1 Reactivation and Resurrection of Organophosphorus Poisoned

Home , Demeton, Obidoxime, Paraoxon, Pralidoxime

Reactivation and Resurrection of Organophosphorus Poisoned Acetylcholinesterase with

Improved Methods of Detection

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in

the Graduate School of The Ohio State University

Stacey Katherine Allen

Graduate Program in Chemistry

The Ohio State University

2020

Thesis Committee

Christopher M. Hadad, Advisor

Thomas J. Magliery, Committee Member

Copyrighted by

Stacey Katherine Allen

2020

Abstract

The inhibition of acetylcholinesterase (AChE) by organophosphorus (OP) nerve agents and pesticides is, thankfully, reversible with the treatment of reactivators, such as

2-pralidoxime (2-PAM). However, if treatment is not administered quickly or if the OP is particularly toxic, these reactivators are rendered useless. After inhibition by an OP, a subsequent dealkylation event can occur at the phosphylated serine residue of AChE. This aged state of the enzyme cannot be revived by reactivators, and there are currently no approved treatments for the aged form of AChE.

The aged form of AChE from OP poisoning was considered irreversible until 2018, when our team demonstrated the only compounds that are capable of reviving, or resurrecting, the aged form of electric-eel AChE using quinone methide precursors

(QMPs). Inspired by this initial set of QMPs, a variety of 2-(aminomethyl)-3- hydroxypyridine QMPs were tested in vitro against inhibited and aged forms of recombinant human AChE that resemble the erythrocyte (dimer) and readthrough

(monomer) isoforms. A modified Ellman’s assay, utilizing the artificial AChE substrate acetylthiocholine (ATC) and 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), was used to detect the reappearance of native AChE during these studies.

After 24 hours, up to 100% of the aged enzyme was resurrected for each isoform with select QMPs and also for specific OP compounds. The same QMP framework is also iii an effective treatment for OP-inhibited AChE and can reactivate up to 70% of recombinant human (erythrocyte) AChE and 20% of the readthrough isoform. Although these QMPs are not as efficient with reactivation as 2-PAM, their lack of a permanent charge makes them more efficient at crossing the blood-brain barrier. QMPs, acting as both a reactivator and a resurrector, will help pave the way to new, more effective treatments for OP poisoning.

This dissertation presents comparative in vitro assays with both isoforms of the enzyme as well as the reactivation and resurrection efficiency with various QMPs in a structure-activity relationship library. To perform in vitro studies that are more representative of a human’s natural AChE, whole blood will be used in resurrection and reactivation studies of the most effective QMPs. Unfortunately, whole blood absorbs at a variety of wavelengths, limiting the practicality of Ellman’s assay. Therefore, new methods of detection must be explored; a variety of absorbance and fluorescence methods for detecting reactivated and resurrected native AChE will be presented herein.

Vita

2020……….………………………………………………….Graduate Research Assistant The Ohio State University

May 27 – June 2, 2020…………………………………………Virtual Science Day Judge The Ohio Academy of Science State Science Day

2018-2019……………………………………………………………....Teaching Assistant The Ohio State University

2018……………………………………………………….Master of Science in Chemistry University of North Carolina Wilmington

2016-2017………..….……………………………………….Graduate Research Assistant University of North Carolina Wilmington

2015-2017……………………………………………………………....Teaching Assistant University of North Carolina Wilmington

2015….………………………………….………………Bachelor of Science in Chemistry Minor in Psychology University of North Carolina Wilmington

2014-2015………..…………………………………….Undergraduate Research Assistant University of North Carolina Wilmington

2013-2015………....………………………………………Certified Pharmacy Technician CVS Pharmacy

Publications

McGorry, R. J.; Allen, S. K.; Pitzen, M. D.; Coombs, T. C. Tetrahedron Lett. 2017, 58

(49), 4623–4627. https://doi.org/10.1016/j.tetlet.2017.10.063.

Allen, S. K.; Lathrop, T. E.; Patel, S. B.; Harrell Moody, D. M.; Sommer, R. D.; Coombs,

T. C. Tetrahedron Lett. 2015, 56 (44), 6038–6042. https://doi.org/10.1016/j.tetlet.2015.09.051.

Fields of Study

Major Field: Chemistry

Table of Contents

Abstract ...... iii Vita ...... v List of Tables ...... viii List of Figures ...... ix Chapter 1. Introduction ...... 1 Chapter 2. Reactivation of Organophosphorus-Inhibited Acetylcholinesterase with Quinone Methide Precursors...... 10 Introduction ...... 10 Results and Discussion ...... 24 Experimental ...... 38 Chapter 3. Resurrection of Organophosphorus-Aged Acetylcholinesterase with Quinone Methide Precursors ...... 42 Introduction ...... 42 Results and Discussion ...... 49 Experimental ...... 61 Chapter 4. Methods for Detecting Native Acetylcholinesterase ...... 65 References ...... 72

vii

List of Tables

Table 1 Rates of Inhibition of Acetylcholinesterase by Various Organophosphorus

Chemical Nerve Agents and Pesticides.* ...... 10

o Table 2 IC50 (μM) measurements (25 C, pH 7.5) of native rhuAChE (973 units of enzyme activity) and CP-AChE (354 units of enzyme activity) for several QMPs...... 28

Table 3 Aging of Acetylcholinesterase by Organophosphorus Nerve Agents and

Pesticides.* ...... 43

viii

List of Figures

Figure 1 G-series organophosphorus nerve agents...... 2

Figure 2 Simplified catalytic hydrolysis of acetylcholine (ACh) by acetylcholinesterase

(AChE)...... 3

Figure 3 Isoforms of AChE (image reproduced from Ref. 7 and 8)...... 4

Figure 4 Catalytic triad of AChE (S203, H447, E334), oxyanion hole (G121, G122, A204), and choline-binding pocket (E202, W86).1...... 4

Figure 5 Membrane anchors for AChE (image reproduced from Ref. 9)...... 5

Figure 6 Inhibition of AChE by sarin (GB)...... 6

Figure 7 V-series organophosphorus nerve agents...... 7

Figure 8 Organophosphorus pesticides...... 8

Figure 9 Parties to the Geneva Protocol.11 ...... 9

Figure 10 Structures of currently approved treatments for OP poisoning: atropine (1), nicotinhydroxamic acid (2), 2-pralidoxime chloride (2-PAM, 3), and diazepam (4)...... 14

Figure 11 Reactivation of sarin-inhibited AChE by an oxime...... 14

Figure 12 Irreversible aging of AChE through O-dealkylation...... 16

Figure 13 Crossing the blood-brain barrier (image reproduced from Ref. 30)...... 17

Figure 14 Other permanently charged oxime reactivators...... 18 ix

Figure 15 Uncharged oxime reactivators...... 19

Figure 16 Non-oxime reactivators...... 20

Figure 17 Reactivation of sarin-inhibited AChE by ADOC (10)...... 21

Figure 18 ADOC derivatives as reported by Cerasoli (11-14)38 and de Koning (15-18).39

...... 22

Figure 19 Proposed reactivation and enzyme degradation by quinone methides...... 23

Figure 20 OP nerve agent surrogate...... 24

Figure 21 Structure activity relationship (SAR) library with a 3-hydroxypyridine framework...... 25

Figure 22 Biological screening of compounds 19-27 (a-e) with the concentration of 250

μM (left) and 1000 μM (right) against CMP-inhibited rhuAChE after 1 hour using 2-PAM as a reference (25oC, pH 7.5, 1216 units of activity for rhuAChE)...... 26

Figure 23 Biological screening of compounds 19-27 (a-e) with the concentration of 250

μM (left) and 1000 μM (right) against EP-inhibited rhuAChE after 1 hour using 2-PAM as a reference (25oC, pH 7.5, 1216 units of activity for rhuAChE)...... 29

Figure 24 Biological screening of compounds 19-27 (a-e) with the concentration of 250

μM (left) and 1000 μM (right) against DFP-inhibited rhuAChE after 1 hour using 2-PAM as a reference (25oC, pH 7.5, 1216 units of activity for rhuAChE)...... 30

Figure 25 Biological screening of compound 26 (a-k) with the concentration of 250, 500 and 1000 μM against OP-inhibited rhuAChE after 1 hour using 2-PAM as a reference

(25oC, pH 7.5, 1216 units of activity for rhuAChE)...... 32

Figure 26 Structure activity relationship (SAR) library with a phenol framework...... 33

Figure 27 Biological screening of compound 28-33 (a-e) with the concentration of 250

μM against CMP-inhibited rhuAChE after 1 hour using 2-PAM as a reference (25oC, pH

7.5, 1013 units activity for rhuAChE)...... 34

Figure 28 Linker QMPs...... 35

Figure 29 Biological screening of compound 34-43 (a-e) with the concentration of 250

μM against CMP-inhibited (top) or EP-inhibited (bottom) rhuAChE after 1 hour using 2-

PAM as a reference (25oC, pH 7.5, 1013 units of activity for rhuAChE during CMP inhibition and 1111 units of activity for rhuAChE during EP inhibition)...... 36

Figure 30 Proposed methods to reverse aging with OP-aged AChE.29,44–46 ...... 46

Figure 31 Screening of three concentrations of various QMPs against PiMP-aged eeAChE.47 ...... 47

Figure 32 Proposed mechanism for resurrection by a QMP...... 48

Figure 33 Biological screening performed by Andrew Franjesevic of compounds 19-27

(a-e) with the concentration of 1000 μM against CMP-aged rhuAChE after 24 hours using

2-PAM as a reference (25oC, pH 7.5, 973 units of activity for rhuAChE)...... 50

Figure 34 Biological screening of compounds 20-27 (a-e) with the concentration of 1000

μM against MP-aged rhuAChE (left) or CP-AChE (right) after 24 hours using 2-PAM as a reference (25oC, pH 7.5, 973 units of activity for rhuAChE and 354 units of activity for

CP-AChE)...... 51

Figure 35 Biological screening of compounds 20-27 (a-e) with the concentration of 1000

μM against DFP-aged rhuAChE (left) or CP-AChE (right) after 24 hours using 2-PAM as

xi a reference (25oC, pH 7.5, 973 units of activity for rhuAChE and 354 units of activity for

CP-AChE)...... 52

Figure 36 Distance map of 26b and the active site of methylphosphonate-aged human

AChE. Multiple intermolecular distance measurements were taken over time by Joseph

Fernandez. For each interaction, the distance at a particular timepoint is mapped to the color bins (red: 2-3 Å; green: 3-4 Å; blue: 4-5 Å; white: >5 Å.) The distance categories are grouped according to noteworthy interactions as defined above...... 54

Figure 37 Biological screening of compound 26a-k with the concentration of 250, 500 and

1000 μM against OP-aged rhuAChE after 24 hours using 2-PAM as a reference (25oC, pH

