Development of a colorimetric and a fluorescence phosphatase-inhibitor assay suitable for drug discovery approaches.

Protein phosphatases (PP) are interesting drug targets. However, their ubiquitous presence and involvement in different, partially opposing signal pathways suggest that specificity may be achieved rather by targeting their interaction with subunits determining substrate specificity than the enzyme itself. An interesting subunit is phosphatase inhibitor-1 (I-1), which, in its protein kinase A-phosphorylated form (I-1(P)), inhibits the catalytic subunit of type 1 phosphatase (PP1c). In the current study, we established a colorimetric and a fluorescence-based assay system for the identification of compounds interfering with the inhibitory effect of I-1(P) on PP1c. The fluorescence assay exhibited 500-fold higher sensitivity toward PP1c. A nine-residue peptide containing the PP1c-binding motif (RVxF) of I-1 stimulated PP1c activity in the presence of I-1(P) (EC50 27 µM and 2.3 µM in the colorimetric and fluorescence assay, respectively). This suggests that the peptide interfered with the inhibitory effect of I-1(P) on PP1c and represents a proof-of-principle. The calculated Z' factor for PP1c (0.84) and the PP1c-I-1(P) complex (0.73) confirmed the suitability of the fluorescence assay for high-throughput screenings (HTS). By testing several thousand small molecules, we suggest the advantages of kinetic measurements over single-point measurements using the fluorescence-based assay in an HTS format.