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。尽管WIP1在大多数正常细胞中以低水平表达，但在细胞暴露于DNA损伤诱导剂（例如电离辐射（IR）或紫外线（UV）光）中，其转录是由p53诱导的。 WIP1蛋白经常过表达或在几种人类癌症中放大了PPM1D基因，并且这种增加的表达与较差的预后有关。人类细胞的研究表明，WIP1的过表达会损害肿瘤抑制功能，并且对缺乏WIP1的小鼠的研究表明它们对肿瘤发生有抵抗力。当前对WIP1的研究集中在理解其调节和功能上，确定其功能靶标并进行小分子文库的高通量筛选（HTS），以识别WIP1磷酸酶活性的特定抑制剂/活化剂。最近，我们通过氢/氘交换质谱法和X射线晶体学构造表征了不稳定金属离子和磷酸化肽底物对人PPM1A的构象的影响。这些结构研究使我们能够更好地理解这种磷酸酶家族中的底物结合，并表征了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特定抑制剂的结合来消除。 WIP1催化结构域的高分辨率结构的确定，该结构包括B环的构象，将极大地有助于进一步发展WIP1磷酸酶活性的特定抑制剂。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研究中鉴定的抑制剂或激活剂的结构活性研究。为此，我们继续优化重组WIP1的表达，鉴定易于聚集的残基，并与DRS合作筛选结晶条件。 NIDDK分子生物学实验室Fred Dyda和Dalibor Kosek。我们预计这些系统的努力将提供WIP1的第一个结构，以帮助更好地了解抑制的特异性和潜力。刺猬 - 基因瘤相关的癌基因同源1（GLI1）信号传导途径调节胚胎发生过程中的关键过程，并与人类癌症发展的几个方面有关。与Barbara Stecca博士（意大利佛罗伦萨ISPRO）合作，我们发现CDK1激酶通过降低的GLI1蛋白水平降低了刺猬途径的转录活性。该信号与WIP1相反，WIP1通过去磷酸化GLI1 THR1045来增强GLI1活性，表明对WIP1的抑制可能是过表达WIP1并激活HedgeHog信号传导的人类癌症的有效治疗方法。与Oleg Demidov博士（法国第洪勃艮第大学）合作，我们使用了合成肿瘤模型来研究免疫系统中消融WIP1肿瘤进展的影响。在合成小鼠模型中，WIP1的髓样特异性缺失延迟了B10黑色素瘤肿瘤和LLC1肺癌肿瘤的生长，证实了WIP1缺陷型先天性免疫细胞在抗肿瘤免疫中的重要作用，并表明WIP1是提高抗癌效率的有前途的抗癌选择靶标。由于WIP1在许多人类癌症中被扩增或过表达，包括乳腺癌，卵巢透明细胞癌，胃癌，胰腺腺癌，髓母细胞瘤和神经母细胞瘤，因此WIP1活性的抑制剂可能是对几种人类癌症的治疗可能是有益的。我们已经开发并验证了两个基于HTS的基于正交板的WIP1活性测定。这两个测定法具有高灵敏度和通过荧光检测或质谱法实现的广泛动态范围，适用于WIP1活性调节剂的筛选化合物文库（Clausse等人J Biol Chem。2019，294：17654-17668）。通过与国家前进的转化科学中心（NCAT）合作，我们使用上述快速消防质谱法HTS方法使用生理上相关的底物从NCATS Genesis库中筛选了100,000多种化合物来识别WIP1调节剂。该库提供了新颖的化学型，结合了各种化学支架和具有良好特征和靶向化合物，具有新的特性，可用于进一步的治疗发展，并且是可申请的化学型的丰富来源。主筛选后，将几百种化合物视为活性，并以剂量​​响应格式进行板条以完善选择。我们实施了正交读数以降低干扰化合物的患病率，随访研究将消除那些非特异性活动的人。最有希望的化合物将使用基于细胞的测定法进行评估，我们优化了通过测量电离辐射后测量γ2AX磷酸化来确定WIP1活性。目前，没有可用的WIP1抑制剂，它们有效并显示出良好的药代动力学。我们旨在发现一种新的WIP1调节器支架，该支架可能适合优化更好的药代动力学特性。