7.5, 1216 units of activity for rhuAChE)...... 55

Figure 38 Biological screening of compounds 28-33 (a-e) with the concentration of 250

μM against OP-aged rhuAChE after 24 hours using 2-PAM as a reference (25oC, pH 7.5,

1048 units of activity for rhuAChE against CMP (top left), 1014 units of activity against

MP (top right), and 322 units of activity against DFP (bottom)). *33a-e was not tested against MP-aged rhuAChE...... 57

Figure 39 Biological screening of compounds 34-43 (a-e) with the concentration of 250

μM against CMP-aged rhuAChE after 24 hours using 2-PAM as a reference (25oC, pH 7.5,

1048 units of activity for rhuAChE)...... 59

Figure 40 Ellman’s assay...... 65

Figure 41 Other compounds paired with ATCI: 6,6'-disulfanediyldinicotinic acid (DTNA,

44), dibromobimane (45), 7-(4-(2,4-dinitrophenylsulfonyl)piperazin-1-yl)-2-oxo-2H- chromene-3-carboxylate (46)...... 67

xii

Figure 42 Two-photon excited fluorescence emission spectra (excitation: 800 nm) for red blood cells, erythrocyte ghosts, and reagent-grade fluorophores (image reproduced from

Ref. 56)...... 68

Figure 43 Hydrolysis of indoxylacetate and formation of indigo...... 68

Figure 44 Hydrolysis of 7-acetoxy-1-methylquinolinium iodide (48)...... 69

xiii

Chapter 1. Introduction

1970-1979. India. 220,000 killed annually. 3.0 million hospitalized annually.1 1980-1989. Iran-Iraq War. 5,000 killed. 100,000 injured. 1988. Halabja, Iraq. 5,000 killed. 7,000 injured. March 20, 1995. Tokyo, Japan. 12 killed. 5,500 hospitalized. 2013. India. 23 children killed. August 21, 2013. Damascus, Syria. 1,429 killed. February 13, 2017. Kuala Lumpur. Kim Jong-nam assassinated. April 4, 2017. Syria. 89 killed. 541 injured. March 4, 2018. United Kingdom. Russian spy and his daughter hospitalized. June 30, 2018. United Kingdom. 1 killed. 1 hospitalized.

All of these incidents had one thing in common: organophosphorus (OP) compounds. Dating back to World War I, OP compounds have been used as pesticides and chemical warfare agents. Their potency and volatility vary between OP structure, but their effects are similar: SLUDGE (salivation, lacrimation, urination, defecation, gastrointestinal distress, emesis), muscle spasms, respiratory arrest, seizures, and, eventually, death.2 There are only a handful of ways that OP poisoning can be treated effectively, so the discovery of a universal “cure” is vital.

Post-World War I, Gerhard Schrader, a chemist at I.G. Farben in Germany, was inventing new insecticides containing phosphorus and cyanide when suddenly he and his coworkers were hospitalized with similar symptoms – difficulty breathing, impaired vision, and dizziness.3 Fortunately, they all recovered after their exposure to ethyl-(N,N-

1 dimethylamido)-phosphorocyanidate, or tabun (GA), an organophosphorus compound they synthesized successfully (Figure 1).

Figure 1 G-series organophosphorus nerve agents.

Upon testing, Schrader discovered that tabun was ineffective towards insects, but it could kill a mammal within 20 minutes. Once I.G. Farben contacted the German military, tabun became the first OP chemical warfare agent in the G-series and sparked Germany’s exploration into the development of chemical weapons.

A few years after the discovery of tabun, more potent OP nerve agents were synthesized: isopropyl-methyl-phosphono-fluoridate or sarin (GB) and 3,3-dimethyl- butan-2-yl-methyl-phosphono-fluoridate or soman (GD) (Figure 1).3 Germany stockpiled these nerve agents, and, fortunately, Adolf Hitler decided against their use during World

War II due to fear of retaliation with similar chemical weapons.4 His fear was not completely unjustified. At the University of Cambridge in 1941, Bernard Charles Saunders synthesized diisopropyl fluorophosphate (DFP), a fluoride-containing OP which had some toxicity.2 The British shared their information on DFP with the American military, who started synthesizing derivatives to increase its adverse effects. The US and UK were on the right track to synthesizing more toxic OPs, like tabun or sarin. If Hitler had employed his chemical weapons during WWII, it would not have taken the Allies long to decipher the structures of his OPs. 2

Until 1943, the mechanism of OP poisoning was unknown. Richard Kuhn, hired by the Germans, discovered that exposure to OPs resulted in the accumulation of the neurotransmitter acetylcholine (ACh) in synapses.1 This buildup of ACh causes overstimulation of muscle cells, and the only relief comes from the hydrolysis of ACh by acetylcholinesterase (AChE, Figure 2).5,6

Figure 2 Simplified catalytic hydrolysis of acetylcholine (ACh) by acetylcholinesterase (AChE).

AChE is an enzyme that comes in a variety of isoforms, and it can be found in several different mammals. For humans, AChE resides in synapses of the central nervous central, neuromuscular junctions of the peripheral nervous system, and erythrocyte membranes in blood. Though its native function remains the same, the location of AChE influences how the enzyme is transcribed from RNA (Figure 3).7

Figure 3 Isoforms of AChE (image reproduced from Ref. 7 and 8).

Found in all isoforms of AChE, exon 1 (noncoding) and exons 2-4 (coding) transcribe the core domain of AChE: the active site, an oxyanion hole, a choline-binding pocket, an acyl binding pocket, and the peripheral anionic site (Figure 4).

Figure 4 Catalytic triad of AChE (S203, H447, E334), oxyanion hole (G121, G122, A204), and choline-binding pocket (E202, W86).1

Hydrolysis of ACh occurs in the active site, a catalytic triad containing serine, histidine, and glutamate. Within the active site of the enzyme, the oxyanion hole (glycine 4 and alanine), the choline-binding pocket (glutamate and tryptophan), and the acyl binding pocket (phenylalanine and tyrosine) collectively help align and stabilize ACh for effective binding as well as all of the transition states and intermediates for hydrolysis. The erythrocyte (AChE-E), brain/muscle (AChE-T), and readthrough (AChE-R) isoforms materialize through alternative splicing after RNA transcription. AChE-E contains exon 5, while AChE-T only contains exon 6. In addition to exon 5, AChE-R also contains intron 4 in its RNA sequence. The addition of an intron prevents AChE-R from dimerizing once translated into a protein, leaving it as a monomer, and the absence of exon 6 prevent both

AChE-R and AChE-E from tetramerizing. AChE in the blood can dimerize and, in the brain and muscles, tetramerize, with their respective isoform by forming disulfide bonds between cysteine residues.9 These multimers are anchored to neuronal cell membranes or neuromuscular junctions through a proline-rich membrane anchor (PRiMA) or a collagen

(ColQ) tail or to red blood cell membranes by glycosylphosphatidyl inositol (GPI-G2) moieties (Figure 5).

Figure 5 Membrane anchors for AChE (image reproduced from Ref. 9). 5

Two recombinant human isoforms of AChE were used in this study. Expressed in HEK-

293 cells, researchers under Dr. Zoran Radić at the University of California San Diego donated their isoform of AChE (rhuAChE), containing exons 1-4 only and representing dimeric AChE. The second isoform was provided by Dr. Douglas Cerasoli and Dr. C Linn

Cadieux (United States Army Medical Research Institute of Chemical Defense), who purchased it from Chesapeake PERL Inc (CP-AChE). CP-AChE is the readthrough isoform containing exons 1-5, intron 4, and a polyhistidine-tag (His-tag) at the C-terminus.

Unlike some proteins, the active site of AChE is not on the surface but buried within the interior.1 ACh must traverse a 20 Å gorge from the exterior peripheral anionic site to reach the active site. Despite the long journey, AChE is still able to hydrolyze 25,000

9 -1 -1 molecules of ACh per second with a catalytic efficiency (kcat/Km) of 1.50*10 M s .

Unfortunately, when someone is exposed to an OP agent, such as sarin, the catalytic serine residue of AChE covalently binds the phosphorus atom instead of the carbonyl carbon of

ACh (Figure 6).

Figure 6 Inhibition of AChE by sarin (GB).

This competitive inhibition causes the accumulation of ACh to occur and can lead to death by asphyxiation. Depending on the OP and method of exposure, nerve agents cause

6 asphyxiation through different mechanisms: the constriction of bronchial tubes in the lungs, suppression of the brain’s respiratory center, and paralysis of breathing muscles.2

The race to synthesize the most potent chemical warfare agent did not stop at

WWII. During the Cold War, the United States and the United Kingdom worked together in synthesizing sarin derivatives, such as cyclosarin (GF, cyclohexyl-methyl-phosphono- fluoridate), as shown in Figure 1.2 Although less toxic than soman, sarin was cheaper to synthesize; therefore, sarin and cyclosarin were stockpiled. Imperial Chemical Industries, based in the UK, discovered (O,O-diethyl-S-(2-(diethylamino)ethyl)-phosphoro-thioate or

VG while developing new OP pesticides containing nitrogen and sulfur (Figure 7).

Figure 7 V-series organophosphorus nerve agents.

The US developed a similar compound ethyl-N-2-diisopropyl-aminoethyl-methyl- phosphono-thiolate (VX), spawning the V-series of chemical nerve agents. Unlike the G- series, these OPs had no odor, a long shelf-life, and only required 20 μg/kg to be lethal.

VX was also stable in cold temperatures, rendering it the US’s weapon of choice for the

Cold War. Simultaneously, Russia started developing their own chemical weapons with the synthesis of a VX isomer: N,N-diethyl-2-(methyl-(2-methylpropoxy)phosphoryl)- sulfanyl-ethanamine (VR or “Russian VX).

With the threat of war comes the need for a country to be self-sufficient. Research on pesticides and insecticides gained popularity in many countries who wanted to decrease their reliance on imported foods. In the 1950s, the US discovered diethyl-(dimethoxy- phosphino-thioyl)-thio-butanedioate or malathion, and Schrader, working for Bayer in

Leverkusen, Germany, synthesized demeton (Figure 8).2

Figure 8 Organophosphorus pesticides.

Other OPs, such as methyl and ethyl paraoxon as well as diisopropylfluorophosphate

(DFP), were also used as pesticides, especially after the ban of organochlorine insecticides in the 1970s. Although still toxic to humans, low concentrations of these OP pesticides were used to reduce the potential harm to consumers and agricultural workers. Despite precautions, 3 million people, mainly in developing countries, are poisoned by OP pesticides every year due to “poor working conditions, improper handling, inadequate regulations, or even intentional self-harm.”1

Thanks to the Geneva Protocol of 1925 and increasing governmental regulations, the utilization of OPs as chemical weapons and pesticides is minimal in most countries

(Figure 9).10

Figure 9 Parties to the Geneva Protocol.11

However, many countries have developed reservations toward the protocol, allowing the use of chemical weapons if an enemy country is a non-party or if a participating country violates the protocol.12 Unfortunately, developing countries continue to use OPs in agriculture, and many countries continue to develop new and improved OP nerve agents.

