Discovery of non-opioid receptor protein targets of fentanyl across tissues from animal models and humans using photoaffinity probes

Synthetic opioids such as fentanyl and related analogs have been widely used for pain management. However, their negative side effects, including respiratory depression and high potential for addiction, underscore the need for a deeper understanding of fentanyls interactions with proteins throughout the human body. Fentanyl analogs bind and activate opioid receptors in the central and peripheral nervous systems, triggering numerous downstream signaling pathways. Increasingly, fentanyl has been shown to interact with non-opioid receptors, and elucidation of these non-canonical fentanyl-protein interactions may provide insights into the mechanisms contributing to fentanyls adverse effects and illuminate novel countermeasure strategies. To identify proteins in mammalian tissues that may interact with fentanyl, we designed and synthesized three affinity-based probes (AfBPs) that include the fentanyl core and feature a diazirine photoaffinity group and alkyne handle for click chemistry at different positions. Molecular docking simulations predicted that these AfBPs bind the mu opioid receptor similarly to fentanyl. Affinity-based protein profiling using the FA-T1 probe in vitro in tissues from six animal species identified histamine N-methyltransferase (HNMT), endophilin-B1 (SH3GLB1), fructosamine-3-kinase (FN3K), cutA divalent cation tolerance analog (CUTA), and monoamine oxidase B (MAOB) among the top proteins that bind fentanyl in multiple species and tissue types. Molecular docking of fentanyl and remifentanil with these protein structures identified putative binding sites. The interaction of fentanyl with specific proteins was empirically assessed through protein structural analyses. These findings highlight potential fentanyl-protein interactions that may contribute to the acute and long-term impacts of fentanyl exposures.