Inhibitor Development Against the Wip1 Phosphatase

该项目是与Ettore Appella博士（LCB/NCI）实验室长期合作的一部分，在该实验室中发现了Wip 1蛋白。初步表征导致确定两类磷酸化底物，涉及许多蛋白质参与细胞生长调节。第一个具有二磷酸化序列基序（pT-X-pY），如在p38 MAP激酶中，而第二个具有单磷酸化序列基序（p（S/T）Q），如在p53、Chk 1/2和ATM蛋白中。通过开发Wip 1的扩展活性位点的原子级计算机模型和一系列诱变实验，我们能够揭示底物特异性范围的结构基础。这导致了竞争性抑制Wip 1的环肽分子的发展，Wip 1是该酶家族的第一种抑制剂。然后，我们与专门研究合成化学的丹尼尔阿佩拉博士（LBC/NIDDK）合作，开发了一种更像药物的小分子抑制剂。所得小分子基于吡咯环支架，具有5个不同的发散侧链以模拟环肽的氨基酸。虽然成功，但最终抑制常数仍然仅在低微摩尔范围内。为了进一步推进这项工作，去年我们重新优化了环肽抑制剂。通过多次迭代的设计和测试，我们能够大幅增加结合亲和力，导致抑制常数为110 nM。在这一过程中所涉及的结构建模揭示了重要的新的相互作用，在扩展的活性位点，以及近端的B-环在结合底物和调节活性的作用。由于B环是PP 2C家族Wip 1成员所独有的，因此它的作用以前是未知的。我们现在正在将这些经验应用于设计新一代吡咯基抑制剂。我们还在寻求产生足够的Wip 1蛋白，以通过X射线晶体学确定结构，这将大大有助于抑制剂的优化。

This project is part of a long-standing collaboration with the laboratory of Dr. Ettore Appella (LCB/NCI), in which the Wip1 protein was discovered. Initial characterization resulted in determining two classes of phosphorylated substrates, involving many proteins involved in cell growth regulation. The first has a diphosphorylated sequence motif (pT-X-pY), such as in p38 MAP Kinase, while the second has a mono-phosphorylated sequence motif (p(S/T)Q), such as in the p53, Chk1/2 and ATM proteins. By development of an atomic-scale computer model of the extended active site of Wip1 and a series of mutagenesis experiments, we were able to reveal the structural basis for the range of substrate specificity. This lead to the development of a cyclic peptide molecule that competitively inhibits Wip1, the first inhibitor of any kind for this family of enzymes. We then pursued development of a more drug-like, small molecule inhibitor in collaboration with Dr. Daniel Appella (LBC/NIDDK), who specializes in synthetic chemistry. The resultant small molecule is based on a pyrrole ring scaffold, with 5 different emanating sidechains to mimic the amino acids of the cyclic peptide. While successful, the final inhibition constant was still only in the low micromolar range. To further this effort, this last year we returned to optimizing the cyclic peptide inhibitor. By multiple iterations of design and testing, we were able to drastically increase the binding affinity, resulting in an inhibition constant of 110 nM. The structural modeling involved in this process revealed both important new interactions in the extended active site, and the role of the proximal B-loop in binding substrate and regulating activity. Since the B-loop is unique to the Wip1 member of the PP2C family, its role was previously unknown. We are now applying these lessons to designing a new generation of pyrrole-based inhibitors. We are also pursuing generating sufficient Wip1 protein to determine the structure by X-ray crystallography, which will greatly aid in inhibitor optimization.