Rapid NMR-based functional screening and IC50 measurements performed at unprecedentedly low enzyme concentration

NMR-based functional screening performed with 3-FABS represents a reliable method for identifying inhibitors and measuring with high accuracy their inhibitory activity. Although the method scores high in quality, in its current status it cannot compete with the very high sensitivity of the fluorescence-based techniques. In order to perform ultra-high-throughput screening and to use the small volumes that are possible with fluorescence-based techniques, it would be necessary to further improve the sensitivity of 3-FABS. Two variants of the original experiment with improved sensitivity, namely the 3(x2)-FABS and the 3(x4)-FABS, are presented. The substrate for this purpose is labelled with two and four magnetically equivalent CF3 moieties. Chemical synthesis of novel fragments containing poly CF3 moieties and application of the 3-FABS variants to the rapid screening of the Ser/Thr kinase PKA and the protease trypsin are presented. The high sensitivity of the methods allows performing rapid NMR-based functional screening at an unprecedented low enzyme concentration. Drug Dev Res 64:105–113, 2005. © 2005 Wiley-Liss, Inc.