Nerve agents and acetylcholinesterase

Background

In the current world, there are threats of war and new weapons all the time; it has become a constant in our society. Some of these weapons are ones that the public would be able to detect until it was too late. One nerve agent that has generated much attention recently is Novichok. Despite the name Novichok implying that it is a single chemical nerve agent, it is in fact a group of related molecules. ^[1] There are many different types of nerve agents, the most common being the Novichok family, , tabun, and . Most of these agents were created when researching pesticides and were found to be too toxic to use in agriculture and therefore were passed on to the military in whichever country the chemical was first synthesized. ^[2] The most common novichok in its family, for example, was created somewhere between the 1970s and 1997. ^[3] In 1997, 193 countries signed the Chemical Weapons Convention treaty which banned development, production, and stockpiling of chemical weapons and required that these countries safely dispose of their reported chemical agents. This resulted in more than 67,000 tons of these chemicals being destroyed. ^[4] This is due to the fact that nerve agents are so deadly and world leaders were afraid of a war using only these chemicals.

Importance

Despite the Chemical Weapons Convention treaty, chemical warfare continues to be a threat to countries all over the world. Assassins use nerve agents as they are hard to track back to the person who created it, however it is not impossible to trace due to the residues that are left behind. People who wish to harm members of society often do not give any warning when they attack using nerve agents. Until the symptoms start to appear, it may not be known that an attack has even occurred. This can give the attacker time to escape, so being able to track the precursors is essential from a criminal justice perspective. Chemical warfare agents are classified as organophosphorus-based molecules. ^[5] These chemicals are called warfare agents due to their ability to disrupt the central nervous system communications and cause death to anyone exposed and are commonly used as acts of aggression. The reason these agents are effective is due to the fact that they are colorless, odorless, have no taste, and can be introduced through any respiratory or gastrointestinal tract. ^[4] The skin is also able to absorb nerve agents and it is extremely difficult to treat a person who has been exposed to one. Very small amounts of Novichok are needed in order for it to be lethal, which adds to its already terrifying nature. Nerve agents are also dangerous because it is unknown how long they stay active once they are released. ^[1] This presents a problem to first responders when an incident occurs. This was the case in England in 2018 when Novichok was used on Sergei Skripal and his daughter. The first responder was exposed to the nerve agent and had to undergo treatment in order to save his life. Novichok in particular is more dangerous than sarin or VX as it is 6-10 times stronger and therefore a smaller dose is required to produce the desired effect. ^[1]

What is Acetylcholine and its Enzyme

is one of the most efficient human enzymes that is known. It can hydrolyze around 600,000 molecules each minute which shows how essential it is to human life. ^[6] Acetylcholinesterase has 3 active sites, according to current research, but nerve agents attack the primary site. The that is located on the molecule near the plays an essential role in the function of acetylcholine. ^[7] The active site contains a catalytic triad of histidine, serine, and glutamic acid. The gorge allows the active site to open and close in order to control the flow of substrates that come to acetylcholine. Acetylcholinesterase was found to have 14 aromatic amino acids located around the opening to the gorge and this plays a role in the dipole moment within the molecule and it leads to a more symmetric charge distribution within the molecule. In 2017, it was found that acetylcholinesterase was a very effective catalyst and when a substrate interacts with an enzyme, that becomes the rate-limiting step. ^[7]

Parts of Nerve Agents

Many nerve agents that are synthesized by methylphosphonic dichloride (DC) and use hydrogen fluoride (HF) to introduce a fluorine to the molecule, a residue of phosphorus hexafluoride (PF6) was always found. This residue was not found when any other fluorine molecules were used in the synthesis. This can help identify the starting chemicals that were used to create the nerve agents. ^[4] Researchers have found that the various side chains that are added, which leads to the differences in naming, can affect the potency and how long the agent will last. ^[3]

Synthesis

A nerve agent is formed by combining two precursors to form a cyclic oxime ester. In this new molecule, the phosphorus atom is contained in a five-membered ring. When heated above zero, the ring that contains chlorine becomes destabilized and opens, allowing the Novichok to be formed. The process has a 30-60% efficiency. There are around 50 chemicals that are considered precursors and these are chemicals that are toxic and are not stable in water. ^[5] These chemicals are illegal in the United States, but it does not stop people from illegally synthesizing these agents. ^[4]

Image of Synthesis

How it works

Nerve agents are effective due to their interaction with acetylcholinesterase . This is significant because the body uses this enzyme to remove acetylcholine as it can be dangerous if it builds up in the body. Without the removal of acetylcholine, the muscles are continually contracting and spasming. Nerve agents work by interrupting communication between nerves and muscles or communication between nerves in the brain. ^[2] These agents work within minutes of a person being exposed to them and symptoms appear right away. When using X-ray crystallography to try and understand the structure of sarin, it was found that the isopropyl component becomes a closed conformation in order to shield the phosphorus atom so that it cannot be attacked . This was found in both human and nonhuman subjects and so it was determined that this was due to the preferred conformation being closed rather than being due to the crystal packing. ^[8] This is significant as it gives researchers insight as to how a nerve agent protects itself from other chemicals in the body that may try to attack it.

Binding to Acetylcholinesterase

Nerve agents bind to acetylcholinesterase at one site, the active center which is a narrow gorge on the enzyme. ^[6] Nerve agents contain a phosphorus group that binds to the hydroxyl group that is located on this enzyme. This is the location where acetylcholine usually binds, therefore these agents act as a competitive inhibitor of acetylcholine. The body will need to synthesize more enzymes so that it does not build up, but since the human body cannot do it fast enough, most people who are exposed to a nerve agent will die. The main reason that nerve agents are so deadly is that when the phosphorus group of the agent binds to they hydroxyl group, it forms a covalent bond, with the serine residue, so strong that it cannot be broken

Current Treatment

Due to the real possibility of an attack using these deadly chemicals, researchers are working on finding antidotes or treatments that can save a person’s life or even just delay death so that researchers have more time to find a way to cure this. The main focus for these treatments is to get the nerve agent to release the acetylcholinesterase, even if the reaction mechanism is unknown. The United States Army requires its soldiers to carry an anticonvulsant called Diazepam with them in case of a nerve agent attack. However, there is a push to carry midazolam which acts faster than what is currently used. ^[6] When a victim is being treated in a hospital, a mixture of two chemicals are used to treat the poisoning. These are Atropine, which blocks the acetylcholine receptors, and a reactivator, which is used to restore acetylcholinesterase to its original function, therefore negating the effects of a nerve agent. ^[9] More research is being done on treatments that allow an oxime to become neutral so that it can cross the blood-brain barrier, which is where a nerve agent does most of its work. ^[6]

Conclusions

While the chemical structures of some of these nerve agents are not known due to the synthesis of these chemicals being illegal, there are proposed structures since researchers know the essential chemical groups that are a part of nerve agents. Due to the unknown structures, it is difficult to synthesize antidotes to these nerve agents, but researchers can try to find treatments that will work without knowing the structure. The threat of a nerve agent attack is a possibility in our world today and so it is essential to understand how these agents affect the body so that measures can be taken to keep the public as safe as possible.