A novel fluorescent intensity uHTS assay that utilizes physiological protein substrates
Targeting the neddylation machinery is attractive due to the role of this machinery in indirect regulation of cell cycle progression, DNA replication and DNA damage response mechanisms.
The application is straightforward enough involving detection of deconjugated aminocoumarin from a Nedd8-AMC substrate. The released aminocoumarin is readily detected with ex/em at 350/450 using a microplate reader such as the PHERAstar FS from BMG LABTECH. Using the full length Nedd8 should reveal inhibitors that bind to the actual conformation of SENP8 when it is bound to substrate.