PROJECT SUMMARY The hypocretin/orexin (Hcrt) system is a hypothalamic neuropeptide system that is involved in behavioral arousal, metabolism, addiction, stress and neuroendocrine function. Defects in this system, either presynaptically or postsynaptically, result in narcolepsy in both humans and animals. A convergence of results led to the recognition that human narcolepsy is a neurodegenerative disorder in which the Hcrt neurons, located exclusively in the perifornical and lateral hypothalamus, specifically degenerate. Thus, narcolepsy is due to Hcrt insufficiency. The Hcrts are a pair of peptides that differentially bind the G protein-coupled receptors Hcrt receptor 1 (HcrtR1 or OX1R) and HcrtR2 or OX2R. Based in part upon the excessive daytime sleepiness (EDS) that characterizes narcolepsy, it was recognized that the Hcrt system is centrally involved in the maintenance of wakefulness, which led to the development of HcrtR antagonists for the treatment of insomnia and FDA approval of suvorexant (Belsomra?) in 2015. In contrast, current narcolepsy treatments are purely symptomatic: cataplexy is treated with antidepressants or anti-cholinergics which have undesirable side- effects, and the debilitating EDS is often treated with amphetamine or other stimulants which have abuse potential. The goal of our research is to develop HcrtR/OXR-directed narcolepsy therapeutics that are both wake-promoting and cataplexy-inhibiting. Based on the screening of UT Southwestern's ~220,000-compound library, we have developed new HcrtR/OXR agonists with novel scaffolds and carried out structure-activity relationship studies to improve their potencies. Having optimized these initial HcrtR/OXR agonist hits using an iterative process of analog synthesis, biological testing, and molecular modeling using HcrtR/OXR crystal structures, we will now determine the pharmacokinetic (PK) properties of drug-like small molecule HcrtR/OXR agonists to select the best candidates for in vivo efficacy experiments. Thus, we will conduct PK studies on each molecule for metabolic stability, intestinal permeability, plasma concentration over time, and blood-brain- barrier (BBB) permeability using a combination of in vitro and in vivo assays. We will then test the most promising compounds in series of in vivo screens, culminating in evaluation in orexin/tTA; Tet-O DTA mice, a state-of-the-art narcolepsy model in which ablation of Hcrt neurons is controlled through the tetracycline transactivator (Tet-off) system. Using this model, the effects of HcrtR/OXR agonists on arousal state will be evaluated before and after induction of Hcrt cell loss to determine whether HcrtR/OXR agonism normalizes the narcoleptic phenotype; dose-response tests will also be conducted in the DTA model. Given that the Hcrt system is centrally involved in the maintenance of wakefulness, Hcrt agonists may have a broader indication than narcolepsy, e.g., to prolong wakefulness in civilian, industrial or military situations where sustained alertness is required. In addition to these and other potential therapeutic indications, subtype-specific Hcrt agonists would be useful to help elucidate the biological functions of HcrtR1/OX1R and HcrtR2/OX2R.