ABSTRACT Angiotensin-converting enzyme (ACE) plays a well-established role in regulating blood pressure in the periphery. ACE is also expressed in the brain, with high levels in the striatonigral pathway formed by medium spiny projection neurons that express the Drd1 dopamine receptor (D1-MSNs). We have found that ACE degrades an unconventional enkephalin heptapeptide, Met-enkephalin-Arg-Phe (MERF), in the nucleus accumbens of mice. Captopril, a prototypical ACE inhibitor used to manage hypertension, enhances extracellular levels of MERF in the nucleus accumbens. The resulting enhancement of mu opioid receptor activation by MERF causes a cell type-specific long-term depression of glutamate release onto D1-MSNs. This mechanism of action has great therapeutic potential, as our preliminary data indicate the decrease in excitatory drive to D1-MSNs can diminish the rewarding effects of fentanyl. However, neither captopril nor modern ACE inhibitors routinely used in the clinic have been optimized to regulate endogenous opioid signaling. For example, MERF may be specifically degraded by the catalytic N-domain of ACE, whereas most clinically-approved ACE inhibitors have greater selectivity for the catalytic C-domain of ACE. There is thus a need to identify optimal chemical entities that inhibit MERF degradation by ACE in brain tissue. The goal of this project is to identify new compounds that inhibit ACE and regulate endogenous opioid signaling in the nucleus accumbens. In AIM 1, we will screen a selected library of ~2,000 small molecules for domain-selective inhibition of recombinant ACE protein. This will include candidate molecules drawn from rationally selected libraries using a pharmacophore-based approach, as well as molecules with diverse chemical scaffolds identified through a virtual screen. In AIM 2, we will validate the domain selectivity and efficacy of lead compounds on MERF degradation in nucleus accumbens tissue. This will include direct measurement of extracellular MERF using liquid chromatography- tandem mass spectrometry, as well as whole-cell voltage-clamp recordings to measure functional effects on excitatory input to D1-MSNs. These experiments will include knockout mice to test the necessity of each catalytic domain of ACE in MERF degradation, as well as the involvement of mu opioid receptor signaling in physiological changes. In AIM 3, we will evaluate the ability of lead compounds to attenuate fentanyl reward and reinforcement. This will include measurement of fentanyl-conditioned place preference and intravenous self- administration of fentanyl. At the conclusion of these experiments, we expect to identify chemical motifs that may serve as leads to generate novel ACE inhibitors that block MERF degradation in the nucleus accumbens. We may observe a double-dissociation between catalytic domains of ACE that degrade angiotensin (C-domain) and MERF (N-domain), raising the exciting possibility of selectively manipulating endogenous opio...