “mostly interested in postexposure treatment,” notes David A. Jett, CounterACT’s program director.
Organophosphate nerve agents such as sarin target the enzyme acetylcholinesterase (AChE). The neurotransmitter acetylcholine binds to receptors in muscle fibers, causing muscles to contract. Immediately afterward, AChE moves in and breaks down acetylcholine to terminate the contraction signal. But if a nerve agent ties up AChE, the acetylcholine is free to continue transmitting signals, causing muscles to contract uncontrollably and leading to dramatic symptoms such as convulsions and foaming at the mouth, and finally death.
On the face of it, BChE seems to be an ideal protectant. Large quantities of BChE could flood the system and bind any circulating nerve agent before it had a chance to get to AChE, says John Troyer, vice president of chemical defense product development at PharmAthene, a biotech firm that focuses on national security medical issues.
BChE binds almost any organophosphate nerve agent or pesticide and appears to be nontoxic, even in large quantities. An injection can last for days in the body, making it prophylactically useful for military and first responders.
However, BChE has proven difficult to synthesize in large quantities. It’s resistant to production via recombinant techniques in bacteria or yeast. PharmAthene was able to produce large amounts of BChE, which it calls Bioscavenger, in the milk of transgenic goats. Phase I clinical safety trials of Bioscavenger in humans showed it was well tolerated. But that project was ultimately deemed too expensive and was recently shelved.
Troyer says PharmAthene has now discovered a way to produce significant quantities of BChE less expensively in a human cell line. Yet BChE still has a problem: dose size. The enzyme acts like a sponge, so one molecule is needed to inactivate every nerve-agent molecule. That means enormous doses between 500–750 mg. And they would have to be injected intramuscularly in advance of nerve-agent exposure, a relatively impractical means of administration.
Enzymes that break apart one organophosphate molecule and then move on to disable many others could be used in much smaller quantities. To that end, a number of groups are pursuing the development of enzymes that could turn over many times in this way.
Most of these engineered enzymes, however, are specific to a particular nerve agent. Therefore, researchers envision administering a cocktail of enzymes that could potentially disarm any number of possible nerve agents.
However, some researchers are working to broaden the activity of high-turnover enzymes. For example, after obtaining the crystal structure of human carboxylesterase 1 (hCE-1) 10 years ago, Matthew R. Redinbo, a chemistry professor at the University of North Carolina, Chapel Hill, noted that the enzyme functions very similarly to AChE and BChE. Using molecular modeling and protein design techniques, his group synthesized a variant of hCE-1—differing in some cases by only two amino acids from the wild-type enzyme—that destroyed sarin, soman, and cyclosarin and reactivated itself significantly faster than the wild type (PLOS One 2011, DOI: 10.1371/journal.pone.0017441). Redinbo started a company, Identizyme, to further develop these enzymes.
Dan S. Tawfik, biological chemistry professor at Weizmann Institute of Science, in Israel, has been using directed evolution to develop variants of the enzyme paraoxonase 1 (PON1) that can neutralize all so-called G-type nerve agents, which include tabun, soman, sarin, and cyclosarin (Chem. Bio. 2012, DOI:10.1016/j.chembiol.2012.01.017). “I think the challenge is to get broad specificity,” Tawfik says.
Additionally, a bacterial enzyme, organophosphorus hydrolase, or OPH, shows promise as a catalytic multiple-nerve-agent neutralizer. Frank M. Raushel, chemistry professor at Texas A&M University, designed libraries of OPH mutants using rational design and random mutagenesis. On the basis of tests at the U.S. Army’s Aberdeen Proving Ground, he and his coworkers hit pay dirt with a couple of variants: one that swiftly hydrolyzes sarin, soman, and cyclosarin, and another, Raushel says, that is the “best ever reported for the hydrolysis of VX” (J. Am. Chem. Soc. 2013, DOI: 10.1021/ja405911h).
Richard L. Rotundo at the University of Miami, on the other hand, thinks AChE itself could serve as both protectant and antidote. His lab has developed peptides that induce production of AChE at the site of neurotransmitter action to replace AChE molecules already incapacitated by nerve agents. Although his work has yet to be published, Rotundo has presented results at meetings showing that AChE treatment saved 90% of mice exposed to two times the lethal dose of a nerve-agent surrogate.
Rotundo is launching a project with CounterACT to develop this approach. “Hopefully within one to two years, we’ll be able to start doing clinical trials,” he says.
