PROJECT SUMMARY Canavan disease (CD) is an autosomal recessive inherited leukodystrophy caused by the mutations in ASPA gene encoding the aspartoacylase enzyme, leading to loss of enzyme activity and increased concentrations of its substrate N-acetylaspartate (NAA) in the brain. The abnormally high levels of NAA result in spongiform degeneration of white matter, aberrant myelination, brain edema, macrocephaly, and severe cognitive and motor deficits. Currently, there is no cure or disease-modifying treatment for CD. There is an urgent need for effective therapies for patients with CD. Aspartate N-acetyltransferase encoded by the NAT8L gene catalyzes biosynthesis of NAA from aspartate and acetyl-CoA. We and others postulated that brain-penetrant NAT8L inhibitors could reduce brain NAA levels and alleviate the NAA-driven pathology in CD. Indeed, genetic disruption of NAT8L in CD mouse model (Aspanur7nur7) was found to show strong therapeutic benefit. Currently, however, there are no small molecule NAT8L inhibitors that penetrate the blood-brain barrier. Through a large HTS screening campaign followed by preliminary medicinal chemistry efforts, however, our team identified a novel sub-micromolar and brain penetrant NAT8L inhibitor structurally distinct from previously reported inhibitors. To capitalize on this discovery, herein we propose to advance this project to the next stage, where the focus will be on optimizing the lead compound for desirable pharmacological properties (R61 Phase), enabling in vivo efficacy studies in a murine model of CD (R33 Phase). We are poised to seize this opportunity and establish in vivo proof of concept using a potent, selective, brain-penetrable NAT8L inhibitor to treat CD by executing the following three Specific Aims: (R61 Phase Aim 1) Conduct structure-activity relationship (SAR) studies on the lead NAT8L inhibitor; (R61 Phase Aim 2) Conduct in vitro characterization and in vivo pharmacokinetic/pharmacodynamic (PK/PD) profiling of potent NAT8L inhibitors from Aim 1; (R33 Phase Aim 3) Assess in vivo efficacy of the optimized NAT8L inhibitor in an established murine model of CD.