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 and drug discovery expertise of our team and is a critical step towards our goal of developing therapeutics to facilitate recovery in individuals with OUD.

PROJECT NARRATIVE Misuse of opioid drugs and opioid overdose pose major public health concerns. Existing medications for opioid use disorder (OUD) are inadequate, as evidenced by the high rate of relapse to opioid seeking and opioid- associated deaths. Here, we propose to validate the neuropeptide receptor neurotensin receptor 1 (NTSR1) as a therapeutic target in OUD and to discover small molecule probes for this receptor that may serve as the basis for novel medications for relapse prevention.