Project Abstract Opioid-related overdoses account for almost half of all drug overdose deaths in the United States and cause more preventable deaths every year than car crashes. Fentanyl, a highly potent mu opioid receptor (MOR) agonist, and its analogs (fentalogs) are increasingly found cut into illicit drug samples, both where the primary drug of abuse is an opioid and in cases where it is not. The prevalence of fentalogs in the illicit drug market is thought to be the primary driver of increased opioid-related overdose deaths since 2016. The standard opioid overdose rescue therapy, naloxone is often insufficient to reverse opioid overdoses caused by fentalog agonists under current treatment paradigms. It has been reported that naloxone is either not potent enough or has too short a duration of action to effectively reverse fentalog overdose and resuscitate patients. The objective of this proposal is to design novel opioid antagonists on the fentanyl scaffold based on previously identified fentalog antagonists and improve their pharmacokinetic properties (limited metabolism, high blood-brain penetration, and rapid absorption) relative to naloxone. We propose that the fentanyl scaffold is a good starting point as fentanyl and its analogs already bind tightly to MOR and display rapid onset of action in vivo. The overarching hypothesis is that antagonists on the fentalog scaffold will be more effective than naloxone in blocking opioid overdose. We will explore this hypothesis in two aims: 1) we will examine the ability of two lead fentalog antagonists to block fentanyl induced antinociception and respiratory depression and examine their distribution and metabolism in whole animal models and 2) design novel metabolically stable analogs which we will characterize both in vitro and in vivo. Overall, this project will develop new fentalog antagonists for the treatment of opioid overdose and characterize their in vitro and in vivo pharmacological properties.