Characterization of Small Molecule Therapeutic Agents for a Lysosomal Leukodystrophy

ABSTRACT Krabbe disease, known as globoid-cell leukodystrophy (GLD), is a classical neurological lysosomal storage disease (LSD) caused by a genetic deficiency of b-galactocerebrosidase (GALC), lysosomal hydrolase responsible for removing galactose residues of galactocebroside and galactosylsphingosine, mostly known as psychosine. This sphingolipid is increased in brain of patients with GLD. Psychosine is physiologically present in most brain cells at low concentrations, however, at high concentrations, it becomes extremely cytotoxic, especially oligodendrocytes resulting in diffuse demyelination and subsequent devastating leukodystrophy. Given the rapidly progressive nature of the neurological manifestations in GLD, small molecule therapies, which are more likely to cross the blood-brain barrier (BBB), become an attractable therapeutic approach. Previously, from newborn brain cortices of GLD murine model, we established a specific brain-derived cell line that recapitulates cellular phenotypic aspects of GLD, in particular the increased psychosine levels, not observed in GLD primary cells. In 2 different small molecule screenings, we identified 2 classes of potential therapeutic molecules for GLD. First, in the parent grant, we developed and implemented a live cell-based throughput LC-MS/MS assay for psychosine and identified small molecules that reduce its levels to almost normal physiological range. Second, expressing a common misfolded GALC-G270D in a brain-derived line, we implemented another cell-based high-throughput screening (HTS) screening and identified molecules able to assist the folding and increased the mutant GALC activity. I propose to test the hypothesis that the psychosine- reducing and GALC-folding assistant molecules are therapeutic agents that reduce psychosine levels resulting in the arrest and even prevention of the rapid demyelination in GLD. Based on our previous studies in GLD and our expertise assembled, our goals are to use induced-neural stem cells (iNSC) from patients with wide GLD spectrum and examine and prioritize the small molecule ?hits? that efficaciously reduce psychosine levels; (ii) perform SARs and generate different analogs to optimize their efficacy and improve their access to the central nervous system (CNS); (iii) examine selected small molecules agents in the murine GLD models monitoring established neurobehavioral outcomes and BBB penetration to prioritize the compounds for further studies. Due to initial encouraging results of hematopoietic stem cell transplantation (HSCT), newborn screening (NBS) for GLD has been implemented in several states. However, the HSCT is unable to prevent later onset neurological symptoms 8. As GLD is currently screened in all newborns in several states, the validation of small molecule candidates proposed in this project becomes highly important and urgent. Therefore, a burgeoning number of asymptomatic newborns are now, and will be, diagnosed with GLD throughout these state NBS programs. Most, if not all, of them will eventually benefit from the small molecule therapies coming from the proposed project.