Abstract Fentanyl’s high potency, facile synthesis, abuse liability, and propensity to cause profound respiratory depression via the mu opioid receptor (MOR) have combined to create the opioid epidemic. Fentanyl now accounts for ~80% of opioid overdose deaths and is predicted to contribute to more than 1.2 million deaths during the current decade. The rapid onset of fentanyl-induced respiratory depression requires a fast-acting therapy. For practical purposes, this needs to be a pharmacological treatment. The primary pharmacological treatment for opioid overdose is naloxone. Naloxone is a competitive orthosteric antagonist of MOR and thus its effectiveness depends on the dose of naloxone given, naloxone’s affinity for MOR compared to the opioid, and the rate at which the opioid leaves the receptor. While naloxone can be lifesaving, there are numerous examples of naloxone failing to reverse an opioid overdose, emphasizing the importance of improving the pharmacological treatment of opioid overdose. One way to improve naloxone’s efficacy at MOR is by using a negative allosteric modulator (NAM) of MOR. NAMs decrease agonist signaling by either increasing the rate at which the agonist leaves the receptor or by decreasing the efficiency of signaling. Either could be beneficial combined with naloxone to reverse an opioid overdose. Interestingly, cannabidiol (CBD) is a low potency MOR NAM. In preliminary experiments, we screened a CBD analog library for their ability to reverse fentanyl-mediated inhibition of adenylyl cyclase (AC). We identified potent NAMs that reversed fentanyl inhibition of AC and fentanyl analgesia, but they failed to prevent fentanyl induced respiratory depression or enhance naloxone reversal of respiratory depression. These results suggest that pathways other than MOR inhibition of AC mediate fentanyl-induced respiratory depression. Thus, in the proposed work, our first goal is to screen our candidate NAMs for attenuation of fentanyl/MOR signaling across a broad range of signaling pathways with the goal of identifying CBD analogs that inhibit MOR signaling pathways in addition to AC. Even though our potent MOR NAMs didn’t decrease respiratory depression, an important second goal is to determine if they affect other key behaviors mediated by fentanyl/MOR: reward, dependence, and tolerance. We will address these two goals with two specific aims. Specific Aim 1. Screen the CBD analog library for attenuation of fentanyl/MOR modulation of the following pathways: adenylyl cyclase, GIRK, MOR internalization, ERK1/2, phospholipase C, and arrestin. Specific Aim 2. Determine the ability of the novel MOR NAM, JGC8, to attenuate key fentanyl/MOR-mediated behaviors: reward, dependence, alleviation of neuropathic pain, and tolerance. We feel Aim 1 will likely identify compounds with unique signaling profiles. Aim 2 will determine if the already identified MOR NAM, JGC8, affects therapeutically important fentanyl-elicited behaviors and, combined...