Assays
Figure 13: Proteolytic cleavage of Puretime14-labelled peptide. Various enzyme concentrations between 0nM and 25nM, substrate concentration 600nM. Each point depicts the mean value and standard deviation from 40 samples, sample volume 25uL in a 384-well microplate
Thus the signal change develops in real-time together with the biochemical reaction under scrutiny. Determining kinetic parameters of enzy- matic reactions becomes the norm without further assay development effort. In order to enable users of this technique to further optimise their proto- cols, AssayMetrics are developing their own dedi- cated fluorescence lifetime reader, focusing on short read times while keeping it highly flexible for a wide variety of applications. The Protease Platform at Novartis Pharma AG (Basel, Switzerland) uses this assay principle routinely for large scale inhibitor profiling and medium- throughput screening (MTS) campaigns on pro- teases. In comparison to assays employing stan- dard protease substrates that are dual-labelled with UV-active fluorophore/quencher pairs, the FLT assay format led to an increase in the robust- ness against background fluorescence. Additionally, the work-flow was significantly sim- plified due to the universal assay format, allowing for running automated assay on broad panels of endo- and exopeptidases with just one common reader setup. The high flexibility in substrate design facilitated the in-house development of assays for approximately 80 different proteases from all four classes to date5-7 (Figures 13 and 14).
Figure 14: Protease assay principle. The peptidic substrate carries a Puretime14 label and a tryptophan either side of the scissile bond. While PT14’s fluorescence is dynamically quenched in the parent structure, after cleavage label and quencher are separated, with a concurrent increase in the fluorescence lifetime
80
Almac (
www.flexyte-assays.com) has drawn on its peptide sciences expertise in a collaboration with Dundee University and Edinburgh Instruments to develop an economical, homogeneous, antibody- free assay platform branded as FLEXYTE®™. This new platform exploits the potential of FLT to offer robust approaches to screening and profiling that minimise background interference. Key to the success of the FLEXYTE® platform has been the development and application of 9-aminoacridine (9AA) as a long lifetime fluorescent reporter (τ = 17ns)8. This photostable fluorophore has a high quantum yield and its lifetime can be modulated in a defined fashion through interaction with specific aromatic moieties. By exploiting the fluorescence properties of this dye, a series of 9AA labelled sub- strates have been developed, which produce a sig- nificant change in fluorescence lifetime upon mod- ification by a variety of target enzymes. The initial focus was on Ser/Thr protein kinases. The FLEXYTE® protein kinase platform uses three generic peptide substrates, developed through the collaboration with the University of Dundee, to configure assays for a broad panel of kinases9. The assay uses a phosphoserine/phosphothreonine chelating agent to induce a change in the fluores- cence lifetime of the 9AA label. Typical lifetimes
Drug Discovery World Summer 2010
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