If the threat of OP poisoning continues to exist, then methods to treat exposure must be explored.

In this thesis, we will discuss our team’s efforts to develop novel therapeutics to treat both the OP-inhibited and the OP-aged form of AChE, including new methods to evaluate the activity of recovered (native) AChE from tissues and blood.

Chapter 2. Reactivation of Organophosphorus-Inhibited Acetylcholinesterase with

Quinone Methide Precursors

Introduction

Whether it enters through the skin, lungs, or blood, organophosphorus (OP) chemical nerve agents and pesticides inhibit acetylcholinesterase at different rates (ki) due to their overall size and the strength of the leaving group (Table 1).

Table 1 Rates of Inhibition of Acetylcholinesterase by Various Organophosphorus Chemical Nerve Agents and Pesticides.* k ∙106 Inhibited Serine Category OP OP Structure i (M-1min-1) Residue

Sarin, GB 39.813

Methyl Soman, GD 19313 phosphonates

Cyclosarin, 43913 GF

*Against human AChE unless noted otherwise †Racemic §Bovine AChE Continued

Table 1 Continued

VX 11513

VR 45813

Tabun, GA 18.213

Novichok 29†14 A230

Phosphor- amidates Novichok 120†14 A232

Novichok 73†14 A234

*Against human AChE unless noted otherwise †Racemic §Bovine AChE Continued

Table 1 Continued

Malaoxon 0.0036§15

Demeton 0.006416

No data VG found

Phosphates

DFP 0.1317

Methyl paraoxon, 1.113 MP

Ethyl paraoxon, 3.313 EP

*Against human AChE unless noted otherwise †Racemic §Bovine AChE

As WWII progressed, Germany made more potent analogues (GB, GD, and GF) of their first OP, tabun (GA), and these new analogues possessed rates of inhibition that were up to 20 times more effective than GA. Upon the discovery of Germany’s G-series nerve

12 agents, the Allies developed the V-series of agents (VX, VR, VG) which had similar rates of inhibition. Phosphates, such as methyl and ethyl paraoxon, are still used today as pesticides in developing countries, but usually delivered as the thion (P=S) forms – with formation of the toxic metabolite (the oxon form, P=O) being created by cytochrome P450 oxidation in vivo.18 Their lower concentrations and rates of inhibition are not strong precautions in preventing OP poisoning completely.1 Knowing of their toxicity, many people use OP pesticides to induce self-harm, and OP exposure, by a variety of routes, kill over 200,000 people per year.19

The most recently publicized OP series are the Novichok agents, a group of phosphoramidates developed by Russia in the 1990s.20 In 2018, Sergei and Yulia Skripal and, a few months later, Charlie Rowley and Dawn Sturgess were poisoned by a Novichok agent.21 Sturgess eventually died from her OP exposure, while the others slowly recovered.

Information on the toxicity of the Novichok series of OP nerve agents is not fully available, as some aspects about these compounds remain classified.

When AChE is inhibited by an OP (Figure 6), the poisoning can, thankfully, be reversed or the symptoms can be managed. During the tabun human trials in 1938, it was discovered at the Military Medical Academy in Berlin that atropine (1) helped relieve some effects of OP poisoning by binding to muscarinic acetylcholine receptors (Figure 10).2,22

Figure 10 Structures of currently approved treatments for OP poisoning: atropine (1), nicotinhydroxamic acid (2), 2-pralidoxime chloride (2-PAM, 3), and diazepam (4).

Since atropine works through competitive inhibition with AChE in the peripheral nervous system, some side effects, such as muscle spasms, persist, while others, such as hypersalivation, are alleviated. Unfortunately, atropine does not provide much help to

AChE that has already been inhibited by an OP, and it does not detoxify the OP itself.

The concept of reactivation was not introduced until 1951 when Irwin Wilson at

Columbia University incubated nucleophiles with DFP-inhibited AChE.23,24 After 24 hours, Wilson found that hydroxamic acids, such as 2, could restore 96% of the inhibited enzyme to its native state. This sparked the idea that other oxime (N–OH) nucleophiles could act as reactivators (Figure 11).

Figure 11 Reactivation of sarin-inhibited AChE by an oxime.

After deprotonating the hydroxyl group of the oxime, the resulting oximate anion attacks the phosphorus center, and the electrons of the phosphylated oxyanion push toward the

14 phosphorus.1,25 This releases the serine residue, which is reprotonated by the neighboring histidinium, bringing the enzyme back to its native state.

2-Pralidoxime chloride (2-PAM, 3) is currently the only approved treatment in the

United States for detoxification of an OP after exposure, and characteristics of 2-PAM facilitate binding in the active site (the positive charge) and as a good nucleophile (oxime).

The positive pyridinium interacts with the choline binding site, which helps position the nucleophilic oxime into the active site.25 The global standard of treatment for OP poisoning includes 2-PAM, atropine, and diazepam (4).26,27 As an anticonvulsant, diazepam can prevent permanent damage to the central nervous system.

Between these three compounds, most symptoms of OP poisoning are treated by 1 and 4 and the OP-inhibited enzyme is reactivated by 3. Unfortunately, as a reactivator, 2-

PAM has its limits. For moderate to severe cases of OP poisoning, a 1 g dose 2-PAM must be given by intravenous infusion over 20-30 minutes, followed by 0.5 g every hour.26 At this dosage, 2-PAM can reactivate sarin-inhibited AChE by 40% and VX-inhibited by

70%.28 However, its efficacy plummets when the enzyme is inhibited by tabun or soman.26

Even with the less toxic OP pesticides, 2-PAM is only effective if administered soon after exposure. Once AChE is inhibited by an OP, the phosphylated serine residue can undergo a spontaneous O-dealkylation event, an irreversible process called aging (Figure 12).

Figure 12 Irreversible aging of AChE through O-dealkylation.

Once the enzyme ages, oxime reactivators, such as 2 and 3, can no longer reverse the phosphylation of the serine residue in AChE. The resulting phosphylated oxyanion reduces the electrophilicity of phosphorus and retards the approach of the oximate anion, making the aged form to be less prone to nucleophilic attack. The phosphylated oxyanion of the aged form also develops a stable salt bridge with the neighboring histidinium ion. To bring the aged AChE back to its native state would constitute “resurrection” of the enzyme, a term coined by Dr. Daniel Quinn (Iowa).29 Currently, there are no approved therapeutics which can resurrect OP-aged AChE. However, some promising compounds will be presented in Chapter 3.

A high dosage and the inability to resurrect aged AChE are not the only complications exhibited by 2-PAM. Its permanent positive charge prevents the molecule from crossing the blood-brain barrier (BBB) efficiently. A tightly fused lining of endothelial cells surrounds the brain’s blood vessels, preventing any unwanted, outside materials from entering.30,31 The delicate central nervous system is easily influenced by small changes in its environment, and any major disruptions in neuron signaling can cause a variety of health issues. Over 95% of drugs cannot cross the BBB efficiently and are

16 removed, or effluxed, from the BBB.30 In order to allow select molecules to cross over, the endothelial cells contain transport pathways (Figure 13).

Figure 13 Crossing the blood-brain barrier (image reproduced from Ref. 30).

Compounds with higher lipophilicity can traverse the BBB more easily than water- soluble compounds so long as the molecules remain free and unbound to each other. The permanent positive charge on 2-PAM increases its water solubility, and only 10% of 2-

PAM is able to cross the BBB.32 Transport proteins and vesicles can grip or adsorb molecules and transport them from the blood to the brain.30,31 They can also be used to export waste out of the central nervous system. Engineered transcytosis, as a Trojan horse- type method, has become a popular way to move drugs across the BBB. To develop an OP treatment better than 2-PAM, the charge and lipophilicity of the drug candidate must be considered and using a natural or engineered vesicle to aid in penetrating the BBB should not be ruled out.

To find an universal treatment for OP poisoning, the compound must work against all OPs, efficiently cross over the BBB and remain in the central nervous system, have a reasonable dosage for efficacy, and the potential to reactivate OP-inhibited and to resurrect

OP-aged AChE.

For oximes, many researchers have worked to increase the affinity of an oxime for

OP-inhibited AChE by use of an additional anchor, or linker, to interact with the peripheral anionic site. Obidoxime (5) was found in 1964 to reactivate tabun-inhibited AChE, unlike

2-PAM (Figure 14).26

Figure 14 Other permanently charged oxime reactivators.

Similarly, 5 is most efficient with reactivation when paired with atropine, and it also works against pesticides, sarin, and VX but not soman. When given with atropine, 5 is initially administered by slow intravenous injection at 250 mg, then an infusion of 750 mg over 24 hours.

In 1966, HI-6 (6) was reported to be the first, effective treatment for soman poisoning while also reactivating sarin- and VX-inhibited AChE.26 Unfortunately, 6 has acute toxicity with an LD50 of 781 mg/kg in rats, but, thankfully, the dosage needed to

18 reactivate AChE is lower (~4 mg/L).33 HI-6 is the antidote used in the Czech Republic,

Slovakia, Sweden, and Canada despite its LD50 and its lack of stability in aqueous solutions.

Despite being able to treat multiple OP poisonings, 5 and 6, with their multiple, permanent charges, have difficulty crossing the BBB. Re-purposing FDA-approved drugs or using them as a template is a popular and potentially efficient way to develop new reactivators. Launched in the United States as an Alzheimer’s drug in 1996, donepezil (7) is known to bind to the peripheral site of AChE as a noncompetitive inhibitor (Figure 15).34

Figure 15 Uncharged oxime reactivators.

Using this as inspiration, a variety of oxime derivatives of donepezil were invented.

In 2016, Renard and coworkers tested the binding affinity and reactivation ability of 8 and three other oximes.35 This compound had a similar binding affinity toward AChE as 5 and

6, but it was outperformed in reactivation against VX and sarin. Compound 8 did reactivate

VX-inhibited AChE to a better degree than 2-PAM.

The antimalaria drug, amodiaquine (9), was first synthesized in 1948 and works by preventing DNA and RNA production in the parasite (Figure 16).36,37

Figure 16 Non-oxime reactivators.

