Abstract There is a significant need for new treatments for opioid use disorder (OUD) to manage patients abusing heroin, fentanyl, oxycodone and related drugs. Buprenorphine and methadone are both effective medications for OUD, but are agonists with potential for abuse and risk of respiratory depression. Moreover, they are not effective in relapse situations. The antagonist naloxone is an alternative that does prevent relapse, but has very poor patient compliance due to its complete blockade of the mu-opioid system. We have discovered compounds that act as antagonists of the mu-opioid receptor (MOR), by a non-competitive mechanism. Such compounds are allosteric antagonists also called NAMs or Negative allosteric modulators and reduce the activity of MOR agonists in a fashion that is non-surmountable and that does not completely shut down the receptor, thereby providing the ability to reduce opioid actions in dependent individuals, potentially without negative side-effects of competitive antagonists like naloxone. Our long term goal is to identify NAMs as an alternative, non-agonist treatment for OUD. Based on our previous work with positive modulators it is likely that NAMs will also: a) show “probe dependence”, such that it could be possible to block opioid drugs without affecting the activity of the endogenous opioid peptides and b) show bias, by blocking certain pathways downstream of MOR associated with the rewarding and respiratory depressant-like actions without preventing analgesia. Our long term hypothesis is that NAMS of MOR could be developed as effective non-agonist treatments for OUD by directly targeting the site of action of abused opioids in a novel way. The objective of the current application is to develop our original lead molecule to obtain a NAM for MOR that has high affinity and IP potential. We will 1) Identify novel, improved NAMs by chemistry with HTS and 2) Determine the mechanism of NAM action and demonstrate probe dependence and bias as well as perform preliminary proof of concept studies in vivo. Completion of this project will initiate preclinical studies on the potential of NAMs for the management of OUD.