Structural Basis for Small Molecule-Mediated Allosteric Activation of Protein Phosphatase 2A

小分子介导的蛋白磷酸酶 2A 变构激活的结构基础

• 批准号: 10191568
• 负责人: Ken Morita
• 金额: $ 13.61万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-03 至 2021-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10191568
• 关键词: 3-Dimensional; Acute T Cell Leukemia; Advisory Committees; Affinity; Amino Acids; Antipsychotic Agents; Applications Grants; Automobile Driving; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Biology; Cell Cycle Arrest; Cells; Chemicals; Clinical Research; Complex; Crystallization; Drug usage; Energy Transfer; Enzymes; Family; Fluorescence; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Goals; Grant; Holoenzymes; Human; Immunoprecipitation; Knowledge; Label; Leadership; MYBL2 gene; Malignant - descriptor; Malignant Neoplasms; Manuscripts; Mass Spectrum Analysis; Mediating; Mentors; Mentorship; Mission; Molecular; Molecular Conformation; Mutation; Outcome; Patient-Focused Outcomes; Perphenazine; Phosphoproteins; Phosphoric Monoester Hydrolases; Physiological; Physiology; Protein Family; Protein phosphatase; Proteins; Proteomics; Public Health; Reporter; Research; Research Personnel; Resolution; Role; Scientific Advances and Accomplishments; Signal Transduction Pathway; Specificity; Structure; Technology; Testing; Therapeutic Clinical Trial; Time; Toxic effect; Training; Tumor Suppressor Proteins; Work; Writing; X-Ray Crystallography; acute T-cell lymphoblastic leukemia cell; analog; anti-cancer therapeutic; cancer cell; cancer therapy; career; design; fluorophore; improved; neoplastic cell; novel; novel strategies; novel therapeutic intervention; novel therapeutics; overexpression; protein activation; protein function; protein structure; recruit; response; small molecule; structural biology; survival outcome; three dimensional structure; tool; transcription factor; tumor

The group of phosphatases collectively referred to as PP2A (protein phosphatase 2A) represent an important family of tumor suppressors. As a research fellow working under the mentorship of Dr. Thomas Look, the candidate has recently discovered that perphenazine (PPZ), a classic antipsychotic drug, and its related small molecule iHAP1 selectively drive the formation of heterotrimeric PP2A complexes containing the B56ϵ (PPP2R5E) subunit out of 15 possible distinct regulatory B subunits (K. Morita et al. Cell. 2020). Defining the structural mechanisms through which these small molecules allosterically activate PP2A with extreme specificity is of paramount importance because such knowledge will fundamentally advance the field of phosphatase biology, while forming the basis for medicinal chemists to optimize these tool compounds as novel anticancer therapeutics for clinical study. In this grant proposal, the candidate seeks to understand the fundamental mechanisms responsible for this allosteric activation of PP2A phosphatase from a structural perspective, working under the primary mentorship of Dr. Eric Fischer, an emerging leader in biochemistry and the field of structural biology. The central hypothesis is that PPZ/iHAP1 binds to the AC heterodimer and allosterically alters its conformation -- in ways that will be evident from the high-resolution crystal structure -- so that the B56ϵ subunit is readily recruited, thus driving the formation and activation of the PP2A-B56ϵ phosphatase in the cell. The immediate objectives are to determine the three-dimensional protein structures of the PP2A holoenzyme complexes formed in response to PPZ/iHAP, and to define the molecular contacts of the PP2A subunits with PPZ/iHAP compounds by X-ray crystallography. This goal will be pursued in two specific aims: 1) Determine the three-dimensional structure of the PP2A holoenzyme complex in the presence of PPZ or iHAP compounds to decipher the mechanisms underlying allosteric activation of specific PP2A subunit complexes by these small molecules, and 2) Determine the biochemical mechanisms underlying the ability of allosteric small-molecule PP2A activators to drive dynamic exchange of the PP2A regulatory B subunits with the AC heterodimer, from the Striatin family proteins in control cells to the B56ϵ or B55α proteins for iHAP/PPZ and SMAP, respectively. This strategy is significant because the results could reveal new mechanisms underlying allosteric activation of specific PP2A phosphatase complexes, which in turn will be key to dissecting the normal physiologic roles of this diverse family of phosphatases and to understanding their roles as tumor suppressors and how they are subverted in malignant transformation. This proposal has been designed to provide the candidate with advanced training in structural biology, as well as mentoring in manuscript/grant writing and leadership qualities. Bolstered by the guidance of a new primary mentor, Dr. Eric Fischer, together with the ongoing contributions of co-mentor Dr. Thomas Look, and the scientific advisory committee composed of experts necessary to complete this work, this proposal should significantly enhance the candidate's career goal of becoming an independent investigator.

统称为PP 2A（蛋白磷酸酶2A）的磷酸酶组代表一种磷酸酶。 重要的肿瘤抑制因子家族。作为一名在导师制下工作的研究员， 托马斯博士的候选人最近发现，奋乃静（PPZ），一个经典的 抗精神病药物及其相关的小分子iHAP 1选择性地驱动 在15种可能的PP 2A复合物中，含有B56 β（PPP 2 R5 E）亚基的异源三聚体PP 2A复合物 不同的调节B亚基（K. Morita等人，Cell. 2020年）。确定结构机制 这些小分子通过其以极端特异性变构激活PP 2A， 因为这些知识将从根本上推动磷酸酶领域的发展 生物学，同时形成药物化学家的基础，以优化这些工具化合物作为新的 用于临床研究的抗癌治疗剂。在这份拨款申请中，候选人寻求 了解PP 2A变构激活的基本机制 磷酸酶从结构的角度来看，在埃里克博士的主要指导下工作 费希尔是生物化学和结构生物学领域的新兴领导者。中央 假设PPZ/iHAP 1与AC异二聚体结合并变构改变其构象 从高分辨率的晶体结构中可以明显看出， 亚基很容易募集，从而驱动PP 2A-B56的形成和活化。 细胞中的磷酸酶。近期目标是确定三维 PP 2A全酶复合物的蛋白质结构响应于PPZ/iHAP而形成， 通过X射线确定PP 2A亚基与PPZ/iHAP化合物的分子接触 结晶学这一目标将在两个具体目标追求：1）确定三维 在PPZ或iHAP化合物的存在下分析PP 2A全酶复合物的结构， 这些小分子化合物对特异性PP 2A亚基复合物的变构活化的潜在机制 分子，和2）确定生物化学机制的能力变构 小分子PP 2A激活剂，以驱动PP 2A调节B亚基与 AC异源二聚体，从对照细胞中的Striatin家族蛋白到B56 α或B55α蛋白， iHAP/PPZ和SMAP。这一策略意义重大，因为结果可能揭示新的 特异性PP 2A磷酸酶复合物的变构激活机制， 将是解剖这个不同的磷酸酶家族的正常生理作用的关键， 了解它们作为肿瘤抑制因子的作用，以及它们在恶性肿瘤中是如何被破坏的， 转型本建议书旨在为候选人提供以下方面的高级培训： 结构生物学，以及指导手稿/赠款写作和领导素质。 在新的主要导师Eric Fischer博士的指导下， 共同导师托马斯看博士的贡献，和科学咨询委员会组成的 完成这项工作所需的专家，该提案应显着提高候选人的 他的职业目标是成为一名独立调查员。