PROJECT SUMMARY Submitted in response to RFA-DA-24-063, we propose to develop novel, brain-penetrant, small molecule biased allosteric modulators (BAMs) of the neurotensin receptor 1 (NTSR1) to attenuate relapse to opioid seeking in individuals with opioid use disorder (OUD). NTSR1 is a G protein coupled receptor (GPCR) that is highly expressed in dopamine (DA)-rich brain regions and modulates brain DA signaling. NTSR1 ligands counter the effects of multiple classes of misused drugs. As a GPCR, NTSR1 signals via heterotrimeric G proteins and β- arrestin proteins. While NTSR1 has long been recognized as a promising target for the treatment of chemical addictions, development of balanced NTSR1 agonists that active both pathways is precluded by on-target side effects (i.e., hypothermia, hypotension). Our collaborative team developed a series of first-in-class β-arrestin BAMs of the NTSR1, which attenuate psychostimulant drug effects without the side effects characteristic of balanced NTSR1 activation. While NTSR1 is a well-established therapeutic target for stimulant use disorders, and its mechanism of action suggests utility that spans drug class, its validity as a target for OUD has not been rigorously established. The limited data available on NTSR1’s effect on opioid action is promising. Our data suggest that first generation β-arrestin NTSR1 BAMs act via a reward mechanism conserved across drug classes and attenuate both stimulant and opioid drug self-administration. In aim 1, we will validate NTSR1 as a drug target for the treatment of OUD with our optimized lead BAMWe will leverage a mouse model of relapse to intravenous (IV) opioid seeking, NTSR1 knockout (NTSR1-/-) mice, and our extensive knowledge of β-arrestin BAM pharmacology. Recently, we discovered that these compounds block NTSR1 signaling via some G proteins, but permit signaling via others. Because balanced NTSR1 agonists promote drug seeking, we hypothesize that this G protein permissiveness detracts from the therapeutic utility of these BAMs in OUD. In aim 2, we will discover next generation β-arrestin BAMs for NTSR1 with improved β-arrestin selectivity to test this hypothesis. We conducted comprehensive signaling characterization for a panel of ligands from our lead series. In addition, we have robust cell-based assays (and appropriate counter-screens) to reliably monitor NTSR1 activation of more than 14 transducers. Leveraging these assets, we will conduct a medicinal chemistry campaign to increase the potency and β-arrestin selectivity of BAM scaffold, with a flow scheme consisting of cell-based receptor signaling assays and early assessment of ADME, brain penetration, and central NTSR1 engagement. We have stringent criteria for second-generation leads. Compounds that match this profile will be advanced to efficacy testing in a model of relapse to IV remifentanil seeking in wild-type and NTSR1-/- mice. This multidisciplinary research plan capitalizes on the unique scientific an...