Project Summary Suppressing glycogen storage with small molecule inhibitors as a therapeutic approach to Lafora disease Lafora disease is a fatal childhood-onset, progressive epilepsy caused by mutations at either the EPM2A or EPM2B genes and for which no cure is currently available. Much has been learned of the disease in the last decade that can help guide new approaches to effective therapies. A consistent feature is the accumulation of insoluble deposits, called Lafora bodies, in many tissues and, of special significance, in neurons in the brain. Lafora bodies contain polyglucosan, an abnormal glycogen-like compound that is less branched and less soluble than normal glycogen. In mouse models of Lafora disease, laforin-/- and malin-/- mice, genetic depletion or elimination of glycogen was shown to decrease the formation of Lafora bodies and to alleviate indicators of the disease. Therefore, current thinking is that the formation of Lafora bodies is causative of the disease and suppression of their formation would represent a promising approach as a therapy. In this project, we propose to reduce glycogen stores in the brain by developing small molecule inhibitors of its accumulation. Aim 1: Identification and validation of small molecule glycogen synthase inhibitors. We are searching for active site and allosteric site inhibitors of glycogen synthase by high-throughput screens (HTS) using novel assays. Positives from the above screens will be validated in vitro and analyzed by the Medicinal Chemistry Core for favorable ADME their potential to cross the blood brain barrier (BBB). Aim 2. Medicinal chemistry optimization. Prioritized hits from Aim 1 will be further validated for activity through direct synthesis and a limited set of related analogs will either be purchased or synthesized to evaluate a range of sub-structures, substituents, and/or substitution patterns for activity by the in vitro analyses described in Aim 1. Preliminary structure-activity relationships (SAR) will inform on the potential for further pre-clinical development. Aim 3: Cell-based assay of glycogen accumulation, toxicity and preliminary pharmacokinetics. We have developed two novel plate-based assays to monitor cellular glycogen levels in glycogen accumulating cell lines. The assays will be applied to evaluate and validate glycogen synthase inhibitors identified in Aim 1 and could also be developed as an alternative for HTS of compound libraries to identify novel inhibitors at the cellular level. The most promising candidates will be advanced for toxicology and pharmacokinetic analyses in mice. Aim 4: Testing inhibitors of glycogen accumulation in mouse models of Lafora disease. Compounds identified in previous Aims that show most promise will be tested in laforin-/- and malin-/- mice to assess their effects on Lafora body formation, neurodegeneration and behavioral impairment associated with the disease. Any compounds making it to this stage will be candidates for further development via NINDS drug development programs of the IGNITE/BLUEPRINT initiatives, such as PAR-15-071.