Photoaffinity and Multi-Omic Analysis of Targets Pathways Driving Synthetic Opioid-Induced Toxicity and Reversal - PROJECT SUMMARY/ABSTRACT Ultra-Potent Synthetic (UPS) opioids, including 4-anilinopiperidines such as fentanyl and carfentanil and structurally dissimilar benzimidazoles (nitazenes), are high-consequence chemicals of concern that produce rapid and severe respiratory, cardiac, and neurological toxicity. Current mu-opioid receptor (MOR) antagonists may incompletely reverse these effects, implicating non-MOR targets and pathways and highlighting the need for new post-exposure reversal/mitigation strategies. Our preliminary larval zebrafish studies identify a high-dose fentanyl phenotype characterized by profound locomotor and cardiac suppression with minimal naloxone responsiveness, while in vivo photoaffinity labeling (PAL), proteomic, kinomic, and transcriptomic studies nominate noncanonical protein and signaling networks. The objective of this focused one-year project is to establish a causal, genotype-resolved mechanistic map of fentanyl toxicity and translate prioritized naloxoneinsensitive mechanisms into testable post-exposure reversal agents. In Aim 1, we will generate and validate a stable oprm1 loss-of-function zebrafish line and use genetic loss of MOR as a mechanistic perturbation to define which acute fentanyl-induced behavioral, cardiac, protein-engagement, kinase-signaling, and transcriptional responses require MOR. In Aim 2, wild-type and oprm1-deficient datasets will be integrated to construct a mechanistic map that classifies pathways as MOR-dependent, MOR-independent, or shared/adaptive. RNA-seq signatures will undergo 3-POD and LINCS/iLINCS connectivity analysis for translational prioritization of discordant perturbagens predicted to reverse the fentanyl-associated molecular state and selected concordant perturbagens as mechanistic probes. In parallel, prioritized proteins and pathway nodes will be matched to commercially available agonists, antagonists, inhibitors, or activators. A focused set of top-ranked candidate reversal agents will then be tested as post-exposure interventions in wild-type zebrafish after fentanyl-induced suppression is established. This mechanistic-to-translational workflow will deliver a validated genetic model, an integrated mechanistic map, prioritized actionable targets and reversal agents, and proof-of-concept pharmacologic data. Although fentanyl serves as the DHS chemical-of-concern experimental anchor, the resulting framework is designed to inform subsequent target prioritization and reversal/mitigation studies across UPS opioids, including carfentanil and nitazenes.
