User:Jacob Rubin/Sandbox 1

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Austin Remington and Jake Rubin Video Project Transcript April 12, 2013

Examining the Mechanism of Action and Treatment of Sarin, an Organophosphate Nerve Gas that Irreversibly Inhibits Acetylcholinesterase Group Member Role: Austin Remington Primary - Literature search, image selection, transcript construction, and narration Secondary - Video editing, bibliography

Group Member Role: Jake Rubin Primary - Video editing, bibliography, transcript construction, and narration Secondary - Literature search, image selection

1. History and Introduction

In 1937 German scientist Gerhard Schrader spilled a drop of Tabun while researching pesticides.A1 Within minutes the lab staff was asphyxiated. This led to the adoption of Tabun as the first nerve agent. The Germans synthesized Sarin a year later. While nerve agents never saw the battlefield during WWII, Sarin was used in terrorist attacks in Japan in the mid 1990’s. Sarin targets acetylcholinesterase, the enzyme that metabolizes acetylcholine in the synaptic cleft. The chemistry involved in acetylcholinesterase function, poisoning, and treatment integrates a broad spectrum of intermolecular interactions: hydrophobic, electrostatic, and covalent.

2. Outline

In this presentation, we will discuss: 1. The acetylcholinesterase enzyme and its normal physiological role 2. The mechanism of action of organophosphate inhibitors 3. The mechanism of action for Pralidoxime Chloride (2-PAM), a treatment for nerve agent victims 4. Advances and challenges in this field

3. Background: The Acetylcholinesterase Enzyme

This is the 3-d structure of acetylcholinesterase. The major form of AChE is a hydrophilic species found in the brain and muscle. It forms oligomers with disulfide linkages to collagen or lipid-containing structural subunits.A2 Acetylcholine (ACh) is a principal neurotransmitter. The concentration--and thus the activity--of ACh is regulated by the acetylcholinesterase enzyme (AChE), which hydrolyzes it to choline and acetic acid.A3

4. The Electrostatic Mechanism for Substrate Guidance Research was conducted to explain substrate guidance in AChE, uncovering an electrostatic mechanism.A4 The active site of AChE is located deep within the molecule at the end of a long gorge, which measures 5 angstroms wide and 20 angstroms deep.A5 The enzyme itself has a strong dipole aligned exactly with the gorge that leads ACh down to the active site.A4 The gorge is lined with 14 aromatic amino acid residues, suggesting that these residues serve dual roles. First, they are low-affinity binding sites for the quaternary ammonium of ACh. Second, they shield ACh from the negatively charged, acidic residues that create the dipole--these residues would otherwise interact strongly with ACh and cause it to get ‘stuck.’ Thus, a much larger part of the enzyme’s surface is responsible for trapping substrate molecules than the active site alone.

5. The Active Site: The Catalytic Triad and Kinetics

The active site encompasses two subsites, the anionic site and the esteratic site. The anionic subsite is negatively charged to complement the positive quaternary amine of ACh. The esteratic subsite contains the catalytic triad--three amino acid residues paramount to the function of the enzyme. The residues that comprise the catalytic triad are Ser 200, His 440 and Glu 327. This catalytic triad is essentially identical to that of other serine proteases studied in class, except that here Glu replaces Asp as the third residue; additionally the ‘handedness’ of the triad is reversed relative to other serine proteases. AChE is one of the fastest known enzymes, close to the diffusion limit; although mixed findings exist, one recent study found the turnover number to be close to 5000 (ACh/AChE)/second.A6

6. Effects of Organophosphate Poisoning The activity of AChE is compromised by toxins both synthetic and natural. This is the green mamba, which has AChE inhibitors in its venom. Enter the organophosphate, which prevents the degradation of ACh. Consequently, ACh builds up in the synaptic cleft and continuously activates the receptors. Muscle contraction and breathing are thus prevented, causing death.

7. Irreversible Inhibition Mechanism: Sarin Sarin is highly lipophilic and it is readily absorbed through the skin and distributed to lipophilic tissues.A1 A nerve gas acts through molecular mimicry. We will explore this in the context of the action of Sarin on AChE. This is the mechanism of Sarin. The serine of the catalytic triad becomes phosphorylated because the phosphorous possesses a partial positive charge.A3 Mechanistically, the serine acts as a nucleophile to attack phosphorous with fluoride as a stable leaving group. Sarin acts as a “hemisubstrate,” meaning the transition state resembles the normal deacylation transition state.A7 This forms a stable product that is slow to hydrolyze back to its original state.

8. Pralidoxime Chloride “2-PAM” & The Aging Process

Administration of Pralidoxime Chloride or 2-PAM reactivates AChE by cleaving the covalent bond between the enzyme and nerve agent. Specifically, the charged ammonium on 2-PAM is first attracted to the anionic site.A3 Once 2-PAM has attached at the active site, it executes nucleophilic attack on the phosphorus moiety bound to AChE, forming an oxime-phosphate bond and regenerating the free enzyme. Over time the alkoxy group on the nerve gas is hydrolyzed to yield a hydroxyl group. This process is termed “aging.” A7 Because the hydroxyl group is more inductively electron-donating than the alkyl group, the aged serine-phosphorus bond is stronger than the original covalent bond, and 2-PAM can no longer cleave the phosphate group from the enzyme. The leaving group of the aging reaction is a carbocation; more highly substituted cations are more stable, thus more substituted organophosphates are more susceptible to hydrolysis. Therefore the rate of aging, and thus the time frame that the victim may be treated with 2-PAM, depends on the degree of alkyl substitution.A8

