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Screening inhibitors for the ubiquitination regulator and anticancer target CSN5 using an HTS fluorescence polarization method

U. Hassiepen, M.D. Jones, R. Assenberg, I. Bechtold, C. Logel, M. Renatus, B. Martoglio B. (Novartis Pharma AG, Switzerland), 04/2017

Cullin-RING E3 ubiquitin ligases (CRLs) are activated by neddylation and inactivated by de-neddylation that is accomplished by the COP9 signalosome (CSN). Trapping CRLs in their inactive state is reported to elevate tumor suppressors. Therefore, inhibition of CSN is a potential modality for cancer treatment.

Here, we show the development of a high-throughput de-neddylation assay for a CRL substrate that was used to find inhibitors of CSN. The fluorescence polarization assay was optimized using a fluorophore with comparatively long fluorescence lifetime.

Performing the assay in a 384 well plate and reading it on a PHERAstar® microplate reader provided an assay window of 130 mP. The reader’s capability of simultaneous detection of the two polarization channels accelerated the measurement and enabled kinetic reads. Hence, the assay is applicable for both CSN inhibitor finding and characterization.

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