A High-Throughput Screening Strategy for Development of RNF8-Ubc13 Protein-Protein Interaction Inhibitors

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A detailed method to identify RNF8-Ubc13 PPI Inhibitors

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Are you interested in identifying specific inhibitors to disrupt RNF8-Ubc13 protein-protein interaction? A multilevel strategy, from hit identification up to hit confirmation and validation, is presented in this interesting article by Elisabeth Weber from Helmholtz Zentrum München.Ubiquitination is a highly complex enzymatic cascade that involves a sequential assembly of proteins in a highly structured scaffold. RNF8-Ubc13 interaction is a key component of the ubiquitin-proteasome system. RNF8 has recently been associated with many pathological disorders, including different types of cancers. There is enough evidence for scientists to put it under the spotlight, sufficient rationale to rank it as a potential clinical target, and ample reasons to screen for inhibitors.This is where things get complicated: specific challenges arise when for screening inhibitors that disrupt protein-protein interactions. Which assays should be implemented to monitor RNF8-Ubc13 PPI disruption? Should they use small molecules or recombinant protein competitors?Discover, step-by-step, how Elisabeth Weber designed the screening path, from HTS to secondary biochemical counter and confirmation assays, and even further to cell-based assays for assessing hit potency.Enjoy a 360° view into PPI screening strategy revolving around TR-FRET.


The ubiquitin-proteasome system plays an essential role in a broad range of cellular signaling pathways. Ubiquitination is a posttranslational protein modification that involves the action of an enzymatic cascade (E1, E2, and E3 enzymes) for the covalent attachment of ubiquitin to target proteins. The emerging knowledge of the molecular mechanisms and correlation of deregulation of the ubiquitin system in human diseases is uncovering new opportunities for therapeutics development. The E3 ligase RNF8 acts in cooperation with the heterodimeric E2 enzyme Ubc13/Uev1a to generate ubiquitin conjugates at the sides of DNA double-strand breaks, and recent findings suggest RNF8 as a potential therapeutic target for the treatment of breast cancer. Here, we present a novel high-throughput screening (HTS)-compatible assay based on the AlphaScreen technology to identify inhibitors of the RNF8-Ubc13 protein-protein interaction, along with a follow-up strategy for subsequent validation. We have adapted the AlphaScreen assay to a 384-well format and demonstrate its reliability, reproducibility, and suitability for automated HTS campaigns. In addition, we have established a biochemical orthogonal homogeneous time-resolved fluorescence (HTRF) assay in HTS format and a cellular microscopy-based assay allowing verification of the primary hits. This strategy will be useful for drug screening programs aimed at RNF8-Ubc13 modulation.


SLAS Discovery 2017 Mar;22(3):316-323.

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