Although it cannot be used to prevent malaria due to its hepatotoxicity, 9 is used alongside artesunate for combination therapy to treat malaria. In 2015 at the Columbia University

Medical Center, Katz and coworkers tested this drug, along with 2000 other FDA-approved drugs, against OP-inhibited AChE for reactivation ability.25 Amodiaquine was able to reactivate paraoxon-inhibited AChE more quickly than 2-PAM; however, in vivo studies showed that a high dosage of 9 would be needed for reactivation to be effective. Due to cases of hepatotoxicity and agranulocytosis, using 9 as a treatment for OP poisoning was considered unsafe. Upon studying the structural components of 9, the group found that the

Mannich phenol, 4-amino-2-(diethylaminomethyl)phenol (ADOC, 10), was responsible for reactivation, and because ADOC lacked the hydrophobic anchor of 9, its toxicity on white blood cells should decrease. This new reactivator was able to reactivate DFP-, NIMP-

(sarin analogue), and SIMP-inhibited (soman analogue) AChE better than 2-PAM and, in some cases, HI-6. The reactivation mechanism is thought to be similar to 2-PAM (Figure

17), but after some investigation, the results were inconclusive.

Figure 17 Reactivation of sarin-inhibited AChE by ADOC (10).

Additional studies by Cerasoli38 and de Koning39 looked into a variety of ADOC derivatives (Figure 18). During their study on ADOC’s substituents, Cerasoli and coworkers found that each component, the alcohol, aniline, and benzylic amine, played a role in its reactivation ability.38 Removal of the amine groups (11 and 12) resulted in a decrease in magnitude of reactivation and inhibition of native AChE, indicating that either or both amines play a role in binding to the enzyme. When the alcohol group was modified

(13) or removed (14), the magnitude of reactivation decreased significantly compared to

ADOC.

Figure 18 ADOC derivatives as reported by Cerasoli (11-14)38 and de Koning (15-18).39

In the publication by de Koning et al., 17 derivatives of ADOC were synthesized by changing the benzylic amine (such as 15-18),39 which showed the smallest changes in reactivation in the previous study.38 They found that 17 had a better rate of reactivation against GB, GF, VX, and paraoxon ethyl than ADOC; however, reactivation by 17 was initially very rapid but then the enzyme activity decreased over time.39 It was hypothesized that the byproducts from reactivation were re-inhibiting the enzyme or that, at high concentrations, the reactivator caused modifications to AChE. A possible mechanism of reactivation involves forming a quinone methide, and it is possible that this species can alkylate both the OP adduct and the nucleophilic amino acid residues on the enzyme, which could cause the modification (Figure 19).

Figure 19 Proposed reactivation and enzyme degradation by quinone methides.

Since 2-PAM was first synthesized in 1955, oximes have been the main force against OP poisoning.26 However, the discovery of quinone methides (QMs) as efficient reactivating agents has opened new ways to treat exposure. Drawing inspiration from the structure of 2-PAM and acetylcholine, quinone methide precursors (QMPs) were synthesized by researchers under Dr. Christopher M. Hadad at The Ohio State University

(OSU), and their potential to reactivate OP-inhibited AChE will be discussed herein.

Results and Discussion

Because of the extreme toxicity of OP nerve agents, a methyl phosphonate derivative of cyclosarin, CMP (Figure 20), was synthesized to mimic the inhibited serine residue of AChE, but pesticides, such as ethyl paraoxon (EP) and diisopropylfluorophosphate (DFP), as shown in Figure 8, were available for purchase due to their lower toxicity.

Figure 20 OP nerve agent surrogate.

To avoid the enzyme’s aged-state for these reactivation studies, recombinant human AChE

(rhuAChE or CP-AChE) was incubated for only an hour with excess OP, which was subsequently removed through filtration. Due to its relatively rapid half-time for aging (see

Chapter 3), the pesticide, methyl paraoxon (MP), was not used to inhibit AChE for these reactivation studies.

Combining the structures of ADOC and 2-PAM, a variety of QMPs were synthesized from substituted 3-hydroxypyridines using the Mannich reaction (Figure 21).

Figure 21 Structure activity relationship (SAR) library with a 3-hydroxypyridine framework.

Cerasoli38 and De Koning39 both showed the importance of the benzylic amine and its size in both reactivating and binding to inhibited AChE. Amines a-e were chosen based on their size and were relative successful in previous studies on ADOC derivatives. With its oxime moiety in the 2-position of pyridine, 2-PAM is the most successful reactivator to date. It was hypothesized that converting ADOC into a pyridine with the 2,3-substitution pattern would generate a more potent reactivator. Cerasoli had concluded that the aniline in ADOC also played an important role in reactivation and binding.38 With compounds 19-27 at either

250 or 1000 μM, a structural activity relationship (SAR) could be built with their ability to reactivate rhuAChE inhibited by CMP, EP, or DFP (Figures 22-24, respectively). Once inhibited by an OP, the enzyme, rhuAChE, was incubated with compounds 19-27 a-e for

1 hour then analyzed by Ellman’s assay upon a 100-fold dilution.

250 M 1000 M 120 120

100 100

80 80

) 70 70

(

y t

i 60 60

c a

50 50

i t

a 40 40

l e

R 30 30

20 20

10 10

0 0 e e e e e e e e e - + e e e e e e e e e - + – – – – – – – – – M – – – – – – – – – M a a a a a a a a a A a a a a a a a a a A 9 0 1 2 3 4 5 6 7 -P 9 0 1 2 3 4 5 6 7 -P 1 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2

Figure 22 Biological screening of compounds 19-27 (a-e) with the concentration of 250 μM (left) and 1000 μM (right) against CMP- inhibited rhuAChE after 1 hour using 2-PAM as a reference (25oC, pH 7.5, 1216 units of activity for rhuAChE). radic

If rhuAChE was reactivated spontaneously (negative control, –) or by a QMP, an increase in absorbance at 412 nm over time would be detected. By relating this velocity

(absorbance/time) for each QMP sample to a positive control (AChE which was never exposed to an OP), a relative percent of reactivation was obtained.

When combined with CMP-inhibited rhuAChE, only compounds 20, 21, and 27 showed reactivation potential at both concentrations, and there was no obvious trend between the different benzylic amines (Figure 22). These three motifs, along with compounds 19 and 25, are 5-substituted pyridines. Although their ability to reactivate was low, 19 and 25 did show slight reactivation potential at 1000 μM. Electron-withdrawing groups in 19, 20, and 21 were unable to reactivate CMP-inhibited rhuAChE as well as compound 27 with its 5-methoxy substituent, which is an electron-donating group.

Interestingly, 27a-e performed best at the lower concentration, and 27b even outperformed

2-PAM at 250 μM. This may be due to the same phenomenon de Koning observed in 2018, when his ADOC derivatives or byproducts from reactivation re-inhibited the enzyme.39 At a range of concentrations, select QMPs were combined with native rhuAChE and native

CP-AChE to determine their half maximal inhibitory concentrations (IC50, Table 2).

o Table 2 IC50 (μM) measurements (25 C, pH 7.5) of native rhuAChE (973 units of enzyme activity) and CP-AChE (354 units of enzyme activity) for several QMPs. IC50 (μM) QMP rhuAChE* CP-AChE 25b 946.7 ± 226.4 602.0 ± 80.7 25d 419.3 ± 30.1 213.6 ± 50.6 26b 976.6 ± 226.4 264.7 ± 5.2 26c 923.2 ± 118.3 358.7 ± 26.0 26d 404.3 ± 12.1 295.9 ± 1.2 27a 1154.0 ± 483.8 829.3 ± 26.2 27b 412.7 ± 70.0 244.6 ± 33.5 *Determined by Andrew Franjesevic.

For rhuAChE, all QMPs listed in Table 2, except 27a, have an IC50 value that is lower than

1000 μM, suggesting a potential to re-inhibit the enzyme after reactivation. When reactivation was performed at 250 μM, 27b was at a concentration lower than its IC50 value, justifying why its effectiveness decreased at a higher concentration. Similar IC50 values were obtained using CP-AChE; however, comparisons between the two isoforms are difficult with some QMPs due to high standard deviations with rhuAChE.

When reactivating EP-inhibited rhuAChE, a shift in selectively was observed: compound 27 was no longer effective at either concentration (Figure 23). However, compounds 19, 20, and 21 had a higher reactivation potential against EP-inhibited rhuAChE, reaching up to 25% reactivation. Unfortunately, 2-PAM dominated as it fully reactivated EP-inhibited rhuAChE at both concentrations after 1 hour. Similar to the results against CMP, there was no obvious trend between the different benzylic amines.

250 M 1000 M 120 120 100 100 80 80

30 30

)

% (

25 25

i t

c 20 20

i t

a 15 15

e R 10 10

5 5

Figure 23 Biological screening of compounds 19-27 (a-e) with the concentration of 250 μM (left) and 1000 μM (right) against EP- inhibited rhuAChE after 1 hour using 2-PAM as a reference (25oC, pH 7.5, 1216 units of activity for rhuAChE).

250 M 1000 M 120 120 100 100 80 80

40 40

) 35 35

(

y t

i 30 30

c a

25 25

i t

a 20 20

l e

R 15 15

10 10

5 5

Figure 24 Biological screening of compounds 19-27 (a-e) with the concentration of 250 μM (left) and 1000 μM (right) against DFP- inhibited rhuAChE after 1 hour using 2-PAM as a reference (25oC, pH 7.5, 1216 units of activity for rhuAChE).

2-PAM also out-performed compounds 19-27 with DFP-inhibited rhuAChE, giving

100% reactivation after 1 hour at 1000 μM (Figure 24). Compound 20b was able to achieve

15% reactivation at 250 μM and almost 35% at 1000 μM after 1 hour. Against DFP, compounds with electron-donating groups (24-27) had poor reactivation potential, while bromo-containing compounds (21-23) were only effective at the 5-position (21). Based on these reactivation results, DFP-inhibited rhuAChE has stronger affinity for 5-chloro substituted 3-hydroxypyridines (20) compared to 5-fluoro (19) and 5-bromo (21).

In an attempt to increase the effectiveness of electron-donating QMPs, a variety of benzylic amines were coupled to compound 26 (Figure 25). Unfortunately, changing the benzylic amine on 3-hydroxy-6-methylpyridine (26) did not change the molecules’ ability to reactivate CMP- or DFP-inhibited rhuAChE. Against EP, 26i had comparable reactivation (~25%) with 26a despite the addition of a large, bulky substituent. 26g and 26j also demonstrated a reactivation potential similar to 26b and 26d. But again, 2-PAM dominated reactivation against all OPs tested, and at all concentrations.

