Multiple Sulfatase Deficiency (MSD) is a yet incurable disease characterized by a combined loss of all intracellular sulfatase activities. This loss results from mutations in the SUMF1 gene encoding the formylglycine-generating enzyme (FGE). FGE drives the posttranslational activation of sulfatases and displays impaired function in MSD. For a variety of different rare diseases, high throughput screening approaches of repurposing collections are a feasible approach to identify potential therapeutic agents amongst approved drugs or late-stage drug candidates for future therapies. We established a miniaturized arylsulfatase A (ARSA) activity assay using an immortalized MSD patient derived fibroblast cell line and used restored ARSA activity as primary readout. We successfully screened a library of 800 FDA-approved drugs and identified effective drugs, one of which we studied in detail and that proved its effect further secondary assays including analysis of different sulfatase activities, lysosomal size and positioning as well as glycosaminoglycan in multiple primary MSD cell lines with different SUMF1 mutations. Our results led to a US patent application (No. US20210100756A1 and WO2021064159A1) and a manuscript is currently under revision at EMBO Molecular Medicine. In our project we propose to further develop and optimize the existing MSD-targeting ARSA assay towards miniaturization and automation (384-well microplate format compatibility), screen a library of 5600 substances (Fraunhofer Repurposing Library) with and without the already discovered drug to identify synergistic effects. We will follow up on hits using a systematic experimental program to determine additional bioactivities and annotate their impact on transcriptomic, (phospho-) proteomic, morphological and viability characteristics, including hiPSC-derived neuronal precursor cells (NPCs) from MSD patients.