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

项目总结。

Project Summary. Organophosphate (OP) insecticides (e.g., chlorpyrifos, Naled, parathion) and chemical weapons (e.g., sarin, VX, Novichok) are potent inhibitors of acetylcholinesterase (AChE) that can trigger cholinergic crises presenting as muscle contractions, seizures, and in extreme exposures death. Particular OP compounds like parathion and sarin can rapidly (within minutes) irreversibly inhibit AChE, resulting in an “aged” enzyme that is refractory to existing AChE reactivators, namely 2-pralidoxime (2-PAM), which is part of the current OP treatment in the United States. Current strategies to reactivate aged AChE have shortcomings such as insufficient blood-brain barrier permeability and limited efficacy in vivo. Consequently, there is an urgent need to identify and refine novel approaches to rescue AChE activity following OP exposure to mitigate the damaging effects of OPs. The long- term goal of the proposed research is to develop viable, lasting treatments for acute and repeated OP exposures. Previous efforts to rescue aged AChE have focused on producing new classes of reactivators that will alkylate the phosphorylated enzyme allowing them to react with oximes like 2-PAM to recover enzyme function. 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. 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 hypothesis will be investigated by undertaking the following specific aims: (1) Evaluate the in vitro therapeutic benefit of targeting proteins in the AChE complex for degradation, and (2) Examine the impact of acute MuSK inhibition on aged AChE levels and enzyme activity. In the first aim, Pz-1, a known inhibitor of MuSK shown in preliminary studies to induce turnover of aged AChE, will be used to treat Long-Evans rats exposed to diisopropyl fluoride (DFP), an OP known to age AChE quickly. Measures of DFP neurotoxicity, AChE levels, and cholinesterase activity recovery will be used to determine Pz-1 efficacy. In the second aim, individual proteins comprising the AChE will be selectively targeted for degradation using pharmacological approaches to assess the contribution of each protein to aged AChE stability in human cells, so that individual complex components may be evaluated as drug targets in future 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.