CMP-inhibited rhuAChE EP-inhibited rhuAChE DFP-inhibited rhuAChE 200 26a 0.2 0.2 0.7 26a 7.7 13.7 26.0 26a 0.3 0.5 1.2 180 26b 0.2 0.2 0.9 26b 6.8 10.0 13.3 26b 0.2 0.3 0.7 160 26d 0.2 0.3 1.1 26d 7.1 8.2 13.3 26d 0.3 0.4 1.1 140 26f 0.2 0.2 0.8 26f 5.8 7.2 10.3 26f 0.2 0.4 0.9 120 26g 0.2 0.2 0.7 26g 5.6 7.4 13.4 26g 0.2 0.3 0.8 100 26h 0.2 0.3 1.8 26h 5.9 7.3 9.1 26h 0.2 0.3 0.7 80 26i 0.1 0.2 0.5 26i 7.8 13.7 24.7 26i 0.3 0.4 1.1 60 26j 0.1 0.2 0.6 26j 5.9 9.2 14.4 26j 0.2 0.4 1.1 40 26k 0.1 0.2 0.7 26k 4.4 4.9 4.3 26k 0.2 0.3 0.6 20 2-PAM 23.1 45.9 182.0 2-PAM 139.7 112.6 127.7 2-PAM 46.3 64.2 117.6 0 250 500 1000 250 500 1000 250 500 1000 M M M

Figure 25 Biological screening of compound 26 (a-k) with the concentration of 250, 500 and 1000 μM against OP-inhibited rhuAChE after 1 hour using 2-PAM as a reference (25oC, pH 7.5, 1216 units of activity for rhuAChE).

Upon studying the effectives of substituents and benzylic amines on 3- hydroxypyridines, the purpose of the pyridine nitrogen was questioned. Thus, compounds

28a-e to 33a-e (Figure 26) were synthesized by several undergraduate students (Jacob

Weaver, Cassandra Miller, and Christopher Codogni) in order to compare them to their pyridine counterparts (19-27). Unfortunately, some of the phenols needed for comparison still remain to be synthesized.

Figure 26 Structure activity relationship (SAR) library with a phenol framework.

Unfortunately, these new compounds were not effective in reactivating DFP- or EP- inhibited rhuAChE. However, against CMP, compound 28b reactivated over 20% of the inhibited enzyme at 250 μM (Figure 27).

120 100 80

)

% (

i t

c 20

i t

a 15

e R 10

0 ) e ,e e e e e M -) – c – – – – ( (+ a – a a a a A 8 a 0 1 2 3 -P 2 9 3 3 3 3 2 2

Figure 27 Biological screening of compound 28-33 (a-e) with the concentration of 250 μM against CMP-inhibited rhuAChE after 1 hour using 2-PAM as a reference (25oC, pH 7.5, 1013 units activity for rhuAChE).

This reactivation potential is better than its pyridine counterpart 20b which had less than

5% reactivation of CMP-inhibited rhuAChE (Figure 22). It is possible that the pyridine nitrogen does not play a predominate role in reactivation, but further study is needed to reach this conclusion. Thus far, the QMPs which have been synthesized and tested in vitro are rather small, so they could easily traverse the 20 Å gorge of AChE. To improve the binding and stability in the active site of AChE, extensions were added to QMPs in the meta or para position to the benzylic amine (Figure 28).

Figure 28 Linker QMPs.

It was hypothesized that these QMPs would link to the peripheral anionic site and extend down the gorge to the active site of AChE. Unfortunately, these linker QMPs were not effective at 250 μM against DFP-inhibited rhuAChE after 1 hour and were only slightly effective against EP and CMP (Figure 29). It is possible that these QMPs are re-inhibiting the enzyme after reactivation; therefore, IC50 values for each compound will need to be determined. Interestingly, 40b had a slightly higher reactivation than 19b (Figure 23) against EP-inhibited rhuAChE.

CMP-inhibited rhuAChE 120 100 80

) 35

(

y t

i 30

c a

i t

a 20

l e

R 15

0 ) ) e e ,e 7 ,e 9 e 1 2 3 M - – – c 3 a 3 – 4 4 4 ( (+ a a – 8 a A 4 5 a 3 0 P 3 3 6 4 - 3 2

EP-inhibited rhuAChE 120 100 80

) 35

(

y t

i 30

c a

i t

a 20

l e

R 15

0 - e e ,e 7 ,e 9 e 1 2 3 M + – – c 3 a 3 – 4 4 4 a a – 8 a A 4 5 a 3 0 P 3 3 6 4 - 3 2

Figure 29 Biological screening of compound 34-43 (a-e) with the concentration of 250 μM against CMP-inhibited (top) or EP-inhibited (bottom) rhuAChE after 1 hour using 2- PAM as a reference (25oC, pH 7.5, 1013 units of activity for rhuAChE during CMP inhibition and 1111 units of activity for rhuAChE during EP inhibition).

This may be due to its ability to produce both ortho- and para-quinone methides. Gathering other 3-hydroxypyridines, such as 20, 26, 27, and 28, and subjecting them to a di-Mannich substitution would be an interesting study on the effectives of ortho vs para QM formation.

As more QMPs are synthesized by other members of our research team at OSU, the quest in finding a more effective and general reactivator than 2-PAM will continue.

Compounds that show promise in vitro will be distributed to United States Army Medical

Research Institute of Chemical Defense or MRIGlobal for in vivo testing in mice.

Experimental

Caution: Organophosphorus nerve agents and pesticides are extremely toxic and can only be handled after proper training and review of the standard operating procedure.

An Integra AssistPlus Pipetting Robot was used to fill 96-well or 384-well plates. Protocols were made through Integra Biosciences’s ViaLab software program and uploaded to

Voyager Adjustable Tip Spacing Electronic Pipettes. Integra Evolve Pipettes were used for manual dispensing. Corresponding pipette tips were purchase from Integra Biosciences.

Amicon centrifugal ultrafilters (MWCO 30 kDa), 96-well and 384-well clear, flat-bottom microplates, low protein binding collection tubes (1.5 mL), low profile PCR tubes and flat caps were all purchased from Fisher Scientific. Hard-Shell® 96-well PCR plates were obtained from Bio-Rad. Sodium phosphate dibasic dihydrate, sodium phosphate monobasic monohydrate, and sodium hydroxide pellets were purchased from Fisher

Scientific. Reagents used for Ellman’s assay, acetylthiocholine and 5,5-dithio-bis-(2- nitrobenzoic acid) (DTNB), were purchase from Fisher Scientific and Millipore Sigma, respectively. Bovine Serum Albumin was obtained as a lyophilized powder from Fisher

Scientific. The absorption obtained from Ellman’s assay at 412 nm was monitored with a

Molecular Devices SpectraMax i3 or a BioTek Synergy H1 Hybrid Multi-Mode Reader using a monochromator and kinetic scan. Recombinant human AChE was provided by Dr.

Douglas Cerasoli and Dr. C Linn Cadieux (United States Army Medical Research Institute of Chemical Defense, purchased from Chesapeake PERL Inc.) or by Dr. Zoran Radić

(University of California, San Diego, expressed in HEK-293 cells).40

Reactivation of OP-Inhibited Acetylcholinesterase—Representative Procedure:

Inhibition of rhuAChE. 1.71-2.12 µL* of 0.1 mM recombinant human AChE (rhuAChE) from Zoran Radic (16 ug/mL) was added to 96.3-95.9 µL of 1 g/L BSA (in 0.2 M sodium phosphate buffer at pH 7.5, with 0.02% NaN3) along with 2 µL of 10 mM ethyl paraoxon

(EP) solution (in acetonitrile). A positive control was prepared in parallel with the OP solution replaced by blank acetonitrile. The mixed solutions were incubated at 37 °C for 1 hour, then washed with 0.2 M sodium phosphate buffer (pH 7.5) in centrifugal ultrafilters

(Amicon, MWCO 30 kDa) at 14,000 g for 4 minutes (x 3). Recovered protein concentrate

(~50 µL) was mixed with 50 µL of 0.2 M pH 7.5 phosphate buffer. For larger screens, the ratio was kept constant, and the volumes were increased by a factor of 2. A single point inhibition check was conducted using Ellman’s assay to ensure the protein was completely inhibited. If so, the sample was used in the following experiments. (*Volume of rhuAChE varied between experiments to obtain the same relative enzyme activity in all assays. An activity test was performed before each experiment to determine the proper ratio to a reference rhuAChE sample with 973 U relative activity. 2 µL of this reference rhuAChE was previously used to make 100 µL controls.)

Recovery of EP-inhibited rhuAChE with 1000 μM QMP. 10 µL of 10 mM solution of each compound (QMP) was mixed with 89 µL of 1 g/L BSA (in 0.2 M sodium phosphate buffer at pH 7.5, with 0.02% NaN3) and 1 µL of 2 µM rhuAChE inhibited with EP. In the

39 positive control, the inhibited rhuAChE and the QMP solution were replaced by native rhuAChE and blank water, respectively. All samples were incubated at 37 °C for 1 hour.

At each time interval, the samples were diluted by 10-fold with 0.2 M sodium phosphate buffer (pH 7.5) prior to their use in Ellman’s assay.

Recovery of EP-inhibited rhuAChE with 500 uM QMP. 5 µL of 10 mM solution of each compound (QMP) was mixed with 94 µL of 1 g/L BSA (in 0.2 M sodium phosphate buffer at pH 7.5, with 0.02% NaN3) and 1 µL of 2 µM rhuAChE inhibited with EP. In the positive control, the inhibited rhuAChE and the QMP solution were replaced by native rhuAChE and blank water, respectively. All samples were incubated at 37 °C for 1 hour.

At each time interval, the samples were diluted by 10-fold with 0.2 M sodium phosphate buffer (pH 7.5) prior to their use in Ellman’s assay.

Recovery of EP-inhibited rhuAChE with 250 uM QMP. 2.5 µL of 10 mM solution of each compound (QMP) was mixed with 96.5 µL of 1 g/L BSA (in 0.2 M sodium phosphate buffer at pH 7.5, with 0.02% NaN3) and 1 µL of 2 µM rhuAChE inhibited with EP. In the positive control, the inhibited rhuAChE and the QMP solution were replaced by native rhuAChE and blank water, respectively. All samples were incubated at 37 °C for 1 hour.

At each time interval, the samples were diluted by 10-fold with 0.2 M sodium phosphate buffer (pH 7.5) prior to their use in Ellman’s assay.

Ellman’s Assay using the Molecular Devices SpectraMax Multi-Mode Reader. Each diluted sample was tested in four wells on a 96-well microplate. Each well contained 142

µL of 40 mM sodium phosphate buffer (pH 7.5), 10 µL of 10 mM acetylthiocholine solution, 10 µL of 20 mM DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)), 18 µL of 1 g/L

BSA (in 40 mM sodium phosphate buffer at pH 7.5, with 0.02% NaN3), and 20 µL of diluted sample. The plate was read at 37 °C and 412 nm every 29 seconds for 10.5 min.

The rates of absorbance increase were recorded. The initial slopes were taken and compared to that of the positive control to calculate the relative activity of rhuAChE.

