Development of a HTS Assay for a Neurological Lysosomal Disease

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DESCRIPTION (provided by applicant): Lysosomal storage diseases (LSDs) are inherited genetic conditions caused by the defect in a specific protein that is essential for lysosomal function. Krabbe disease, or globoid-cell leukodystrophy (GLD), is a LSD caused by ¿-galactocerebrosidase (GALC) deficiency, resulting in accumulation of glycosphingolipids including psychosine. High levels of psychosine are toxic to oligodendrocytes, resulting in apoptosis and subsequent demyelination, which correlates to the wide spectrum of neurodegeneration in GLD. Therefore, reducing the levels of psychosine, found physiologically at low concentrations, is an attractive approach to prevent oligodendrocyte apoptosis, which can translate in arrest or stabilization of disease progression. Hematopoietic stem cell transplantation (HSCT) prevents the fulminant neurological course of the infantile form of GLD, but fails to prevent further neurodeterioration. Small molecules are more likely to cross the blood-brain barrier and consequently can be used as agents to reduce psychosine levels in the brain. In my previous work screening FDA-approved compounds, I was able to identify and characterize small molecules as therapeutic agents for two LSDs. My central hypothesis is that specific small molecules that reduce the levels of psychosine will have significant therapeutic potential for GLD to prevent oligodentrocyte apoptosis and ultimately controlling or arresting the progression of neurological symptoms. These small molecules are most likely to be identified through cultured brain-derived cells with GALC deficiency, which show increased levels of psychosine. To test this hypothesis, I plan to: (i) develop of a robust cell-based high-throughput screening (HTS) assay to identify small molecules that reduce the elevated psychosine levels in cultured brain-derived cells from mouse model of GLD; (ii) optimize secondary assays including assays with cultured oligodendrocyte precursor cells to select small molecules from the primary screening that more effectively reduce the psychosine; (iii) perform a comprehensive evaluation of sphingolipids in cells from GLD mouse model and in induced-neuronal (iN) cells from GLD patients treated with candidate small molecules selected by HTS assay. Upon the development of the HTS and secondary assays here proposed, the implementation will be done against the NCGC Pharmacological Compound Collection, which includes several small molecules approved by several regulatory bodies. Animal models of GLD are available for testing the potential compounds including the mouse (whose cells will be used in HTS assay) and dog models. Cell-based HTS will also identify small molecules that reduce psychosine by indirect mechanisms, which ultimately collaborate to a better understanding on the pathogenesis of GLD. The screening of approved pharmacological small molecules will make the translation to clinical studies faster based on previous safety and pharmacokinetics records of the screened compounds.