Medical Countermeasures to Organophosphorus Pesticides and Chemical Warfare Agents
The central mechanism of action of organophosphorus (OP) pesticides and chemical warfare nerve agents is the covalent modification of serine within the active site of acetylcholinesterase (AChE). This modification prevents the catalytic hydrolysis of acetylcholine (ACh) at the synaptic cleft or neuromuscular junction, leading to cholinergic crisis. To further complicate the situation, an enzyme catalyzed process called aging occurs whereby the OP becomes dealkylated yielding an anionic phosphoryl group in the AChE active site for which there are currently no fielded therapeutics. The currently available therapeutics for OP exposure address the symptoms of the cholinergic crisis (benzodiazepines for seizure prevention and atropine to antagonize the muscarinic acetylcholine receptors) or reactivate inhibited (but not aged) AChE (pyridinium or bis-pyridinium oximes), but further prophylactic and therapeutic interventions are desperately needed.
Positive Allosteric Modulation of Acetylcholinesterase
If a compound could be discovered which increases the efficiency of AChE hydrolysis of ACh, such a compound could easily be incorporated into the current standard of care and provide therapeutic benefit as the remaining uninhibited AChE and the AChE reactivated by oximes would be more efficient, thereby reducing the concentration of active AChE required to prevent cholinergic crisis. If such a compound could also decreasing inhibition rates by OPs, increase reactivation rates of OP-inhibited AChE, and/or decreasing aging rates of OP-inhibited AChE, such a compound would present an incredible advancement in the standard of care for OP poisoning. An earlier screening of a 5000-compound library of drug-like compounds provided ~40 compounds that functioned as positive allosteric modulators of AChE with some of these compounds showing enhanced ACh activity by a factor of 10. Some of these compounds are also capable of protecting AChE from inhibition with OP compounds such as paraoxon (PON), as can be seen from shifts in the IC50 of PON with AChE. We continue to work towards discovering positive allosteric modulators that are capable of increasing the catalytic efficiency of AChE cleavage of ACh and with good drug-like properties. We are also pursuing compounds that are capable of decreasing the inhibition rate by OPs, increasing the reactivation rate by oximes, and/or decreasing the aging rate of inhibited AChE.
Human Metabolism of Organophosphorus Compounds
In this effort, we will evaluate the metabolism of organophosphorus (OP) compounds by human drug metabolizing enzymes using a hybrid computational and in vitro approach. Over the past 20 years, large data repositories on human drug metabolism have been formed, enabling the development of predictive computational models that can aid in predicting metabolism and drug-drug interactions from a molecular structure. Combined ligand and protein-based methods can help us predict a compound’s likely sites of metabolism, rate of metabolism, and the degree of involvement of specific enzymes. Using both computational and in vitro approaches, it will be possible to predict the likely metabolites for OPs and generate data to validate the predictions. This will enable us to improve models that can be used for predicting metabolism of new OPs. Additionally we propose evaluating the effects of known inhibitors and activators of the metabolizing enzymes and inducers of nuclear hormone receptors that affect the regulation of the enzymes responsible for OP metabolism. This could lead to additional prophylactic or therapeutic options for exposure to OP pesticides and chemical warfare agents.
The only currently approved prophylaxis, pyridostigmine bromide (PBr), has a number of drawbacks including pharmacokinetics that require frequent dosing, inhibition of acetylcholinesterase (AChE) which can lead to or exacerbate the signs and symptoms of OP exposure, and a lack of extended protection as would be required for a percutaneous exposure with extended agent influx. The newer prophylactics in development include both stoichiometric as well as catalytic bioscavengers. Stoichiometric bioscavengers, such as butyrylcholinesterase, provide a high specificity and affinity for OPs; however, they suffer from a number of issues – the most egregious of which is a high mass imbalance. Effective prophylaxis with a stoichiometric scavenger requires one molecule of protein with a mass on the order of 64 kDa per one molecule of OPNA with a mass on the order of 0.2 kDa. This mass imbalance requires massive doses of scavenger to provide adequate protections. Catalytic bioscavengers attempt to circumvent this mass imbalance problem by providing catalytic degradation of OPs, whereby each molecule of protein can degrade multiple molecules of OP. However, catalytic bioscavengers are not without their issues. Most of the catalytic bioscavengers are more effective at degrading the less toxic stereoisomers of OPs and struggle to provide broad-spectrum protection (i.e., they are typically more effective at turning over a few of the OPs, such as G-agents, but provide little turnover for others, such as VX). Additionally, the most effective and broadest spectrum catalysis comes from organophosphate hydrolase (OPH) which is a protein of bacterial origin which exacerbates the immunity issue discussed next. Both stoichiometric and catalytic bioscavengers suffer from the issue of repeat dose host immunity; the repeated dosing required by the poor pharmacokinetics discussed next, leads to a severe host immune response precluding extended protection for warfighters and first responders. Stoichiometric and catalytic bioscavengers also suffer from poor pharmacokinetics profiles which demonstrate relatively rapid clearance of the bioscavengers even when they are PEGylated or other appending groups are added to slow the clearance.
