|MKP-3 (PYST1) is a dual-specific phosphatase that dephosphorylates MAPK on threonine and tyrosine residues (Groom et al., 1996). The study of MKP biology has thus far been limited to genetic approaches as small molecule inhibitors are lacking. The most popular approach to identifying potential phosphatase inhibitors are in vitro assays using recombinant enzymes small molecule synthetic substrates, such as paranitrophenyl phosphate or O-methyl fluorescein phosphate. These small molecule substrates have been employed because they provide a robust output signal, are inexpensive and are more convenient to use than the phosphorylated protein substrate. Nonetheless, we now know that the activity of MKPs depends on their interaction with other proteins in the cell. This has been documented in great detail for MKP-3, which requires binding to its activated substrate, Erk2, for full enzymatic activity (Camps et al., 1998). Thus, it would seem desirable to develop a method to analyze MKPs in their physiological environment. The availability of a cell active, selective MKP-3 inhibitor would make an immediate impact on the dissection of the complex regulatory processes involved in the attenuation of mitogenic and stress signaling. Selective inhibitors of MKP-3 would also help define the contributions of MKP-3 and its cellular targets to cell growth, death, and organism development. Importantly, an assay for MKP-3 would be an extremely important counterscreen for MKP-1 inhibitors. This is because MKP-1 appears to be a valid anticancer target, whereas although high levels of MKP-3 have been found in cells transformed with the Ret oncogene (Colucci-D'Amato et al., 2000),
MKP-3 has attributes of a tumor suppressor (reviewed in (Ducruet et al., 2005). Despite the existence of a crystal structure, no potent specific inhibitors of MKP-3 have been reported. Sodium orthovanadate, phenylarsine oxide, and a NSC 357756, a 3-aminoindole derivative (Vogt et al., 2003), are the only inhibitors of MKP-3 described to date. This is partly due to ambiguities and technical challenges in vitro enzyme assays as well as a lack of specific cellular assays for phosphatase inhibition. We have begun to address the problems in phosphatase inhibitor discovery by developing specific assays for phosphatase inhibition in intact mammalian cells based on a “chemical complementation” strategy, which we previously used to prove inhibition of the Cdc25A cell cycle phosphatase in the cell by small molecules (Vogt et al., 2001; Lazo et al., 2001). More recently, we have translated this concept into a single-cell multiparametric assay for MKP-3 activity (Vogt et al., 2003). The inception and development of the Chemical Complementation assay, including its conversion from cell population average format (i.e. Western blot) to a single-cell multiparameter HCS assay have been described in detail (Vogt et al., 2001; Lazo et al., 2001; Vogt et al., 2003; Vogt et al., 2005; Vogt and Lazo, 2005).