Unmasking a neurotoxic duo: a comprehensive overview of fentanyl-xylazine crosstalk in kinase pathways.

Introduction: Fentanyl is the main cause of the US overdose crisis, responsible for over half of overdose deaths, totaling around 75,000 lives each year. The contamination of the illegal drug supply with xylazine has been linked to an increase in overdose fatalities. Adding xylazine to fentanyl causes additional health issues, including tissue necrosis, more severe withdrawal symptoms, and higher overdose lethality, while also potentially reducing the effectiveness of naloxone. Therefore, understanding the molecular signaling events when fentanyl is combined with xylazine is crucial. Methods: In these experiments, we treated the human neuronal cell line SH-SY5Y with fentanyl and xylazine and conducted detailed analyses using the PamGene PamStation to measure kinase activities across over 500 pathways, thereby stratifying and identifying affected signaling pathways. Results: Our results show that fentanyl and xylazine impact kinase activity differently when either is used alone, and often, xylazine diminishes the kinase activity changes caused by fentanyl alone. We also found that the combination of these drugs uniquely increases the phosphorylation of diverse substrates compared with each drug alone. Discussion: These are the first kinome analyses performed on human cells with fentanyl and xylazine, which may uncover new therapeutic targets to counteract their harmful combined effects.