Category: Advances in Bioanalytics and Biomarkers

1221-B - Screening inhibitors of the ubiquitination regulator and anticancer target CSN5 using an HTS fluorescence polarization method

Monday, February 5, 2018
5:00 PM - 6:00 PM

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 microplate de-neddylation assay for a CRL substrate that was used to find inhibitors of CSN. A conventional fluorescence polarization approach was pursued based on the CSN5-mediated release of fluorophore-labelled NEDD8 from the CRL-substrate. Fluorescence polarization assays exploit the change in molecular weight of fluorophore-labeled molecules. The molecular weight influences the rotational speed of a molecule and consequently the polarization of emission light, the readout of fluorescence polarization assays. However, molecular weight difference from substrate-bound NEDD8 to free NEDD8 was not sufficient to provide an acceptable assay window for a reliable assay.

A further determinant of the fluorescence polarization change is fluorescence lifetime of the fluorophore used. This opened up the possibility for assay optimization of the de-neddylation assay: A fluorophore with a lifetime of 22 ns (PT22) was coupled to NEDD8. This way, a difference in fluorescence polarization of substrate-bound to free NEDD8 of 130 mP was achieved. In order to provide the required speed for a screening assay, the method was performed in 384 well plates with a microplate reader that measures two polarizations simultaneously. The assay was qualified to be used for screening and characterization of CSN5 inhibitors.

Tobias Pusterla

International Marketing Manager
Ortenberg, Baden-Wurttemberg, Germany

Tobias Pusterla is International Marketing Manager with BMG LABTECH. He graduated in Veterinary Biotechnology at the University of Milan, Italy. After an experience in the Core Facility for Mouse Mutagenesis in Milan, he obtained a Ph.D. in Cellular and Molecular Biology at the Open University of London, UK, in a joint program with the Hospital and Scientific Institute San Raffaele, Milan, Italy. Subsequently, he joined as a postdoc fellow the German Cancer Research Center (DKFZ) in Heidelberg, Germany. He joined BMG LABTECH in January 2013.