Chemogenetic control of kinase and phosphatase activity by modulating autoinhibition

通过调节自抑制对激酶和磷酸酶活性进行化学遗传学控制

• 批准号: 10195182
• 负责人: Michael Z. Lin
• 金额: $ 23.37万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10195182
• 关键词: Active Sites; Affinity; Animals; Behavior; Binding; Biomedical Research; CREB1 gene; Ca(2+)-Calmodulin Dependent Protein Kinase; Calcineurin; Calcium; Calmodulin; Catalytic Domain; Cell Surface Receptors; Cell Therapy; Cells; Cellular biology; Chemicals; Clinical; Complement; Complex; Dissociation; Drug Controls; Drug usage; Elements; Engineering; Enzyme Activation; Enzymes; Genetic Transcription; Heterodimerization; Holoenzymes; IL2 gene; Interleukin-2; Kinetics; Length; Lymphocyte; Mediating; Methods; Outcome; Pathway interactions; Peptides; Performance; Permeability; Pharmaceutical Preparations; Pharmacology; Phosphoric Monoester Hydrolases; Phosphotransferases; Positioning Attribute; Protein phosphatase; Proteins; Regulation; Role; Signal Pathway; Signal Transduction; Signaling Protein; Structure; System; T-Lymphocyte; Techniques; Testing; Work; base; calcineurin phosphatase; design; dimer; engineered T cells; enzyme activity; experimental study; gene therapy; improved; in vivo; inhibitor/antagonist; innovation; interest; next generation; novel; prevent; protein function; small molecule; tool

ABSTRACT Signaling enzymes such as kinases and phosphatases control multiple aspects of cellular differentiation and behavior, and are especially important in transducing signals from cell surface receptors to changes in cell fate or function. The ability to activate signaling proteins of interest using validated cell-permeable drugs would be immensely useful for studying the functions of these proteins in cells or animals, and could provide much-needed control over gene and cell therapies. Here, we propose a novel method for conferring chemical control over kinases and phosphatases based on drug-induced displacement of a tethered autoinhibitory domain (AID) from the active site. We will test and validate this method using the phosphatase calcineurin (CaN) and calcium/calmodulin kinase IV (CaMKIV), two enzymes that are natively inhibited by an AID and activated by a mechanism involving AID dissociation. In our method, we will use fused heterodimerizing elements to position the AIDs near the enzyme active site, then use small-molecule drugs to disrupt this interaction and displace the AIDs from the active site. We will carry out the following specific aims: (1) Creating drug-activated CaN using a chemically-dissociable autoinhibitory peptide, (2) Creating drug-activated CaMKIV using a chemically-dissociable autoinhibitory peptide, and (3) Examining roles of CaN and CaMKIV in IL-2 transcription in T cells using drug-activated proteins. Our design has several unique and innovative features. The single-chain design should improve reliability and reduce complexity over multi-component systems. The ability to rationally modulate linker length, heterodimer affinity, and AID-enzyme affinity provides multiple avenues for construct optimization. Multiple chemically dissociable interactions are known, allowing for multiplexed drug-controllable proteins. Finally, given that intramolecular AIDs should be low rather than high affinity, peptide inhibitors can be selected or designed for signaling enzymes that lack native AIDs. Our method of protein control by drug-induced displacement of an autoinhibitory domain should thus be uniquely useful, robust, and generalizable.

抽象的 信号酶如激酶和磷酸酶控制细胞分化的多个方面 行为，并且对于将细胞表面受体的信号转导至细胞命运的变化尤其重要 或函数。使用经过验证的细胞渗透性药物激活感兴趣的信号蛋白的能力将是 对于研究细胞或动物中这些蛋白质的功能非常有用，并且可以提供急需的 控制基因和细胞疗法。 在这里，我们提出了一种基于化学控制激酶和磷酸酶的新方法 药物诱导的束缚自抑制结构域 (AID) 从活性位点移位。我们将测试并 使用磷酸酶钙调神经磷酸酶 (CaN) 和钙/钙调蛋白激酶 IV (CaMKIV) 验证此方法，两个 天然被 AID 抑制并通过涉及 AID 解离的机制激活的酶。在我们的 方法，我们将使用融合的异二聚化元件将 AID 定位在酶活性位点附近，然后使用 小分子药物可以破坏这种相互作用并从活性位点取代 AID。我们将开展 以下具体目标：(1) 使用化学可解离的自抑制肽创建药物激活的 CaN， (2) 使用化学解离的自抑制肽创建药物激活的 CaMKIV，以及 (3) 检查 使用药物激活蛋白研究 CaN 和 CaMKIV 在 T 细胞 IL-2 转录中的作用。 我们的设计有几个独特和创新的特点。单链设计应提高可靠性和 降低多组件系统的复杂性。合理调节接头长度、异二聚体的能力 亲和力和 AID-酶亲和力为构建体优化提供了多种途径。多重化学 可解离的相互作用是已知的，允许多重药物可控蛋白质。最后，考虑到 分子内AID应该是低亲和力而不是高亲和力，可以选择或设计肽抑制剂 缺乏天然 AID 的信号酶。我们通过药物诱导的位移来控制蛋白质的方法 因此，自抑制域应该是独特有用的、稳健的和可推广的。