An OP vaccine that produces both abzymes for the catalytic cleavage of OPs (from haptens of transition state analogs, TSAs) as well as antibodies with high affinity binding of OPs (from haptens resembling the parent OP) for the protection against OP exposure would provide several advantages over the currently approved prophylaxis, PBr, and the current stoichiometric and catalytic bioscavengers in development. First, the antibodies would not inhibit acetylcholinesterase (AChE) as PBr does, preventing potential side effects and further inhibition of AChE upon OP exposure. Additionally, high affinity binding antibodies can provide immediate protection against a high dose of OP. Moreover, catalytic abzymes could provide continued protection against multiple or continuous exposures to OP through catalytic hydrolysis of the OP toxicant. Furthermore, because production of antibodies (whether high affinity binding or catalytic abzymes) actually utilizes the host immune system for production, repeat-dose host immunity would actually be a benefit rather than a liability as it is for other bioscavengers. The protection provided by antibodies should also be much longer lived (months to years rather than hours to days) requiring boosters much more infrequently than the dosing of PBr or other bioscavengers. Additionally, because multivalent vaccines provide additive protection, the incorporation of multiple different OP specific haptens which provide the best protection for each OP into a multivalent vaccine will allow for ideal broad- spectrum protection against OPs. Finally, this strategy, if effective, can be used for warfighters as well as for civilians, thereby providing a prophylactic solution for broad populations, including first responders.
Resurrection of Aged Acetylcholinesterase
OP exposures induce prolonged and uncontrolled excitation of the cholinergic system through inhibition of cholinesterases and in particular acetylcholinesterase. Clinically, OP poisoning is currently treated by a combination of an anti-cholinergic drug (atropine) and an oxime (2-PAM). Atropine acts as an antagonist of muscarinic acetylcholine receptors, while oximes (or more likely, their deprotonated form, oximates) nucleophilically substitute the phosphylated serine to reactivate the OP-inhibited form of AChE. AChE is an enzyme present in both the peripheral and central nervous system (CNS) where it hydrolyzes the neurotransmitter acetylcholine (ACh). The hydrolysis of ACh is accomplished by a catalytic triad consisting of glutamate, histidine, and serine which occupy the bottom of a narrow gorge within AChE. Upon phosphylation of the catalytic serine residue, known as inhibition, there is an accumulation of ACh in the brain synapses and neuromuscular junctions leading to a cholinergic crisis. Without rapid intervention upon exposure, the cholinergic crisis leads to severe symptoms and eventually death. A further complication for treatment of OP poisoning occurs due to a further process called aging, wherein AChE catalyzes the dealkylation of the OP yielding an anionic phosphylated serine which is recalcitrant to all current therapeutics. Although treatments, such as 2-PAM, exist for the reactivation of OP-inhibited AChE, they have only limited effectiveness in the CNS due to inefficient crossing of the blood-brain barrier and furthermore, no current therapeutics address the phenomenon known as aging for OP-inhibited AChE.
In this collaborative project with Christopher Hadad in the Department of Chemistry and Biochemistry, we seek to develop small molecules capable of resurrection of OP-aged AChE as well as reactivation of OP-inhibited AChE in the CNS. Our approach includes computational drug design and discovery, chemical synthesis /chemical library development, and biochemical assays to demonstrate resurrection activity as well as determine the mechanism of resurrection, which we hypothesize takes place via two distinct steps: first, realkylation of the anionic aged form back to a neutral, phosphylated (inhibited) serine residue, and then reactivation of the inhibited form back to the native AChE. Our small molecules are based upon a quinone methide precursor (QMP) framework, which we have demonstrated are both resurrectors of OP-aged AChE as well as reactivators of OP-inhibited AChE. Therefore, a single QMP therapeutic may be capable of recovering the activity of AChE no matter if the enzyme is inhibited or aged. These results are extremely promising not only because these QMPs are the first compounds ever reported to demonstrate recovery of aged-AChE, but also because they are non-permanently charged reactivators of inhibited AChE and should lead to increased recovery of both aged and OP-inhibited AChE in the CNS.