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Wip1蛋白。初步的表征结果是确定了两类磷酸化底物，涉及许多参与细胞生长调节的蛋白质。第一个是二磷酸化的序列基序(PT-X-Py)，如p38 MAP Kinase；第二个是单磷酸化的序列基序(p(S/T)q)，如在P53、Chk1/2和ATM蛋白中。通过建立Wip1延伸活性部位的原子级计算机模型和一系列诱变实验，我们能够揭示底物专一性范围的结构基础。这导致了环肽分子的发展，它竞争性地抑制Wip1，Wip1是这个酶家族的第一种抑制剂。然后，我们与专门从事合成化学的Daniel Appella博士(LBC/NIDDK)合作，开发了一种更像药物的小分子抑制剂。合成的小分子是基于一个吡咯环支架，有5个不同的发射侧链来模拟环肽的氨基酸。虽然成功了，但最终的抑制常数仍然只在低微摩尔范围内。为了进一步推进这一努力，我们重新优化了环肽抑制剂。通过多次迭代设计和测试，我们能够显著提高结合亲和力，导致抑制常数为110 nM。这一过程中的结构模拟揭示了延伸的活性部位中重要的新相互作用，以及近端B-环在结合底物和调节活性方面的作用。由于B-环是PP2C家族中Wip1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教训应用于设计新一代基于吡咯的抑制剂。我们还在寻求产生足够的Wip1蛋白来通过X射线结晶学确定结构，这将极大地帮助抑制剂的优化。最近，我们验证了结合第三个镁离子对Wip1及其相关的PP2Ca同系物活性的要求，并用氢交换质谱仪研究了其功能构象的变化。我们目前正在测定PP2Ca/环肽抑制剂复合体的晶体结构和核磁共振结构。最近，我们利用氢交换质谱仪研究了Wip1和PP2Ca同系物的功能结构变化。我们还测定了具有结合基片的PP2Ca的晶体结构。我们正在使用我们最近确定的Wip1的晶体结构和难以捉摸的B-环来进行虚拟筛选，以确定抑制剂的先导。

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, 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. Recently, we have verified the requirement of binding a 3rd magnesium ion for activity of Wip1 and the related PP2Ca homologue, and used deuterium exchange mass spectroscopy to study the functional conformational changes. We are currently determining the crystal and NMR structures of PP2Ca/cyclic peptide inhibitor complexes. Recently we used Deuterium Exchange Mass Spectroscopy to study the functional structural changes of Wip1 and the PP2Ca homologue. We have also determine the crystal structure of PP2Ca with a bound substrate. We are using our recently determined crystal structure of Wip1 with the elusive B-loop to perform virtual screening to identify inhibitor leads.