Summary- Significant progress was made on this project, which involves the preclinical development of candidate medications for treating stimulant use disorders. Six relevant papers were published in peer-reviewed journals over the last year. Three articles describe the pharmacology of potential agonist medications acting at monoamine transporters, whereas two other articles report the pharmacology of potential psychedelic medications acting as agonists at serotonin-2A receptors (5-HT2A). One article is a multi-authored scholarly review which explores the utility of psychedelics for treating chronic pain. Emerging evidence suggests the psychedelic-assisted therapy might be useful for treating substance use disorders, including stimulant dependence. In a key original research publication, our team characterized the biological effects of naturally-occurring compounds found in psychedelic mushrooms, as well as a number of synthetic structural analogs. It is well known that the tertiary amine compound, 4-phosphoryloxy-N,N-dimethyltryptamine (psilocybin), is a mushroom constituent and "prodrug" that is converted to its active metabolite, 4-hydroxy-N,N-dimethyltryptamine (psilocin), in vivo. However, there are other psilocybin-like compounds found in mushrooms, such as the secondary amine, baeocystin, and the quaternary ammonium, aeruginascin, that are not well studied. We carried out experiments to investigate structure-activity-relationships for psilocybin, baeocystin, and aeruginascin, as compared to their 4-acetoxy and 4-hydroxy analogs, using in vitro and in vivo methods. Broad receptor screening using radioligand binding assays revealed that secondary and tertiary tryptamines with either 4-acetoxy or 4-hydroxy substitutions display nM affinity for most human 5-HT receptor subtypes, including the 5-HT2A and the serotonin 1A receptor (5-HT1A). The same compounds displayed affinity for 5-HT2A and 5-HT1A in mouse brain tissue in vitro and exhibited agonist efficacy in assays examining 5-HT2A-mediated calcium mobilization and beta-arrestin 2 recruitment. In mouse experiments, only the tertiary amines psilocin, psilocybin, and 4-acetoxy-N,N-dimethyltryptamine (psilacetin) induced head twitch responses (ED50 = 0.11 - 0.29 mg/kg) indicative of psychedelic-like activity. Head twitches were blocked by 5-HT2A antagonist pretreatment, supporting 5-HT2A involvement. Both secondary and tertiary amines decreased body temperature and locomotor activity at higher doses, effects which were blocked by 5-HT1A antagonist pretreatment. Across all assays, pharmacological effects of 4-acetoxy and 4-hydroxy compounds were similar, and these compounds were more potent than their 4-phosphoryloxy counterparts. Results from this study demonstrate that baeocystin and aeruginascin do not induce psychedelic-like head twitches in mice, indicating these compounds do not contribute to psychedelic effects of psilocybin mushrooms in humans. The data also show that only tertiary amine derivatives of psilocybin retain in vivo activity. Finally, the 4-acetoxy analog of psilocybin, psilacetin, appears to be a prodrug for psilocin, suggesting this compound could be used as an alternative to psilocybin. Future studies will explore the in vivo conversion of psilacetin to psilocin, as a means to characterize the potential medical value of the compound.