Discussion

Ubiquitin is covalently linked to many cellular proteins to regulate their activity, stability, localisation or interactions. Ubiquitylation is a reversible process carried out by the opposing activities of ubiquitin ligases and deubiquitylating enzymes (DUBs). The human genome encodes approximately 100 DUBs. Of the 6 families of deconjugating enzymes, five (USP, UCH, MJD, OTU and SENPs) belong to the cysteine peptidase class, while one (the JAMM family) belongs to the metallo-peptidase class. As DUBs have been shown to play critical roles in many pathological processes, particularly cancer, infectious disease and neuro-degeneration, they have begun to attract significant attention as potential targets for therapeutic intervention. Pharmacological modulation of DUBs using a multitude of approaches in the last decade has seen limited success to date. A number of conceptual and technological challenges need to be overcome in order to progress genuine DUB therapies.
A major challenge in characterisation of DUB inhibitors is the development of high throughput assays that accurately represent the native substrate of DUBs and the subsequent detection and removal of false positives from compound screens. Here we have evaluated the use of FLEXYTE® fluorescence lifetime (FLT) assay technology from Almac in combination with the ameon® FLT microplate reader from TTP Labtech and compared to existing assay technologies for screening against a number of DUB targets. The FLEXYTE® technology utilises a full-length isopeptide-linked ubiquitin substrate labelled with Almac’s 9-aminoacridine (9AA) long lifetime fluorophore and incorporating a FLT modulator which reduces the FLT of 9AA in the substrate. Cleavage of the scissile isopeptide bond by the DUB is reported through an increase in FLT. FLT is an intrinsic fluorescence property independent of probe concentration and volume and is unaffected by auto-fluorescence or light scattering. Use of this technology allowed easy identification and removal of a number of types of false positives at an early stage and will reduce the time spent following compounds that are not true inhibitors. The successful and rapid identification of genuine DUB inhibitors through screening cascades determines efficient progression of genuine compounds towards pre-clinical development. Advantages of the fluorescence lifetime technology coupled to the novel generation of FLT plate readers will be discussed.