Assays


Figure 15


Almac FLEXYTE® Ser/Thr kinase assays. A small molecule chelate (SMC) complexes to the phosphate group of 9AA- labelled kinase product resulting in a reduction of fluorescence lifetime compared to the non- phosphorylated substrate. Phosphorylation of the substrate by the enzyme is directly reported through the corresponding decrease in the average fluorescence lifetime


are around 15ns for unmodified labelled substrate and 10ns for the phosphorylated product after reaction with the chelator (Figure 15). Since FLT measurements can be made accurately to within 0.05ns, the 5ns change provides a large assay win- dow. The technology can be expanded to any pro- tein kinase with appropriate peptide substrates. The platform is compatible with a wide range of ATP concentrations and performs very well in head


to head comparisons with radiometric and TR- FRET approaches. The second focus for FLEXYTE® was development of protease assays, opening up the potential of FLT to another thera- peutically important class of targets. In this case 9AA-labelled substrates were designed so that cleavage by the protease causes a significant increase in fluorescence lifetime, typically from 11ns to 17ns (Figure 16), enabling enzyme activity


Figure 16


Almac FLEXYTE® protease assays. The protease substrate incorporates a 9AA dye and an aromatic moiety, upon enzymatic cleavage the aromatic moiety is released from the substrate resulting in an increase in fluorescence lifetime. Protease activity is monitored in real time through the concomitant increase in average fluorescence lifetime


Drug Discovery World Summer 2010


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