Project Summary/Abstract Chronic pain is a debilitating condition that is widely treated with prescription opioid drugs over extended periods of time. The high prevalence of opioid addiction with abuse and death is now a severe national crisis in the US. In Jordan, opioid analgesics are highly restricted in their use, and many people suffer from untreated pain. To address these unmet needs, the goal of this project will be to discover novel natural products that are potent BBB permeable inhibitors of cathepsin L to produce a lead candidate drug molecule(s) that reduces spinal dynorphin and attenuates chronic pain without addiction. This goal is based on compelling evidence showing that spinal dynorphin is a key mediator of chronic pain, and cathepsin L is largely responsible for the production of dynorphin from its inactive prodynorphin. These findings support the hypothesis that inhibition of cathepsin L will lead to reduction of dynorphin and alleviation of chronic pain. An important dimension of this project will be to enrich for BBB permeable natural products and plant extracts early in the discovery process using an in vitro-parallel artificial membrane permeability assay (PAMPA-BBB) in a CNS-targeted workflow. For the purpose of screening, we will choose three distinct sets of natural product extracts and pure compound libraries from various sources and regional areas, thereby enhancing the diversity in this project. The University of Jordan (JU), UC San Diego and the NIH-DTP Repository will be subjected for initial screening for their cathepsin L inhibitory activity at concentrations from 1-10 µg/mL. Active crude extract materials from these screenings will be subjected to the PAMPA-BBB assay. Permeable eluents and impermeable retentates from this assay will be evaluated for cathepsin L activity and profiled by LC-MS/MS metabolomics. Finally, focused/targeted isolation and structure elucidation efforts will be done on fractions that show several positive selection criteria: promising biochemical activity against cathepsin L, BBB+ in the PAMPA permeability assay, novel structural features compared to known cathepsin L inhibitors, and a molecular weight <500. The target fractions will be purified by HPLC, and the pure product structures will be elucidated using the AI-based tools, Small Molecule Accurate Recognition Technology (SMART) and DeepSat. The collaborative effort involving the Almaliti laboratory in Jordan and the Gerwick laboratory in La Jolla offers a remarkable chance to bolster the University of Jordan's staff capabilities and elevate their expertise in discovering natural products in a region renowned for its historical use of plants as traditional remedies. These training initiatives in both La Jolla and Jordan will be further enriched by hosting a natural products symposium in Jordan during the project's second year. Future collaborations will be established to obtain additional funding and expand this work to other projects...