SUMMARY Pantothenate kinase-associated neurodegeneration, PKAN, is a rare progressive neurodegenerative disorder associated with iron accumulation in the brain. The disease causes early immobility and often death by early adulthood. PKAN is caused by mutations in one of four human pantothenate kinase genes, PANK2 gene, which encodes a mitochondrial pantothenate kinase. Consistent with the clinical presentation of PKAN in humans, cell biological analyses from patient-derived cells as well as phenotypic characterization of mouse models of PKAN have demonstrated that the loss of PANK2 activity results in major metabolic, cellular and physiological defects. To date, no specific or established therapy exists for PKAN with most treatments directed towards managing symptoms and to slow disease progression. We hypothesize that activation of the human PANK3 (hPANK3) enzyme would result in stimulation of CoA production in PANK2 mutated cells and would represent an ideal treatment of PKAN. Chemical screening and subsequent medicinal chemistry optimization (SAR) of the lead chemotype identified 9 human PANK3 activators (VTAC1-9) that strongly activate hPANK3 with AC50 values in the nM range. These compounds do not affect the activity of human PANK1 or PANK2, show no toxicity against four human cell lines and one primary human cell, and have desirable functionality and solubility properties. Pharmacokinetics studies in mice with the early leads VTAC1 and VTAC2 demonstrated both plasma and brain exposure, excellent t½, and no apparent toxicity. Together these data indicate that these compounds are ideal candidates for the development of an effective and safe PKAN therapy. The goal of the parent grant and proposed research is to conduct detailed characterization of active VTACs and a library of their analogs to identify late leads that could be advanced towards future clinical development. Towards this end, we will pursue the following three specific aims. In Aim 1, we will complete current SAR on this family compounds by characterizing the biochemical activity, selectivity and physico-chemical properties of an already synthesized 29 analogs and an additional 200 compounds to be evaluated on an iterative basis. In Aim 2, we will conduct cell-based assays to identify compounds with excellent in vitro therapeutic index and can restore biological activity in pank2-deficient cells. In Aim 3, we will conduct ADME and PK analyses and evaluate the in vivo efficacy of VTAC1 and VTAC2 and new leads from Aim 1 in pank2-/- mice and SynCrepank1fl/flpank2fl/fl mice by monitoring important PKAN biological metrics including activation of the CoA metabolic pathway, iron homeostasis, mitochondrial metabolism, and rescue of PKAN-like phenotypes. The success of these studies will set the stage for future clinical evaluation of a lead activator of hPANK3 as a possible effective and safe treatment for PKAN.