Development of an in vitro assay system for protein phosphatase inhibitor-1 antagonists

Reversible protein phosphorylation accomplished by kinases and phosphatases is a major regulatory mechanism. Out of several families of protein phosphatases, serine/threonine phosphatases quantitatively prevail. The functional diversity of the serine/threonine phosphatase type-1 (PP1) in vivo results from the association of its catalytic subunit (PP1c) with different regulatory or inhibitory subunits. One of them is inhibitor-1 (I-1) that inhibits PP1c only in its PKA-phosphorylated form (I-1-Thr35P). Disruption of the I-1 gene in mice resulted in protection from catecholamine-induced lethal arrhythmias and cardiac hypertrophy. This suggested that pharmacological I-1 blockade may represent a therapeutic strategy in heart failure. Therefore, the aim of this study was to screen a library of chemical compounds that inhibit I-1 and to characterize promising candidates in vitro for efficiency and toxicity. We succeeded to establish one colorimetric and one fluorescence assay system using pNPP and DiFMUP as substrates, respectively. The principal feasibility of both assays was confirmed with well-characterized chemical and protein inhibitors of PP1. However, the fluorescence assay exhibited a significantly higher sensitivity (with 500-fold lower requirement for recombinant PP1). We also investigated the effects of mutant I-1 proteins, which implicated the importance of the KIQF motif and Thr-P35 within the conserved N-terminal region of I-1 for binding to and activity against PP1. Screening of selected small peptides derived from I-1 yielded peptides with inhibitory activity against PP1/I-1 interaction. For instance, a nonapeptide containing the KIQF motif antagonized I-1 inhibitory activity with an IC50 value of 3 µM. Notably, a cell-permeable poly-Arg modified peptide attenuated beta-adrenoceptor induced phosphorylation of PLB on Ser16 in neonatal rat cardiomyocytes, indicating biological functionality. Finally, we started to screen a selected, but undirected compound library and a number of in silico designed compounds, which should specifically interfere with the interaction of PP1 with Thr35-phosphorylated I-1. The Z'-factor amounted to 0.86 and 0.73 for PP1 alone and PP1 + I-1P, respectively, which confirmed the suitability of the fluorescence assay for HTS. Nearly 9,000 small molecules were tested in a HTS format using either single-point or kinetic measurements. However, none of them could be validated in secondary assays, suggesting that screening of more compounds is required to identify validated hits. Furthermore, we investigated the aptitude of our fluorescence assay to quantify endogenous phosphatase activities in the cell/tissue extracts. Using okadaic acid (OA) and tautomycin (TTM), this assay enabled us to distinguish the fraction of toxin-sensitive PPP family phosphatases (PP2A, PP4, PP5 and PP6), from insensitive phosphatases (PP2B, PP7, PPM family, PTPases, ACP and AP). Conditions for the specific quantification of PP1 holoenzymes activity still need to be defined. In conclusion, a reliable assay system for the specific detection of I-1 inhibitory compounds has been developed and forms the basis to identify promising small molecule candidates, which could pave the way for the development of clinically applicable drugs to treat patients with chronic heart failure.