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P14023
Identification of Novel Therapeutics for Treatment of Huntington’s Disease – a Phenotypic Screening Cascade
Presenter Wolfgang Fecke, Siena Biotech SpA, Italy
Additional Authors: Freddy Heitz, Salvatore La Rosa, Cesare Federico, Georg C. Terstappen

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by an expansion of multiple polyglutamines (>35) at the N-terminal part of the large protein huntingtin (HTT). Expression of mutant huntingtin (µHTT) induces proteolysis at the 5’end, conferring to the polyQ stretch the capacity to aggregate and ultimately cause cytotoxicity in diverse cell types.
At Siena Biotech, we aim at identifying small molecules which inhibit the aggregation process and therefore should be able to slow, halt or reverse disease progression. To this end, we have implemented a screening cascade which initiates with a primary screen pathway assay based on inducible expression of full-length µHTT in a 293/T-Rex cell model. The assay principle is the sequestration of the transcription factor CBP by polyQ-formed aggregates, which in turn reduces the expression of a CRE-Luc reporter gene. Briefly, addition of doxycyclin induces µHTT expression, followed by formation of aggregates and measurement of reporter activity after 60 hours. Reference compounds, which have been shown to inhibit aggregate formation, are reversing the µHTT-induced decrease of reporter activity. The assay was validated in a HTS format with a signal window of two, a z’ factor between 0.4 and 0.9 and signal stability for at least 25 passages. A diverse library of more than 24.000 compounds was screened, and several hit molecules with specificity for induced µHTT expression could be identified. Hit compounds are being profiled in assays using toxicity and high-content screening readouts, and details of these assays will be presented. Finally, a strategy for identification of the molecular targets from the pathway screening approach will be shown.