High-throughput screening (HTS) for enzyme inhibitors plays an important role in drug discovery, and is thus of great importance to the pharmaceutical industry. Some of the most sensitive and robust methods to monitor enzymatic reactions are radioactive and fluorescence-based assays, among which the latter are by far preferrable in HTS. A particular challenge in fluorescence-based HTS is to account for an artificially increased fluorescence background by the potential inhibitors. We have introduced a genuine line of fluorescent probes, known as DBO fluorazophores (see structures below), which are now marketed as Puretime^TM 325 dyes.

Overview of available dyes for Nano-TRF assays.

These dyes are characterized by an exceedingly long fluorescence lifetime (up to 1 microsecond in water). Since background fluorescence has generally a much shorter lifetime (1-10 ns), this allows to efficiently “gate out” the background fluorescence, which was successfully demonstrated in assays for proteases, kinases and phosphatases (e.g. p60^c-Src Src kinase, EGFR kinase, YOP protein tyrosine phosphatase, acid phosphatase, alkaline phosphatase). In addition, these dyes are efficiently quenched by tryptophan and tyrosine, such that single-labeling of natural peptide recognition sequences generally suffices.

Time-gated background suppression with the Nano-TRF detection window (yellow). Experimental lifetime traces of dyes (red) and background (blue).

Key references: (a) ChemBioChem. 2006, 7, 733-737; (b) Anal. Biochem. 2007, 360, 255-265; (c) J. Am. Chem. Soc. 2007, 129, 15927-15934.