Human plasma platelet activating factor acetylhydrolase engineered to be a catalytic bioscavenger for organophosphorus compounds
Date
2024
Authors
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Publisher
University of Delaware
Abstract
Chemical weapons have cast a long shadow across human history in warfare, and more so in the modern age when organophosphorus compounds (OPs) can have dramatic lethal effects on humans, especially to unprepared or underequipped civilians. Inhibition of acetylcholinesterase by OPs creates a cholinergic crisis which culminates in death by respiratory arrest. Traditional chemotherapeutics can prevent death if given in a timely manner, however, their actions do not directly prevent nerve agent poisoning but attempt to treat the physiological responses. Alternative treatments like catalytic bioscavengers, enzymes which degrade the nerve agent molecules directly before reaching their target tissues, have shown promise in not only preventing death but also in alleviating cognitive deficits and reducing patient recovery times. Although in principle, many potential catalytic scavengers can be developed using existing or modified proteins, current efforts have been focused almost exclusively on human butyrylcholinesterase and other human plasma proteins. Paraoxonase (PON) 1 is one such enzyme anchored to plasma lipoproteins. Research into PON1 as a catalytic scavenger has focus on a chimeric form developed at the Weizmann Institute of Science (Israel) for enhanced expression and secretion in bacteria. While this is helpful in establishing the platform for multiple laboratories worldwide, it creates an immunological problem downstream in development when delivering a protein drug to humans. To this end, we started with human PON1 as our platform and translated successful mutations from the chimera PON platform to our human PON. Unique mutations for improved catalysis against bona fide organophosphorus nerve agents were attempted. We substituted a tryptophan near the putative active site residues (F347W) for enhanced substrate binding and reduced a bulky group at the periphery (Y71A) to encourage substrate promiscuity. By remaining focused on translational mutations from the chimera to the human PON platform, we sought to reduce the potential for immuno-clearance prior to clinical safety trials. Ultimately, the human PON mutants could not maintain the apparent catalytic efficiencies against OP nerve agents observed in the chimera PON platform. Our attention turned towards phospholipase A2s from human plasma and brain since these enzymes shared similar catalytic mechanisms to butyrylcholinesterase. Crystal structures from human plasma group-VIIA phospholipase exhibited non-aged complexes with OP nerve agents and insecticides which is beneficial for catalytic scavenger development. Human brain group-VIII phospholipase A2 also produced non-aged crystal structures. Both enzymes showed limited stereo preference for binding the more toxic OP stereoisomers (typically P(-)) which is preferential for developing catalytic bioscavengers. We are hopeful that discoveries on these platforms will provide protection in both the peripheral (plasma) and central (brain) compartments. Having the protein platform native in each compartment would potentially minimize aggressive immuno-clearance. A catalytic scavenger in the brain would need to have a KM for OP hydrolysis at or near the association constant of acetylcholinesterase for the OP. Human plasma platelet activating factor acetylhydrolase mutant W298H developed catalytic power against the OP nerve agent soman and transformed from being the victim of OP inhibition to the aggressor for OP destruction. To evaluate the significance of our W298H mutant, we must consider the catalytic bioscavenger power needed to overcome lethal concentrations of nerve agents. Recent work has demonstrated that an engineered PON1 needs a kcat /KM of 5 x 107 M-1 min-1 to provide protection to mammals against a lethal dose of OP nerve agents with no other administered therapeutics. This is substantially higher than the kcat /KM for W298H at 8 x 103 M-1 min-1. However, from a historical perspective, human wild type (WT) PON1 has limited catalytic power against G-type OPs and yet through directed evolution, researchers have been able to optimize catalytic efficiency from <198 M-1 min-1 to >1.7 x 107 M-1 min-1 in an evolved chimeric platform. Our success in plasma PAF-AH W298H is just a start and opens the door for future protein engineering projects including a directed evolution approach and exploring substrate promiscuity. Preventing the effects of chemical weapons either on the battlefield or against civilian targets through the advancement of new medical countermeasures stands not only as a moral imperative for mankind but as a paramount concern for global security.
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Keywords
Bioscavengers, Molecular models, Nerve agent, Organophosphorus compound, Soman