Ellman’s Assay using the BioTek Synergy H1 Hybrid Multi-Mode Reader. Each diluted sample was tested in four wells on a 384-well microplate. Each well contained 71

µL of 40 mM sodium phosphate buffer (pH 7.5), 5 µL of 20 mM DTNB (5,5'-dithiobis-(2- nitrobenzoic acid)), 9 µL of 1 g/L BSA (in 40 mM sodium phosphate buffer at pH 7.5, with

0.02% NaN3), and 10 µL of diluted sample. Using the BioTek Dual Reagent Injector

Module, 5 µL of 10 mM acetylthiocholine solution was dispensed into the plate docked into the Synergy H1 Reader. The plate was then read at 37 °C and 412 nm every 20 seconds for 10.5 min. The rates of absorbance increase were recorded. The initial slopes were taken and compared to that of the positive control to calculate the relative activity of rhuAChE.

Chapter 3. Resurrection of Organophosphorus-Aged Acetylcholinesterase with Quinone

Methide Precursors

Introduction

Inhibition of AChE by an OP is not the only stage of enzyme deactivation. Once an

OP, such as sarin, enters the active site and binds to AChE, dealkylation of the phosphylated serine residue can occur over time (Figure 12). This was demonstrated by F.

Hobbinger in 1955 at the Middlesex Hospital Medical School in London.41 As Hobbinger was studying the effects of 2, a reactivator, (Figure 10) on inhibited AChE, he found that the efficacy of 2 decreased over time. This secondary reaction of the OP-AChE adduct is referred to as aging and is irreversible and stable due to the strong hydrogen bond between the histidinium ion and the oxyanion of the phosphylated serine residue. Treatments used to reverse inhibition, such as 2-PAM and ADOC, are no longer effective once the enzyme is aged since the anionic phosphylated serine residue is resistant to nucleophilic attack.1

Thankfully, not all OPs cause aging, and the OP structure facilitates aging at different rates

(Table 3). Unfortunately, with the inability to reverse aged-AChE with current treatments, there is a desperate need to discover new ways to approach OP poisoning.

Table 3 Aging of Acetylcholinesterase by Organophosphorus Nerve Agents and Pesticides.* Aging Deaminated Serine Category OP OP Structure Half- Dealkylated Serine Residue Residue life (h)

Sarin, GB 3.01 -

Soman, GD 0.071 -

Cyclosarin, 7.01 - GF Methyl phosphonates

1 VX 36.5 -

VR 138.61 -

*Against human AChE unless noted otherwise †It has been confirmed that GA ages through O-dealkylation.42 ‡It is not known if the phosphylated serine residue ages. Continued

Table 3 Continued

Tabun, 19.21 GA†

Phosphoramidates ‡ A230 - -

A232‡ -

A234‡ -

Malaoxon 3.71 -

*Against human AChE unless noted otherwise †It has been confirmed that GA ages through O-dealkylation.42 ‡It is not known if the phosphylated serine residue ages. Continued 44

Table 3 Continued

Demeton 31.51 -

VG 31.51 -

DFP 3.043 - Phosphates

Methyl paraoxon, 3.71 - MP

Ethyl paraoxon, 31.51 - EP

*Against human AChE unless noted otherwise †It has been confirmed that GA ages through O-dealkylation.42 ‡It is not known if the phosphylated serine residue ages.

Despite decades of effort, there are currently no approved therapeutics which can return OP-aged AChE back to its native state. In addition to the strong salt bridge between the phosphylated oxyanion at Ser203 and the histidinium ion at His447, it is believed that charge-charge repulsion between this oxyanion and reactivating nucleophiles prevent these nucleophiles from attacking phosphorus.44 Re-alkylating aged-AChE with re-alkylating agents such as sulfoniums, methoxypyridiniums, or halomethylketones, could revert the enzyme to its treatable, inhibited state (Figure 30).45

Figure 30 Proposed methods to reverse aging with OP-aged AChE.29,44–46

However, despite successful in silico evaluations or even conversions in non-enzymatic model systems, all of these approaches were unable to re-alkylate or resurrect OP-aged

AChE in vitro.

In 2018, the Hadad group at The Ohio State University showed that aged electric eel AChE (eeAChE) can be “resurrected” in vitro by neutral QMPs (Figure 31).47

Figure 31 Screening of three concentrations of various QMPs against PiMP-aged eeAChE.47

This was the first demonstration of the reversion, or resurrection, of OP-aged AChE, and with compound C8 resurrecting over 10% of the enzyme at 4.0 mM . The QMPs were incubated with methylphosphonate-aged AChE for 24 hours after which 2-PAM was added to reactivate the (supposedly) re-alkylated enzyme for 1 hour. Through additional kinetic experiments, it was determined that these QMPs both resurrect OP-aged and reactivate OP- inhibited AChE, and the final incubation step with 2-PAM was unnecessary. It is possible that QMPs do not solely act as re-alkylators but as both re-alkylators and reactivators. It is speculated that resurrection occurs by an SN2 mechanism or through QM formation, where the nucleophile (Nuc) is the oxyanion of the phosphylated serine residue (Figure 32).

Figure 32 Proposed mechanism for resurrection by a QMP.

Further research into the mechanism of resurrection is needed to determine how these

QMPs function. Exposing the same QMPs presented in Chapter 2 to OP-aged AChE will demonstrate if these compounds can perform both reactivation and resurrection and how electronic and steric effects influence their ability to revive AChE.

Results and Discussion

To ensure the enzyme is completed aged, rhuAChE or CP-AChE was incubated with excess OP for 24 hours. After filtration, an additional incubation with 2-PAM was conducted for 1-2 hours and an aliquot of the solution was removed and tested via Ellman’s assay. If an absorbance at 412 nm was detected, then 2-PAM was able to revive the enzyme: an effect which should not occur with aged AChE. If this test proved a completely aged enzyme, the experiment continued in a similar manner to the reactivation protocol.

Eventually, it may be valuable to study a QMP’s effects on a mixture of aged and inhibited

AChE.

Expanding upon the research conducted in 2018 (Figure 31), the effects of different substituents on 3-hydroxypyridine were studied using compounds 19-27 with amines a-e at a concentration of 1000 μM (Figure 33). Previously, C8 only resurrected 6% of methylphosphonate-aged eeAChE at 1000 μM, and compounds 19-21 a-e show similar results after 24 hours when rhuAChE was used. Addition of an electron-donating group to

3-hydroxypyridine in either the 5- or 6-position raised its effectiveness; compounds 26d,

27a, and 27b had almost 15% resurrection at 1000 μM after 24 hours. Unfortunately, at least 20% AChE needs to be resurrected for there to be minimal side effects.48 Even at 15% resurrection, this is a major breakthrough in this field of research. No other research group has demonstrated the resurrection of aged AChE, while we continue to increase our compounds’ effectiveness in reversing this aged state.

120

100

) 15

(

e 10

e R

0 e e e e e e e e e - + – – – – – – – – – M a a a a a a a a a A 9 0 1 2 3 4 5 6 7 P 1 2 2 2 2 2 2 2 2 - 2

Figure 33 Biological screening performed by Andrew Franjesevic of compounds 19-27 (a-e) with the concentration of 1000 μM against CMP-aged rhuAChE after 24 hours using 2-PAM as a reference (25oC, pH 7.5, 973 units of activity for rhuAChE).

Compounds 20-27 a-e were also effective resurrectors against MP- and DFP-aged rhuAChE with up to 90% and 50% resurrection respectively (Figures 34 and 35).

Interestingly, a change in selectivity was observed when these QMPs are exposed to inhibited versus aged rhuAChE. While compounds with electron-withdrawing groups, such as 20 and 21, performed well as reactivators, they did not show the same capabilities against the enzyme’s aged state. Instead, compounds with electron-donating groups, such as 25 and 26, showed the best resurrection potential for all three aged forms (CMP-, MP-, and DFP-aged rhuAChE). The exact mechanism of reactivation or resurrection through

QMPs is, currently, unknown, but it is our hope by isolating the byproducts of these reactions that the mechanism can be determined.

rhuAChE CP-AChE 120 120 110 110 100 100

90 90

)

) %

% 80 80

(

t i

i 70 70

c a a 60

i t

t 50 50

e R R 40 40 30 30 20 20 10 10 0 0 e e e e e e e e - + e e e e e e e e - + – – – – – – – – M – – – – – – – – M a a a a a a a a A a a a a a a a a A 0 1 2 3 4 5 6 7 -P 0 1 2 3 4 5 6 7 -P 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

Figure 34 Biological screening of compounds 20-27 (a-e) with the concentration of 1000 μM against MP-aged rhuAChE (left) or CP- AChE (right) after 24 hours using 2-PAM as a reference (25oC, pH 7.5, 973 units of activity for rhuAChE and 354 units of activity for CP-AChE).

rhuAChE CP-AChE 120 120

100 100

60 60

)

% (

( 50 50

t c

c 40 40

a l

l 30 30

R R 20 20

10 10

0 0 e e e e e e e e - + e e e e e e e e - + – – – – – – – – M – – – – – – – – M a a a a a a a a A a a a a a a a a A 0 1 2 3 4 5 6 7 -P 0 1 2 3 4 5 6 7 -P 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

Figure 35 Biological screening of compounds 20-27 (a-e) with the concentration of 1000 μM against DFP-aged rhuAChE (left) or CP-AChE (right) after 24 hours using 2-PAM as a reference (25oC, pH 7.5, 973 units of activity for rhuAChE and 354 units of activity for CP-AChE).

Similar results were also obtained when another isoform of human AChE, CP-

AChE, was aged. Since these QMPs can resurrect multiple isoforms in vitro, it is hopeful that they will behave similarly in vivo in targeting aged AChE in the brain, muscles, and blood. However, due to the stability and expense of CP-AChE, only rhuAChE was used in future studies.

Several interactions between 26b and methylphosphonate-aged AChE have been observed through a series of molecular docking studies performed by Joseph Fernandez, depicting this compound’s binding affinity to and stability within the active site (Figure

35). The hydrogen-bonding network between the phosphylated Ser203 residue and surrounding amino acid residues maintained close interactions with most distances measuring less than 3 Å. In order to be an effective resurrector, a QMP must exhibit a stronger affinity for these residues than the phosphylated serine in order to break up this network and resurrect the enzyme. The hydroxy and diethylamino groups in 26b are stabilized in the choline binding pocket of AChE, where Glu202 forms a potential hydrogen bond to the hydroxy group and Trp86 facilitates a cation-π interaction with the protonated diethylamine. Trapped within the acyl binding pocket, the pyridine ring of the

QMP interacts with variety of aromatic residues through π-π stacking. With its natural substrate ACh, AChE uses the choline binding pocket to stabilize the quaternary amine of

ACh, while the acyl binding pocket aligns the substrate within the active site to help it bind properly. Utilizing both of these binding pockets with 26b, computational models predicted that these QMPs enter and bind to the active site well. If QMPs, such as 26b, resurrect aged

AChE through realkylation, the distance between the benzylic carbon of the QMP and the phosphylated serine residue needs to be small for the reaction to occur.

