Inhibitor Development Against the Wip1 Phosphatase

该项目是与Ettore apella博士（LCB/NCI）实验室长期合作的一部分，该实验室发现了Wip1蛋白。初步鉴定结果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。前者具有双磷酸化序列基序（pT-X-pY），如在p38 MAP激酶中，而后者具有单磷酸化序列基序（p(S/T)Q），如在p53、Chk1/2和ATM蛋白中。通过开发Wip1扩展活性位点的原子尺度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。这导致了一种环状肽分子的发展，这种分子可以竞争性地抑制Wip1，这是该酶家族的第一个抑制剂。然后，我们与Daniel Appella博士（LBC/NIDDK）合作开发了一种更像药物的小分子抑制剂，他专门研究合成化学。合成的小分子基于吡咯环支架，具有5种不同的发散侧链来模拟环状肽的氨基酸。虽然成功，但最终的抑制常数仍然仅在低微摩尔范围内。为了进一步努力，我们回到优化环肽抑制剂。通过多次设计和测试，我们能够大幅提高结合亲和力，从而获得110 nM的抑制常数。这一过程的结构建模揭示了扩展活性位点的重要新相互作用，以及近端b环在结合底物和调节活性中的作用。由于B-loop是PP2C家族中Wip1成员所特有的，它的作用以前是未知的。我们现在正将这些经验应用于设计新一代吡咯基抑制剂。我们也在寻求生成足够的Wip1蛋白，通过x射线晶体学来确定其结构，这将极大地帮助抑制剂的优化。最近，我们验证了Wip1和相关的PP2Ca同源物的活性需要结合第3个镁离子，并利用氘交换质谱研究了功能构象的变化。我们目前正在确定PP2Ca/环肽抑制剂复合物的晶体和核磁共振结构。最近，我们利用氘交换质谱技术研究了Wip1和PP2Ca同源物的功能结构变化。我们还测定了具有结合底物的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环进行虚拟筛选，以确定抑制剂导联。

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.