Identification of SPPL2a Inhibitors by Multiparametric Analysis of a High-Content Ultra-High-Throughput Screen

Abstract

The intramembrane protease signal peptide peptidase-like 2a (SPPL2a) is a potential drug target for the treatment of autoimmune diseases due to an 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 that 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.

Physiological functions of SPP/SPPL intramembrane proteases

“…Based on the growing interest in inhibition of SPP/SPPL protease family members especially for the modulation of the adaptive immune system but also for treatment of viral and plasmodium infections, a substantial effort was invested in the generation of small molecule inhibitors for these proteases [110][111][112]. This process significantly profited from existing compounds initially generated for inhibition of the related γ-secretase for treatment of Alzheimer's disease and leukaemia [113].…”

“…Therefore, the development of novel SPP/SPPL family and ideally, individual protease-specific inhibitors represents an absolute prerequisite for potential therapeutic use. Using several independent strategies, Novartis has generated small molecule inhibitors specifically targeting SPPL2a, which might represent an interesting target for treatment of autoimmune diseases based on its role in dendritic cells and also B cells, though the impact on the latter is not confirmed in humans [110][111][112]. For high throughput compatible assessment of compounds, these studies employed an high-content imaging assay to monitor activity of SPPL2a [110].…”

“…Using several independent strategies, Novartis has generated small molecule inhibitors specifically targeting SPPL2a, which might represent an interesting target for treatment of autoimmune diseases based on its role in dendritic cells and also B cells, though the impact on the latter is not confirmed in humans [110][111][112]. For high throughput compatible assessment of compounds, these studies employed an high-content imaging assay to monitor activity of SPPL2a [110]. This was based on a reporter cell line with stable expression of SPPL2a in combination with inducible expression of a GFP-tagged reporter construct [110].…”

“…For high throughput compatible assessment of compounds, these studies employed an high-content imaging assay to monitor activity of SPPL2a [110]. This was based on a reporter cell line with stable expression of SPPL2a in combination with inducible expression of a GFP-tagged reporter construct [110]. This comprised a membranebound fragment of TNFα, which can be directly cleaved by SPPL2a without the need of preceding ectodomain processing and was fused to GFP at its N-terminus via a nuclear localisation signal (NLS).…”

“…This comprised a membranebound fragment of TNFα, which can be directly cleaved by SPPL2a without the need of preceding ectodomain processing and was fused to GFP at its N-terminus via a nuclear localisation signal (NLS). Cleavage of this construct by the co-expressed protease results in constitutive nuclear transport of the GFP-containing cleavage fragment, whereas inhibition of the protease leads to a retention of the substrate molecule in the plasma membrane and prevents its accumulation in the nucleus [110]. This assay was employed in a multi-parametric image-based screen of a library containing 1.4 million compounds.…”