Figure 36 Distance map of 26b and the active site of methylphosphonate-aged human AChE. Multiple intermolecular distance measurements were taken over time by Joseph Fernandez. For each interaction, the distance at a particular timepoint is mapped to the color bins (red: 2-3 Å; green: 3-4 Å; blue: 4-5 Å; white: >5 Å.) The distance categories are grouped according to noteworthy interactions as defined above.

Thus far, 3-hydroxy-6-methylpyridine (26a-e) has shown the greatest resurrection potential against each OP used. In attempt to increase its effectiveness, a variety of benzylic 54 amines were accessed using this framework (26a-k); however, compounds 26b and 26d still outperformed the other QMPS in all cases (Figure 37).

CMP-aged rhuAChE MP-aged rhuAChE DFP-aged rhuAChE 70% 26a 3.9 6.2 10.4 26a 14.4 28.1 34.5 26a 0.7 0.7 1.0

26b 11.5 17.9 27.0 26b 30.6 52.1 67.7 26b 5.9 6.6 12.9 60%

26d 14.3 19.1 24.3 26d 23.2 22.4 49.7 26d 27.8 19.3 35.4 50% 26f 8.9 11.1 19.2 26f 22.8 29.8 50.1 26f 1.7 2.2 3.0

40% 26g 2.2 2.4 3.0 26g 6.1 5.9 9.2 26g 0.9 1.0 1.0

26h 8.1 9.8 12.9 26h 21.3 27.1 38.7 26h 1.5 1.6 1.5 30%

26i 1.9 2.1 2.6 26i 0.8 1.0 1.0 26i 0.5 0.5 0.5 20% 26j 0.7 0.7 0.7 26j 0.7 0.8 0.6 26j 0.5 0.5 0.5

26k 0.7 0.7 0.6 26k 0.7 0.8 0.6 26k 0.5 0.4 0.4 10%

2-PAM 3.7 4.0 4.6 2-PAM 0.9 1.2 1.2 2-PAM 0.7 0.8 1.1 0% 250 500 1000 250 500 1000 250 500 1000 M M M

Figure 37 Biological screening of compound 26a-k with the concentration of 250, 500 and 1000 μM against OP-aged rhuAChE after 24 hours using 2-PAM as a reference (25oC, pH 7.5, 1216 units of activity for rhuAChE).

Interestingly, 26f had comparable results against CMP- and MP-aged rhuAChE at all concentrations with its asymmetric ethylmethylamino group, while 26a with a dimethylamino group did not perform as well. Perhaps, some steric bulk is needed to help stabilize the orientation of the QMP within the active site or conversely, the basicity of the amino group is very important. However, if the benzylic amine becomes too large, such as 55 with 26i-k, the compounds effectiveness decreases dramatically. This could be due to its inability to traverse the narrow 20 Å gorge of AChE. Resurrecting DFP-aged AChE also proved difficult due to its bulky isopropyl group. This may be preventing any sized QMP from reaching the phosphylated serine residue.

In our team’s efforts to create a medical countermeasure, we are continuously evaluating compounds that can resurrect OP-aged or reactivate OP-inhibited AChE faster and at a lower concentration. After studying the effects of benzylic amines 26a-k, it was decided that future in vitro testing would be conducted at 250 μM or less in order to screen compounds at more medicinally relevant concentrations. 2-PAM is currently administered in a 70 kg patient at 1 g for 30 minutes (~1000 μM) than maintained at 500 mg/h (~500

μM/h) until symptoms subside.26 High dosages of 2-PAM also give similar symptoms to that of OP poisoning, making assessment of a patient more difficult. Reducing the concentration of a treatment could reduce the severity of potential side effects.

Removing the pyridine nitrogen had some effects on resurrection (Figure 38).

Against CMP, 27b resurrected 15% of aged rhuAChE at 1000 μM, while its phenol counterpart, 33b, resurrected 20% when applied at only 250 μM. Even 33b was outperformed by the pyrrolidine Mannich base in 33c, which resurrected almost 25% at

250 μM. It is possible that the increase in percent resurrection was due to a lower IC50 of these compounds, a result observed with 27b during the reactivation of CMP-inhibited

AChE (Figure 22). Since a full comparison between pyridines and phenols is incomplete,

IC50 studies will be conducted once all phenols needed are synthesized by Christopher

Codogni and other members of the research team.

CMP-aged rhuAChE MP-aged rhuAChE 110 110

100 100

30 30

)

( (

t c

c 20 20

t a

a 15 15

R R 10 10

5 5

0 0 ) ) * ) ) e ,e e e e e M (- + e ,e e e e e M (- + – c – – – – ( – c – – – – ( a – a a a a A a – a a a a A 8 a 0 1 2 3 -P 8 a 0 1 2 3 -P 2 9 3 3 3 3 2 2 9 3 3 3 2 2 2 3 DFP-aged rhuAChE 110

100

)

% (

i t

c 20

i t

a 15

e R 10

0 ) ) e ,e e e e e M - – c – – – – ( (+ a – a a a a A 8 a 0 1 2 3 -P 2 9 3 3 3 3 2 2

Figure 38 Biological screening of compounds 28-33 (a-e) with the concentration of 250 μM against OP-aged rhuAChE after 24 hours using 2-PAM as a reference (25oC, pH 7.5, 1048 units of activity for rhuAChE against CMP (top left), 1014 units of activity against MP (top right), and 322 units of activity against DFP (bottom)). *33a-e was not tested against MP-aged rhuAChE. 57

Compounds 28c and 31d, QMPs with an electron-withdrawing group, outperformed phenols containing electron-donating groups (32 and 33 a-e) with more than 20% resurrection against MP-aged rhuAChE. Against DFP, compounds 28, 31, and 33 resurrected at least 10% of aged rhuAChE. Interestingly, the resurrection potential of phenols against DFP-aged rhuAChE is primarily affected by the benzylic amine, where an

(R)-2-methylpyrrolidine group (d) gave the highest amount of resurrection.

Linker QMPs 34-43 a-e did not perform well against DFP-aged rhuAChE after 24 hours at 250 μM with no compound resurrecting more than 5%. Against MP, only compounds 37, 38a, 38e, 39, 41, and 42 were tested, and only 38e showed significant resurrection potential with 55% activity after 24 hours at 250 μM. This may be due to its ability to form both the ortho- and para-QM, but more linker QMPs need to be tested to develop a correlation. As for CMP-aged rhuAChE, compounds 34 and 35 with (R)-2- methylpyrrolidine (d) demonstrated some potential for resurrection (Figure 39).

120 100 80

)

(

y t

i 50

c a

t a

l 30

e R 20

0 - e e ,e 7 ,e 9 e 1 2 3 M + – – c 3 a 3 – 4 4 4 a a – 8 a A 4 5 a 3 0 P 3 3 6 4 - 3 2

Figure 39 Biological screening of compounds 34-43 (a-e) with the concentration of 250 μM against CMP-aged rhuAChE after 24 hours using 2-PAM as a reference (25oC, pH 7.5, 1048 units of activity for rhuAChE).

To determine whether these larger QMP are re-inhibiting resurrected rhuAChE, IC50 studies will be conducted alongside studies on the half maximal effective concentration

(EC50). More of these QMP linkers are being synthesized by members of our team, including Benjamin Clark, Donna Ensey, and Joshua Kaiser, and these linked compounds will be tested in the future.

The threat of chemical warfare remains imminent. With the possibility of encountering unknown OP agents, such as the 2018 Novichok attacks in the UK, it is important to research solutions to undeveloped problems, but it is equally important to

59 preempt future efforts of potential terrorist organizations with research into new OP analogs.

Future in vitro testing will include analogs of tabun and Novichok agents, which will be synthesized by Nathan Ward. New QMPs are constantly being synthesized by members of Dr. Hadad’s research group at OSU. Continuing work on phenols and pyridines, with a variety of benzylic amines, new frameworks, such as quinolinols, naphthols, and indoles, are added to the list. There will always be a need for large-scale in vitro testing of QMPs against OP-inhibited and aged AChE as the hunt for a better treatment continues.

Experimental

Caution: Organophosphorus nerve agents and pesticides are extremely toxic and can only be handled after proper training and review of the standard operating procedure.

An Integra AssistPlus Pipetting Robot was used to fill 96-well or 384-well plates. Protocols were made through Integra Biosciences ViaLab software program and uploaded to

Voyager Adjustable Tip Spacing Electronic Pipettes. Integra Evolve Pipettes were used for manual dispensing. Corresponding pipette tips were purchase from Integra Biosciences.

Scientific. The absorption obtained from Ellman’s assay at 412 nm was monitored with a

Molecular Devices SpectraMax i3 or a BioTek Synergy H1 Hybrid Multi-Mode Reader using a monochromator and kinetic scan. Recombinant human AChE was provided by Dr.

Douglas Cerasoli and Dr. C Linn Cadieux (United States Army Medical Research Institute of Chemical Defense, purchased from Chesapeake PERL Inc.) or by Dr. Zoran Radić

(University of California, San Diego, expressed in HEK-293 cells).40

Resurrection of OP-Aged Acetylcholinesterase—Representative Procedure:

Aging of rhuAChE. 1.71-2.12 µL* of 0.1 mM recombinant human AChE (rhuAChE) from Zoran Radic (16 ug/mL) was added to 96.3-95.9 µL of 1 g/L BSA (in 0.2 M sodium citrate buffer at pH 6.0, with 0.02% NaN3) along with 2 µL of 10 mM methyl paraoxon

(MP) solution (in acetonitrile). A positive control was prepared in parallel with the OP solution replaced by blank acetonitrile. The mixed solutions were incubated at 37 °C for

24 hours, then washed with 0.2 M sodium phosphate buffer (pH 7.5) in centrifugal ultrafilters (Amicon, MWCO 30 kDa) ) at 14,000 g for 4 minutes (x 3). The retrieved protein concentrates (~50 µL) were mixed with 45 µL 0.2 mM sodium phosphate buffer at pH 7.5 and 5 µL of 10 mM 2-PAM. The solution was further incubated for 1-2 hours. The sample was then washed with of 0.2 M sodium phosphate buffer at pH 7.5 in centrifugal ultrafilters (Amicon, MwCO 30 kDa) at 14,000 g for 4 minutes (x 3). Recovered protein concentrate (~50 µL) was mixed with 50 µL of 0.2 M pH 7.5 phosphate buffer. For larger screens, the ratio was kept constant, and the volumes were increased by a factor of 2. A single point aging check was conducted using Ellman’s assay to ensure the protein was completely aged. If so, the sample was used in the following experiments. (*Volume of rhuAChE varied between experiments to obtain the same relative enzyme activity in all assays. An activity test was performed before each experiment to determine the proper ratio to a reference rhuAChE sample with 973 U relative activity. 2 µL of this reference rhuAChE was previously used to make 100 µL controls.)

