Regulation and Function of WIP1 Phosphatase and its Role in Tumor Cells

WIP1磷酸酶的调控、功能及其在肿瘤细胞中的作用

• 批准号: 10262259
• 负责人: ETTORE APPELLA
• 金额: $ 86.97万
• 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/10262259
• 关键词: Binding; Biochemical; Biological Assay; CDC2 gene; Catalytic Domain; Cell Proliferation; Cells; Characteristics; Chemicals; Collaborations; Crystallization; DNA Damage; Detection; Deuterium; Development; Dose; Drug Kinetics; Embryonic Development; Erinaceidae; Family; Family member; Fire - disasters; Fluorescence; Follow-Up Studies; France; GLI Family Protein; GLI gene; Genetic Transcription; Goals; Growth; Human; Hydrogen; Immune; Immune system; Investigation; Ionizing radiation; Ions; Italy; Laboratories; Libraries; MAP Kinase Gene; Malignant Neoplasms; Malignant neoplasm of lung; Mass Spectrum Analysis; Measures; Metals; Methods; Modeling; Molecular Biology; Molecular Conformation; Mus; Myelogenous; National Institute of Diabetes and Digestive and Kidney Diseases; Neuroblastoma; Normal Cell; Outcome; Ovarian Clear Cell Tumor; PPM1D gene; Pancreatic Adenocarcinoma; Pathway interactions; Peptides; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Prevalence; Process; Property; Protein Serine/Threonine Phosphatase; Proteins; Radiation therapy; Recombinants; Regulation; Research; Resistance; Resolution; Role; Signal Pathway; Signal Transduction; Source; Specificity; Stress; Structure; TP53 gene; Translational Research; Treatment Efficacy; Tumor Immunity; Tumor Suppressor Proteins; Ultraviolet Rays; Universities; X-Ray Crystallography; base; cancer immunotherapy; high throughput screening; improved; inhibitor/antagonist; malignant breast neoplasm; malignant stomach neoplasm; medulloblastoma; melanoma; member; mouse model; neoplastic cell; novel; overexpression; p38 Mitogen Activated Protein Kinase; protein phosphatase 2C; response; scaffold; screening; small molecule libraries; smoothened signaling pathway; therapeutic development; tumor; tumor progression; tumorigenesis

The wild-type p53-induced phosphatase Wip1 (PP2Cdelta or PPM1D) is a member of the serine/threonine protein phosphatase 2C (PP2C) family. Although Wip1 is expressed at low levels in most normal cells, its transcription is induced by p53 after exposure of cells to DNA damage-inducing agents, such as ionizing radiation (IR) or ultraviolet (UV) light. The Wip1 protein is frequently overexpressed or the PPM1D gene is amplified in several human cancers, and this increased expression is associated with worse outcomes. Studies on human cells have shown that overexpression of Wip1 compromises tumor suppressor functions, and studies of mice that lack Wip1 show that they are resistant to tumorigenesis. The current research on Wip1 is focused on understanding its regulation and functions, identifying its functional targets and performing high-throughput screens (HTS) of small molecule libraries to identify specific inhibitors/activators of Wip1 phosphatase activity. Recently, we characterized the effects of the binding of the labile metal ion and the phospho-peptide substrate on the conformation of human PPM1A, a family member of Wip1, by both hydrogen/deuterium exchange mass spectrometry and x-ray crystallography. Together these structural studies have allowed us to better understand substrate binding in this family of phosphatases and characterize the labile third metal ion that is essential for catalytic activity, both critical aspects that could be abrogated by the binding of a specific inhibitor. Determination of a high-resolution structure of the Wip1 catalytic domain that includes the conformation of the B-loop would greatly aid further development of specific inhibitors of Wip1 phosphatase activity. Additionally, high-resolution structural information for the Wip1 catalytic site would be useful for further optimization of known inhibitors and to guide structure-activity investigations of inhibitors or activators identified in HTS studies. To that end, we have continued optimizing the expression of recombinant Wip1, identifying aggregation-prone residues, and screening for crystallization conditions in collaboration with Drs. Fred Dyda and Dalibor Kosek, Laboratory of Molecular Biology, NIDDK. We anticipate that these systematic efforts will provide the first structure of Wip1 to help better understand the specificity and potential for inhibition. The Hedgehog-glioma-associated oncogene homolog 1 (GLI1) signaling pathway regulates key processes during embryogenesis and is implicated in several aspects of the development of human cancers. In collaboration with Dr. Barbara Stecca (ISPRO, Florence, Italy), we found that CDK1 kinase decreases the transcriptional activity of the Hedgehog pathway through reduced GLI1 protein levels. This signaling is opposed by Wip1, which enhances GLI1 activity by dephosphorylating GLI1 Thr1045, indicating that inhibition of Wip1 may be an effective therapeutic approach for human cancers that overexpress Wip1 and have activated Hedgehog signaling. In collaboration with Dr. Oleg Demidov (University of Burgundy, Dijon, France), we have used syngeneic tumor models to investigate the effects on tumor progression of ablating Wip1 in the immune system. In syngeneic mouse models, myeloid-specific deletion of Wip1 delayed the growth of both B10 melanoma tumors and LLC1 lung cancer tumors, confirming an important role of Wip1-deficient innate immune cells in anti-tumor immunity and suggesting that Wip1 is a promising target for increasing the efficiency of anti-cancer immunotherapy. As Wip1 is amplified or overexpressed in numerous human cancers including breast cancer, ovarian clear cell carcinoma, gastric cancer, pancreatic adenocarcinoma, medulloblastoma, and neuroblastoma, developing inhibitors of Wip1 activity may be beneficial in the treatment of several human cancers. We have developed and validated two orthogonal plate-based Wip1 activity assays for HTS. The two assays have high sensitivity and broad dynamic range enabled by either fluorescence detection or mass spectrometry and are suitable for screening compound libraries for modulators of Wip1 activity (Clausse et al. J Biol Chem. 2019, 294:17654-17668). In collaboration with the National Center for Advancing Translational Sciences (NCATS), we used the above-mentioned Rapid Fire Mass Spectrometry HTS method to screen more than 100,000 compounds from the NCATS Genesis library using physiologically relevant substrate to identify Wip1 modulators. This library provided novel chemotypes with a combination of diverse chemical scaffolds and well-characterized and targeted compounds possessing new properties for further therapeutic development and is a rich source of patentable chemotypes. Several hundred compounds were considered active after the primary screen and plated in dose response format in order to refine the selection. We implemented orthogonal readouts to reduce the prevalence of interference compounds, and follow-up studies will eliminate those with non-specific activity. The most promising compounds will be assessed using a cell-based assay we optimized to determine Wip1 activity by measuring gammaH2AX phosphorylation following ionizing radiation. Currently, there are no available Wip1 inhibitors which are potent and show favorable pharmacokinetics. We aim at discovering a new Wip1 modulator scaffold that might be amenable to optimization for better pharmacokinetic properties.

