Lin, V. S. | bioRxiv Science

bioRxiv2025

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.

pharmacology and toxicology↗

bioRxiv2023

Profiling sorghum-microbe interactions with a specialized photoaffinity probe identifies key sorgoleone binders in Acinetobacter pittii

Sorghum (Sorghum bicolor) is a major food and bioenergy grass species cultivated worldwide. To promote more robust and sustainable growth of this important crop, we need a deeper understanding of the plant-microbe interactions between sorghum and soil microbial communities that benefit plant host resiliency and enhance nutrient acquisition. The release of specific metabolites from plant roots, or root exudation, drives these plant-microbe interactions, but the molecular pathways by which root exudates shape the sorghum rhizosphere microbiome require further elucidation. To investigate these complex interkingdom interactions in the sorghum rhizosphere, we developed a photoaffinity probe based on sorgoleone, a hydrophobic secondary metabolite and allelochemical produced in sorghum seedling root exudates. Here, we apply a new synthetic sorgoleone diazirine alkyne photoaffinity probe (SoDA-PAL) to the identification of sorgoleone-binding proteins in Acinetobacter pittii SO1, a potential plant growth promoting microbe derived from Sorghum bicolor rhizosphere soil. Competitive photoaffinity labeling of A. pittii whole cell lysates with SoDA-PAL identified 137 statistically enriched proteins that were complementary to a previously identified gene cluster involved in sorgoleone catabolism. Proteins identified by SoDA-PAL included a select set of putative transporters, transcription regulators, and a subset of proteins with lipid and secondary metabolic activities. We confirm binding of SoDA-PAL to a putative hydrolase in the /{beta} fold family (OH685_09420) through structural bioinformatics and in-vitro recombinant protein analysis. This photoaffinity labeling approach using metabolite-based probes can be extended in the future to proteomic profiling of complex rhizosphere microbiomes to discover genes that can be leveraged to promote beneficial plant-microbe interactions. ImportanceHere we demonstrate a photoaffinity-based chemical probe modeled after sorgoleone, a known secondary metabolite released from the roots of sorghum, can be used to dissect complicated plant-microbe interactions. Applying this probe to the sorghum-associated bacterium Acinetobacter pittii identified diverse proteins that directly interact with sorgoleone. We show that probe labeling is dose-dependent and is sensitive to competition with purified sorgoleone, demonstrating the probe is selective for protein targets that directly interact with sorgoleone. By using the probe to broadly profile proteins that interact with sorgoleone, we identified bacterial catabolic pathways, unintuitive transcriptional regulation pathways, and vital exchange mechanisms involving transporters that may be involved in sorgoleone utilization and cellular response toward this plant metabolite. We envision that this workflow will expand our understanding of the sorghum root exudate interactome and elucidate the molecular mechanisms by which specific metabolites shape the sorghum rhizosphere microbiome.

biochemistry↗