Wip1, the product of the PPM1D gene, is a PP2C serine/threonine protein phosphatase that was first identified in my laboratory as a gene whose induction following DNA damage required wild-type p53. In humans and mice, Wip1 is expressed at low levels in most adult tissues, but is expressed at higher levels in proliferating tissues. Wip1 overexpression, either reflecting gene amplification or other mechanisms, is found in a substantial fraction of cases of several human cancers, including breast cancers, ovarian clear cell carcinomas and neuroblastoma, and is generally associated with a worse prognosis. To better understand the connections between Wip1 activity and the disregulation of cellular proliferation that characterizes cancer, we are investigating the regulation of Wip1 expression and activity, identifying targets of Wip1 phosphatase activity, and developing inhibitors of Wip1 phosphatase activity. Regulation of Wip1 expression and activity. The transcriptional induction of Wip1 following exposure to DNA damaging agents requires functional p53 protein. p53 and Wip1 function in a negative feedback loop, with Wip1 phosphatase activity promoting the return to homeostasis by removing activating phosphorylations of p53 itself (Ser15) and those of its upstream kinases, ATM, Chk1 and Chk2. In the several hour interval following exposure to IR, the levels of Wip1 protein change more dramatically than do the Wip1 mRNA levels. The increased dynamic range in Wip1 protein levels may result from a combination of translational and post-translational factors. We are using a variety of techniques to characterize Wip1 expression following stress and during the cell cycle. These studies have allowed us to begin to characterize the regulation of Wip1 in unstressed and stressed conditions. Current observations on the effects of Wip1 deletion in mice are based on the original knockout mouse. Despite the important information that this mouse has provided for understanding the role of Wip1 deletion in tumorigenesis, the non-conditional knockout does not allow us to determine the specific effects of Wip1 deletion in a single tissue. Ubiquitous Wip1 deletion in mice involves the immune system, tumor environment and metabolism, which may affect tumorigenesis in the organ of interest. The possible adaption of signaling pathways during development in the absence of Wip1 may also have effects. To overcome these limitations, we are developing a conditional knock-out mouse in which Wip1 deletion can be directed to a single tissue through the expression of Cre recombinase by a tissue-specific promoter to excise Wip1 only in a specific tissue or through tamoxifen-dependent Cre recombinase to induce deletion at a specified time. We are in the process of generating these conditional knock-out mice, which will be used to study the effects of Wip1 in several mouse tumor models. Wip1 phosphatase activity. Wip1 dephosphorylates serine and threonine residues within pTXpY and pTQ/pSQ motifs, and we have used biochemical methods to characterize its substrate specificity. Many of the known pTQ/pSQ substrates of Wip1 are phosphorylated by ATM. In addition to its roles in the response to DSBs and other forms of DNA damage, ATM regulates the cellular response to insulin through phosphorylation of Ser112 of the inhibitor 4EBP1, resulting in its dissociation from the translation initiation protein, eIF-4E. Since the amino acid context of Ser112 in 4EBP1 resembles those of known Wip1 substrates, we have investigated Ser112 of 4EBP1 as a potential Wip1 substrate. Wip1 Inhibitors. Many human tumors in which the PPM1D gene is amplified or overexpressed contain wild type p53. Our published results indicate that Wip1 phosphatase is a candidate proto-oncogene that promotes tumorigenesis through inactivation of wild-type p53. Phosphatases in general play critical physiological roles in the cell as the antagonists of kinase activity. Because of this, phosphatases represent important targets in a number of diseases, including cancer. We have continued to optimize cyclic peptide inhibitors of Wip1, both as a means to demonstrate the utility of Wip1 inhibition and as a way to gain insight into the structure of the Wip1 catalytic domain. While retaining the core pS-I-pY residues previously shown to be important for inhibition, the other positions of the cyclic peptide were extensively modified. We found that specific interaction of an aromatic ring at the X1 position and negative charge at the X5 and X6 positions significantly increased the inhibitory activity of the cyclic peptide, resulting in an optimized molecule with Ki = 110 nM, an approximately 50-fold improvement in inhibitory activity. To the best of our knowledge, this represents the best activity for an inhibitor of Wip1. We have further incorporated both the data from the optimization study and new structures of related phosphatases into an improved model of the Wip1 catalytic domain. In this model, the Phe at position X1 is buried next to the non-polar regions of the long side chains of Lys19 and Tyr20. Additionally, the side chain of the acidic residue at the X5 position is buried where it interacts with the side chain of His107, allowing for a charge-charge interaction that would increase its affinity for Wip1. We further propose that a highly-basic loop unique to Wip1, formed by residues 239-263, accounts for the increased inhibitory activity of peptides containing an acidic residue at position X6 and may play a role in substrate specificity. We have also continued to pursue the development of pyrrole-based small molecule inhibitors of Wip1. The synthesis of these inhibitors has been considerably improved by decreasing the number of synthetic steps from ten to seven. Additionally, we have optimized the synthetic scheme for solution-phase synthesis as opposed to the previous method in which synthesis was performed on a solid support. The new synthesis is scalable, allowing us to prepare on the order of 100 mg of compound. Additionally, it is more versatile, so that we can now vary the side chain at almost every position around the pyrrole core for optimization of inhibitory activity. We have synthesized 30 different analogs of the original molecule based upon our optimization of the cyclic peptide inhibitor. Thus far, the best pyrrole-based inhibitor has Ki = 1.7 microM while maintaining specificity for Wip1. The new synthesis is also compatible with synthesis of a pro-drug to mask the phosphate groups and improve cellular uptake. We have already begun to synthesize different pro-drug forms of the pyrrole-based inhibitors and we will be testing their activity in cell-based assays of Wip1 activity.
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