ABSTRACT The number of overdose deaths caused by stimulants like cocaine and methamphetamine has increased at an alarming rate over the last decade. Yet, unlike other addictive substances, such as alcohol, nicotine, and opioids, there are currently no FDA-approved medications for stimulant use disorders, leaving millions of Americans without an effective therapeutic treatment. Growing evidence indicates that the α3β4 subtype of the nicotinic acetylcholine receptors (nAChRs) play an integral role in drug addiction and that selective inhibition of these receptors reduces the addictive properties of cocaine, methamphetamine, and other drugs of abuse. However, existing classes of α3β4 nAChR antagonists suffer from poor selectivity, are not brain-penetrant, or induce partial activation of the receptor, which hinder their clinical development. The objective of this proposal is to develop a novel class of α3β4-selective nAChR negative allosteric modulators (NAMs) based on the alkaloid aristoquinoline and demonstrate their effects in animal models of cocaine use disorder. Preliminary data generated in our lab indicate that aristoquinoline and its derivatives potently and selectively inhibit α3β4 nAChR activation and significantly reduce drug-seeking behavior in an animal model of cocaine relapse. The proposed studies will build upon these results by testing the central hypothesis that α3β4 nAChRs NAMs based on the aristoquinoline scaffold can safely and effectively reduce the addictive properties of cocaine. Aim 1 will optimize the α3β4 potency and selectivity of aristoquinoline by generating a collection of rationally designed derivatives and evaluating their affinity and functional activity. Aim 2 will locate the aristoquinoline allosteric binding site, define the mechanism of action, and identify off-target liabilities. Aim 3 will translate these results in vivo by prioritizing lead compounds with optimized drug-like properties and advancing them into preclinical safety studies and efficacy models of cocaine self-administration and relapse. This research is innovative, as it seeks to address and overcome the limitations of existing α3β4 antagonists by investigating an understudied class of nAChR ligands with a unique mechanism of action. The results from these studies will be significant because they will produce the ideal pharmacological probe for investigating the function α3β4 nAChRs in vivo and deliver promising drug leads for the treatment of cocaine use disorder.