野生型P53诱导的磷酸酶Wip1(PP2CDelta或PPM1D)是丝氨酸/苏氨酸蛋白磷酸酶2C(PP2C)家族的成员。虽然Wip1在大多数正常细胞中低水平表达，但在细胞暴露于DNA损伤诱导剂后，如电离辐射(IR)或紫外线(UV)，Wip1的转录可由P53诱导。在几种人类癌症中，Wip1蛋白经常过度表达或PPM1D基因被扩增，这种表达增加与更差的预后有关。对人类细胞的研究表明，Wip1的过度表达会损害肿瘤抑制功能，而对缺乏Wip1的小鼠的研究表明，它们对肿瘤的形成具有抵抗力。目前对Wip1的研究主要集中在了解其调控和功能，确定其功能靶点，并对小分子文库进行高通量筛选(HTS)以确定Wip1磷酸酶活性的特定抑制物/激活剂。最近，我们用氢/氢交换质谱仪和X射线结晶学研究了不稳定金属离子与磷酸肽底物结合对人Wip1家族成员PPM1a构象的影响。综上所述，这些结构研究使我们能够更好地了解这一磷酸酶家族的底物结合，并表征对催化活性至关重要的不稳定的第三金属离子，这两个关键方面都可以通过结合特定的抑制剂来消除。确定Wip1催化域的高分辨结构(包括B-环的构象)将极大地帮助进一步开发Wip1磷酸酶活性的特异性抑制剂。此外，Wip1催化位点的高分辨率结构信息将有助于进一步优化已知的抑制剂，并指导HTS研究中确定的抑制剂或激活剂的结构-活性研究。为此，我们与NIDDK分子生物学实验室的Fred Dyda博士和Dalibor Kosek博士合作，继续优化重组Wip1的表达，鉴定易于聚集的残基，并筛选结晶条件。我们预计，这些系统性的努力将提供Wip1的第一个结构，以帮助更好地了解抑制的特异性和潜力。Hedgehog-胶质瘤相关癌基因同源1(GLI1)信号通路在胚胎发育过程中调节关键过程，并参与人类癌症发展的多个方面。与意大利佛罗伦萨ISPRO博士Barbara Stecca合作，我们发现CDK1激酶通过降低GLI1蛋白水平降低Hedgehog途径的转录活性。Wip1通过使GLI1 Thr1045去磷酸化来增强GLI1的活性，这表明抑制Wip1可能是过度表达Wip1并激活Hedgehog信号的人类癌症的有效治疗方法。与法国第戎勃艮第大学的奥列格·德米多夫博士合作，我们使用同基因肿瘤模型来研究在免疫系统中切除Wip1对肿瘤进展的影响。在同基因小鼠模型中，Wip1的髓系特异性缺失延缓了B10黑色素瘤和LLC1肺癌的生长，证实了Wip1缺陷的先天免疫细胞在抗肿瘤免疫中的重要作用，提示Wip1是提高抗癌免疫治疗效率的一个有前途的靶点。由于Wip1在包括乳腺癌、卵巢透明细胞癌、胃癌、胰腺癌、髓母细胞瘤和神经母细胞瘤在内的多种人类癌症中扩增或过表达，开发Wip1活性的抑制剂可能对几种人类癌症的治疗有利。我们已经开发并验证了两种基于正交平板的HTS Wip1活性检测方法。这两种检测方法具有高灵敏度和宽动态范围，可由荧光检测或质谱仪实现，适合于筛选Wip1活性调节剂的化合物文库(Clausse等人)。J生物化学。2019年，294：17654-17668)。与国家高级翻译科学中心(NCATS)合作，我们使用上述快速火焰质谱学HTS方法从NCATS Genesis文库中筛选出100,000多种化合物，使用与生理相关的底物来鉴定Wip1调节器。该文库提供了新的化学类型，结合了不同的化学支架和具有良好特性和靶向的化合物，具有进一步治疗开发的新特性，是可申请专利的化学类型的丰富来源。在初步筛选后，数百种化合物被认为是有效的，并以剂量反应格式进行电镀，以改进选择。我们实施了正交读数，以减少干扰化合物的流行率，后续研究将消除那些非特定活性的化合物。最有希望的化合物将使用我们优化的基于细胞的方法进行评估，以确定Wip1的活性，方法是测量电离辐射后的GammaH2AX磷酸化。目前，尚无有效的、药代动力学良好的Wip1抑制剂。我们的目标是发现一种新的Wip1调节器支架，可以对其进行优化，以获得更好的药代动力学特性。