Neddylation activates Cullin-RING E3 ubiquitin ligases (CRLs), and de-neddylation which is attained by COP9 signalosome (CSN) deactivates them. Keeping CRLs in their inactive mode enhances tumor suppressors. Hence, for cancer therapy, inhibition of CSN becomes a vital approach.
The development of a high-throughput de-neddylation method for CRL is described here. This method was utilized to spot inhibitors of the de-neddylating CSN. A fluorophore having an extended fluorescence lifetime was used to optimize the fluorescence polarization method.
An assay window of 130 mP was obtained by carrying out the assay in a 384 well plate, and recording it on a PHERAstar® microplate reader. The capability of the instrument for parallel identification of two polarization channels quickened the assessment. It also allowed kinetic reads. Therefore, the technique can be applied to both CSN inhibitor spotting and profiling.
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