Multiparametric analysis of a high-content ultra-high-throughput screen identifies inhibitors of the intramembrane protease SPPL2a

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The intramembrane protease SPPL2a (signal peptide peptidase-like 2a) is a potential drug target for the treatment of autoimmune diseases due to its essential role in B cells and dendritic cells. To screen a library of 1.4 million compounds for inhibitors of SPPL2a, we developed an imaging assay detecting nuclear translocation of the proteolytically released cytosolic substrate fragment. The state-of-the-art hit calling approach based on nuclear translocation resulted in numerous false positive hits, mainly interrupting intracellular protein trafficking. To filter the false positives we extracted 340 image-based readouts and developed a novel multiparametric analysis method which successfully triaged the primary hit list. The identified scaffolds were validated by demonstrating activity on endogenous SPPL2a and substrate CD74/p8 in B cells. The multiparametric analysis discovered diverse cellular phenotypes and provided profiles for the whole library. The principle of the presented imaging assay, the screening strategy and multiparametric analysis are potentially applicable in future screening campaigns.

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