Acetylcholinesterase Complex Protein-Protein Interactions as Drug Targets Against Organophosphate-induced Neurotoxicity.

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Project Summary. This administrative supplement will introduce environmental health sciences research to a summer high school intern. Organophosphate (OP) insecticides (e.g., Naled, parathion) and chemical weapons (e.g., sarin, VX, Novichok) are robust inhibitors of acetylcholinesterase (AChE) triggering cholinergic crises resulting in muscle contractions, seizures, and in extreme exposures death. Certain OPs can rapidly (within minutes) irreversibly inhibit AChE, producing an “aged” species refractory to oxime reactivators, like 2-pralidoxime (2-PAM), which is part of the OP treatment in the United States and whose efficacy has recently been questioned. Consequently, there is an urgent need to discover novel approaches to rescue AChE activity following OP exposure to mitigate the damaging neurological and muscular effects. The long-term goal of the proposed research is to develop viable, lasting treatments for acute and chronic OP exposures. Previous efforts to rescue aged AChE have focused on producing new reactivators that will alkylate the phosphorylated enzyme allowing oximes like 2-PAM to recover enzyme function, but these compounds have shortcomings such as insufficient blood-brain barrier permeability and limited efficacy in vivo. The proposed research is innovative because it employs strategies aimed at increasing the turnover aged AChE and recovery of nascent intracellular AChE. For example, inhibiting or loss of muscle-specific protein kinase (MuSK) destabilizes the AChE complex leading to the degradation of the enzyme. Recent studies demonstrate that Dok7, an adaptor protein in the AChE complex, is necessary for MuSK activity and more importantly, AChE localization on the muscle surface. Therefore, the current research objective is to assess whether targeting proteins in the synaptic AChE complex can increase AChE turnover and restore optimal AChE activity. The central hypothesis in pursuit of this objective is that inhibiting or degrading proteins associated with aged AChE will cause the release of the aged enzyme, making way for new AChE to repopulate the synapse, restore optimal neurotransmission, and mitigate the effects of OP exposures. The rationale for the proposed research is that eliminating aged AChE will alleviate the detrimental effects of OP toxicity and restore proper neurotransmission. Based on published and preliminary studies, the high school intern will investigate elements in Specific Aim 1 of the parent grant: Evaluate the in vitro therapeutic benefit of targeting proteins in the AChE complex for degradation. In the proposed project, the high school intern will use small RNAs to diminish the protein levels of Dok7. Similarly, the student will use a retro-inverso peptide mimicking the Dok7- MuSK interaction site to destabilize the protein-protein interaction, which we hypothesize will result in the release of aged AChE These studies will determine the druggability of Dok7 and determine if it will be evaluated as drug discovery studies. This research is significant because it will provide fundamental mechanistic insights into the stability and turnover of post-synaptic AChE complexes and unearth new targets to treat OP exposures.