Recovery of MP-aged rhuAChE with 1 mM QMP. 10 µL of 10 mM solution of each compound (QMP) was mixed with 89 µL of 1 g/L BSA (in 0.2 M sodium phosphate buffer at pH 7.5, with 0.02% NaN3) and 1 µL of 2 µM rhuAChE aged with MP. In the positive control, the aged rhuAChE and the QMP solution were replaced by native rhuAChE and blank water, respectively. All samples were incubated at 37 °C for 24 hours.Error! Reference source not found. At the time interval, the samples were diluted by 10-fold with 0.2 M sodium phosphate buffer (pH 7.5) prior to their use in Ellman’s assay.

Recovery of MP-aged rhuAChE with 500 uM QMP. 5 µL of 10 mM solution of each compound (QMP) was mixed with 94 µL of 1 g/L BSA (in 0.2 M sodium phosphate buffer at pH 7.5, with 0.02% NaN3) and 1 µL of 2 µM rhuAChE aged with MP. In the positive control, the aged rhuAChE and the QMP solution were replaced by native rhuAChE and blank water, respectively. All samples were incubated at 37 °C for 24 hours. At the time interval, the samples were diluted by 10-fold with 0.2 M sodium phosphate buffer (pH 7.5) prior to their use in Ellman’s assay.

Recovery of MP-aged rhuAChE with 250 uM QMP. 2.5 µL of 10 mM solution of each compound (QMP) was mixed with 96.5 µL of 1 g/L BSA (in 0.2 M sodium phosphate buffer at pH 7.5, with 0.02% NaN3) and 1 µL of 2 µM rhuAChE aged with MP. In the positive control, the aged rhuAChE and the QMP solution were replaced by native rhuAChE and blank water, respectively. All samples were incubated at 37 °C for 24

63 hours.Error! Reference source not found. At the time interval, the samples were diluted by 10-fold with 0.2 M sodium phosphate buffer (pH 7.5) prior to their use in Ellman’s assay.

Ellman’s Assay using the Molecular Devices SpectraMax Multi-Mode Reader. Each diluted sample was tested in four wells on a 96-well microplate. Each well contained 142

µL of 40 mM sodium phosphate buffer (pH 7.5), 10 µL of 10 mM acetylthiocholine solution, 10 µL of 20 mM DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)), 18 µL of 1 g/L

BSA (in 40 mM sodium phosphate buffer at pH 7.5, with 0.02% NaN3), and 20 µL of diluted sample. The plate was read at 37 °C and 412 nm every 29 seconds for 10.5 min.

The rates of absorbance increase were recorded. The initial slopes were taken and compared to that of the positive control to calculate the relative activity of rhuAChE.

Ellman’s Assay using the BioTek Synergy H1 Hybrid Multi-Mode Reader. Each diluted sample was tested in four wells on a 384-well microplate. Each well contained 71

µL of 40 mM sodium phosphate buffer (pH 7.5), 5 µL of 20 mM DTNB (5,5'-dithiobis-(2- nitrobenzoic acid)), 9 µL of 1 g/L BSA (in 40 mM sodium phosphate buffer at pH 7.5, with

0.02% NaN3), and 10 µL of diluted sample. Using the BioTek Dual Reagent Injector

Chapter 4. Methods for Detecting Native Acetylcholinesterase

To determine whether these QMPs can reactivate or resurrect AChE from OP poisoning, colorimetric or photometric methods are typically used due to their quick response and simple procedure. The most common method used for in vitro assays has dominated this field of research since its discovery in 1961: Ellman’s assay.49 Using an artificial substrate, acetylthiocholine iodide (ATCI), and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a yellow color appears when native AChE is present. Like its natural substrate, AChE hydrolyzes ATCI to give thiocholine, which interacts with the disulfide bond of DTNB (Figure 40).

Figure 40 Ellman’s assay.

The resulting product, 2-nitro-5-thiobenzoate (TNB2-), forms the classic yellow color, which absorbs at 412 nm. By measuring the initial change of absorbance over time, a

65 relative percent of reactivation or resurrection can be obtained by comparing the originally poisoned enzyme to a positive control (AChE never exposed to an OP). Despite its common usage, Ellman’s assay has some drawbacks: 1) DTNB is light sensitive, 2) ATCI can hydrolyze spontaneously, 3) oximes can react with both ATCI and DTNB, and 4) hemoglobin absorbs in the same range. If TNB2- is not formed naturally through the hydrolysis of ATCI by AChE and subsequent displacement of TNB- by thiocholine, the absorbance would give a false, positive reading. Using a fresh batch of Ellman’s assay can avoid half of these problems. However, many reactivators contain oximes which can hydrolyze ATCI (oximolysis) or break the disulfide bond of DTNB directly. In addition to side reactions, hemoglobin would have to be removed from a blood sample before proceeding with Ellman’s assay; otherwise, the absorption of TNB2- and hemoglobin would overlap, resulting in an indistinguishable reading. After dominating the realm of

AChE research, Ellman’s assay needs to be conquered by a new method of detecting native

AChE which is unreactive with oximes and allowing detection in whole blood samples.

Before the discovery of Ellman’s reagent, DTNB, manometric and titration methods were used by measuring carbon dioxide (CO2) pressure or pH changes, respectively.50 Allowing the hydrolysis of ACh by AChE to occur in hydrogen carbonate

51 buffer, the byproduct, acetic acid, reacts with the buffer to produce 1 equivalent of CO2.

By using a manometer, the pressure of CO2 can be measured and related back to the amount of ACh hydrolyzed.52 Similarly, a change in pH can be monitored as acetic acid forms from the hydrolysis of ACh or by keeping the pH constant through the titration of a base.51,52

The rate of addition of base is a direct comparison to the rate of hydrolysis.52 Although the

66 calculated enzyme activities were comparable, photometric methods, such as Ellman’s assay, were more precise than manometric or titration methods.

In addition to its combination with DTNB, ATCI can used with other compounds to produce photometric or fluorometric results (Figure 41).50,51

Figure 41 Other compounds paired with ATCI: 6,6'-disulfanediyldinicotinic acid (DTNA, 44), dibromobimane (45), 7-(4-(2,4-dinitrophenylsulfonyl)piperazin-1-yl)-2- oxo-2H-chromene-3-carboxylate (46).

Similar to DTNB, 6,6'-disulfanediyldinicotinic acid (DTNA, 44) reacts with the product of

ATCI hydrolysis, thiocholine, to produce a new disulfide bond and a thiol, which absorbs at 340 nm. Unfortunately, 44 still contains a disulfide bond which could be broken in the presence of an oxime. Dibromobimane (45) reacts with ATCI to form a highly-fluorescent bimane thioether (excitation: 356 nm; emission: 484 nm),53 while the coumarin-derived probe (46) excites at 400 nm and emits at 460 nm upon removal of dinitrophenylsulfonyl by ATCI.54 Unfortunately, these compounds also absorb (300-700 nm)55 or fluoresce in the

67 same region as hemoglobin, but, thankfully, the emission of hemoglobin is difficult to detect through a one-photon excitation (Figure 42).56

Figure 42 Two-photon excited fluorescence emission spectra (excitation: 800 nm) for red blood cells, erythrocyte ghosts, and reagent-grade fluorophores (image reproduced from Ref. 56).

Alas, these methods continue to use ATCI, so the threat of false, positive readings due to oximolysis is still relevant.

There are some detection methods which do not use ATCI or DTNB but, instead, contain an acetate group susceptible to hydrolysis by AChE. With indoxylacetate (47), a non-fluorescent molecule, two molecules of the hydrolyzed product couple to form indigo

(Figure 43).57

Figure 43 Hydrolysis of indoxylacetate and formation of indigo.

At pH 7 or less, indigo white is formed, which is a highly fluorescent compound with an excitation wavelength of 395 nm and emission of 470 nm.52 Unfortunately, over time and at a higher pH, indigo white is oxidized in air to form the non-fluorescent indigo blue.

Although indigo blue does not fluoresce, it does produce an intense absorption band at 670 nm.57 Unlike ATCI, oximolysis does not occur with 47.

Upon studying the regiochemistry of the acetate group in 48, A.K. Prince determined that the 7-position gave the most fluorescence when small quantities of AChE were used (Figure 44).58,59

Figure 44 Hydrolysis of 7-acetoxy-1-methylquinolinium iodide (48).

Between the excitation and emission wavelengths for 48 and 49 is a large Stokes shift, so both intensities can be measured without much overlap. Unlike 47, both the reactant (48) and hydrolyzed product (49) fluoresce, but, thankfully, their excitation and emission wavelengths do not overlap.

To replace the popular combination in Ellman’s assay, ATCI and DTNB, new photometric compounds must be able to bind to AChE better than ATCI (Km) and absorb more than DTNB (molar extinction coefficient, ε). A lower Km designates a high enzyme- substrate binding complex, favoring the binding of the substrate, while a high ε denotes a strong absorption of light at a particular wavelength. After the hydrolysis of ATCI, 69 compounds 44-46 couple to thiocholine to form photometric compound, but, unfortunately, the ε for both 44 and 45 are less than DTNB (Table 4).

Table 4 Binding affinity (Km) and molar extinction coefficients (ε) of acetylcholine (ACh), acetylthiocholine (ATCI), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and photometric compounds 44-48. 3 -1 -1 Compound Km (M) ε ∙10 (M cm ) ACh1 1.67*10-5 - ATCI†57 2.06*10-4 - DTNB60 - 13.6* 4461 - 10 4553 - 4.8 46 No data No data 47†57 3.21*10-3 3.9 48†58,62 0.20-0.35*104 10.3 *At 37oC †Electric eel AChE

This could make detecting the absorption or fluorescence of these compounds more difficult unless a sensitive instrument is used. Indoxylacetate (47) has a comparable Km to

ATCI, but its extinction coefficient is also lower than DTNB. The difference in Km between

ATCI and compound 48 is even greater than that of 47, and it may prove difficult to hydrolyze. This may be due to its larger, less flexible aromatic system; 48 may not be able to enter the catalytic gorge or be stabilized within the gorge easily. Unfortunately, there have been no studies on the Km or ε of compound 46.

Despite these large differences with the components of Ellman’s assay, compounds

44-48 are still of interest. Most of the values presented in Table 4 were conducted using electric eel AChE. Binding affinities are affected by interactions between the substrate and the enzyme’s amino acid sequence, and the different sequences of electric eel and human

AChE could affect the Km of 44-48. Each of these substrates will be combined with human

AChE at varying concentrations in order to determine a Km and Vmax through Michaelis-

Menten kinetics. An assortment of AChE isoforms (recombinant human AChE with exons

1-4 from Dr. Zoran Radić and CPERL’s histidine-tagged readthrough AChE) and samples

(Dr. Steven Ratigan’s recombinant AChE and AChE extracted from whole blood) will also be tested with these new compounds and Ellman’s assay.

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