Inhibitor Development Against the Wip1 Phosphatase

Wip1 磷酸酶抑制剂的开发

• 批准号: 8553140
• 负责人: Stewart Durell
• 金额: $ 7.66万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553140
• 关键词: Active Sites; Affinity; Amino Acids; Binding; Cancerous; Collaborations; Computer Simulation; Cyclic Peptides; DNA Damage; Development; Enzymes; Family; Generations; Goals; Laboratories; Lead; Mono-S; Mutagenesis; National Institute of Diabetes and Digestive and Kidney Diseases; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Phosphotransferases; Process; Protein Serine/Threonine Phosphatase; Proteins; Pyrroles; Role; Series; Structural Models; Structure; Substrate Specificity; Synthesis Chemistry; Testing; Work; X-Ray Crystallography; ataxia telangiectasia mutated protein; base; cell growth; cell growth regulation; cell transformation; design; inhibitor/antagonist; member; mitogen-activated protein kinase p38; protein phosphatase 2C; research study; scaffold; small molecule

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.

该项目是与 Ettore Appella 博士实验室 (LCB/NCI) 长期合作的一部分，Wip1 蛋白就是在该实验室中发现的。 初步表征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。 第一个具有二磷酸化序列基序 (pT-X-pY)，例如在 p38 MAP 激酶中，而第二个具有单磷酸化序列基序 (p(S/T)Q)，例如在 p53、Chk1/ 2和ATM蛋白。 通过开发 Wip1 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 Wip1 的环肽分子的开发，Wip1 是该酶家族的第一个抑制剂。 然后，我们与专门研究合成化学的 Daniel Appella 博士 (LBC/NIDDK) 合作，开发了一种更像药物的小分子抑制剂。 所得小分子基于吡咯环支架，具有 5 种不同的侧链来模拟环肽的氨基酸。 虽然成功，但最终的抑制常数仍然仅在低微摩尔范围内。 为了进一步推进这一努力，去年我们重新开始优化环肽抑制剂。 通过设计和测试的多次迭代，我们能够大幅提高结合亲和力，从而实现 110 nM 的抑制常数。 该过程涉及的结构建模揭示了扩展活性位点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中的作用。 由于 B 环是 PP2C 家族 Wip1 成员所独有的，因此其作用以前未知。 我们现在正在应用这些经验来设计新一代基于吡咯的抑制剂。 我们还致力于生成足够的 Wip1 蛋白，以便通过 X 射线晶体学确定其结构，这将极大地有助于抑制剂的优化。