DiscussionThe FLEXYTE™ platform is an enabling assay technology that uses fluorescence lifetime (FLT) as the reporting modality. By utilising FLT as the readout parameter in primary and secondary screening applications, this platform offers a number of key benefits over other fluorescence based formats, including the ability to minimise compound interference, facile assay miniaturisation, and a robust and sensitive readout. A key step to realising the potential of this approach has been has been the development and application of the long fluorescence lifetime fluorophore, 9-aminoacridine (9AA). Using this simple small-molecule dye, FLEXYTE™ assays have been successfully configured for a number of important targets including protein kinases, proteases, epi-enzymes and the DUB enzyme family.
We have now developed this assay technology to enable protein-protein / protein-ligand interactions (PPIs) to be reported through a change in FLT. PPIs make up an increasing number of therapeutic targets and there is a continuing need for robust, sensitive and HTS compatible assay formats. Currently, the analysis of such systems typically utilises labelled antibodies or other complex secondary reagents, or employs biophysical techniques which, while powerful, do not lend themselves to screening large compound libraries in a high-throughput format. The FLEXYTE™ PPI platform provides a simple solution and enables the benefits of FLT readouts to be realized for this target class.
The utility of this PPI platform is exemplified here through the development of binding assays for the Grb2-SH2 domain and the epi-reader proteins bromodomain BRD4(1) and malignant brain tumour domain L3MBTL1. Using 9AA-labelled proteins, robust FLT PPI assays were readily configured. Validation was provided using reported small molecule inhibitors and high Z’-factors confirm the assays’ suitability for HTS applications.
The combination of this technology with new developments in FLT plate-reader instrumentation and data analysis (ameon™, TTP Labtech) provides complete, HTS compatible, solutions to this challenging target class.