9. Conclusion

Certain limitations exist for 2-PAM therapy. There is little consensus in the literature regarding either the proper drug dosages or the effective time frame of administration. Additionally, 2-PAM only has peripheral action because the charged amine group prevents it from crossing the blood-brain barrier. Furthermore, the most widely studied AChE molecules are taken from erythrocytes, which are replenished faster than neuronal AChE, skewing recovery data. Recent studies have explored nerve gas treatment with Recombinant paraoxonase 1, which is a catalytic bioscavenger that functions to hydrolyse organophosphates before they reach their targets.A9 With REPON1 treatment after nerve gas administration, brain ACHE levels were increased.A10 The role of acetylcholinesterase and its inhibitors is made possible through hydrophobic, electrostatic, and covalent interactions. By expanding our knowledge of biochemistry, we may be able to harness the vast and untapped potential of acetylcholinesterase as an instrument for both disease and warfare.

Works Cited

Audio:

A1 Holstege CP, et al. “Chemical Warfare: Nerve Agent Poisoning.” Critical Care Clinics. 13 (14), 1997. Print.

A2 "Acetylcholinesterase: Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 25 Mar. 2013.

A3 “Cholinesterase Inhibitors: Including Insecticides and Chemical Warfare Nerve Agents.” Agency for Toxic Substances and Disease Registry. 16 Oct. 2007. Web. 25 Mar. 2013.

A4 Ripoll DR, et al. "An Electrostatic Mechanism for Substrate Guidance Down the Aromatic Gorge of Acetylcholinesterase." Proc Natl Acad Sci. 90 (11): 5128-132, 1993. Print.

A5 Silman I, et al. “Acetylcholinesterase: how is structure related to function?” Chem Biol Interact. 175 (1-3): 3-10, 2008. Print.

A6 Tripath AR, et al. "Acetylcholinesterase: a Versatile Enzyme of the Nervous System." Annals of Neurosciences. 15 (4), 2008. Print.

A7 Millard CB, et al. “Crystal Structures of Aged Phosphonylated Acetylcholinesterase:  Nerve Agent Reaction Products at the Atomic Level.” Biochemistry. 38 (22), 1999. Print.

A8 Keyes DC, et al. “CBRNE - Nerve Agents, V-Series - Ve, Vg, Vm, Vx.” Medscape Reference. 9 Nov. 2012. Web. 10 Apr. 2013.

A9 Harel M, et al. “3-D structure of serum paraoxonase 1 sheds light on its activity, stability, solubility and crystallizability.” Arh Hig Rada Toksikol. 58 (3): 347-353, 2007. Print.

A10 Valigyaveettil M, et al. “Recombinant paraoxonase 1 protects against sarin and soman toxicity following microinstillation inhalation exposure in guinea pigs.” Toxicology Letters. 203 (3), 2011. Print.

Figures:

F1 English J. “ Galantamine: A Unique Nutrient for Preserving Memory and Cognitive Function” Figure of Achetylcholine in Synapse <http://www.icdrc.org/documents/Galantamine.pdf>

F2 Katos, Alex. “AChE Cleaving ACh.” Online video clip. Youtube. 3 Nov. 2010. Web. 25 Mar. 2013.

F3 “Cholinesterase Inhibitors: Including Insecticides and Chemical Warfare Nerve Agents.” Agency for Toxic Substances and Disease Registry. 16 Oct. 2007. Web. 25 Mar. 2013.

F4 Ripoll DR, et al. "An Electrostatic Mechanism for Substrate Guidance Down the Aromatic Gorge of Acetylcholinesterase." Proc Natl Acad Sci. 90 (11): 5128-132, 1993. Print.

F5 Kryger G, et al. “Structure of acetylcholinesterase complexed with E2020 (Aricept): implications for the design of new anti-Alzheimer drugs.” Structure. 7 (3): 297-307, 1999. Print.

F6 National Programme on Technology Enhanced Learning - Bioorganic Chemistry of Enzymes Module. Web. 25 Mar. 2013. <http://nptel.iitm.ac.in/courses/104103018/module3/lec3/1.html>

F7 Fromer M, et al. “Chymotrypsin.” Rutgers - Molecular Anatomy Project. 2008. Web. 25 Mar. 2013. <http://maptest.rutgers.edu/drupal/?q=node/48>

F8 Cholinesterase Inhibition Animation, University of Idaho. Web. 25 Mar. 2013. <http://www.webpages.uidaho.edu/etox/resources/animations/flash_cholinesterase_inhibitors/cholinesterase_inhibitors.htm>

F9 Green Mamba. Google Images. Web. 25 Mar. 2013. <http://snakesfb.blogspot.com/2012/06/eastern-green-mamba.html>

F10 Harrison K. Spinning Sarin Animation. Online animation. www.3dchem.com. Web. 25 Mar. 2013. <http://www.3dchem.com/3dmolecule.asp?ID=25>

F11 Harel M, et al. “3-D structure of serum paraoxonase 1 sheds light on its activity, stability, solubility and crystallizability.” Arh Hig Rada Toksikol. 58 (3): 347-353, 2007. Print.

F12 Valigyaveettil M, et al. “Recombinant paraoxonase 1 proects against sarin and soman toxicity following microinstillation inhalation exposure in guinea pigs.” Toxicology Letters. 203 (3), 2011. Print.