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: 10371123
• 负责人: Michael Z. Lin
• 金额: $ 19.5万
• 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/10371123
• 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; innovation; interest; next generation; novel; prevent; protein function; rational design; 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.

摘要 信号传导酶如激酶和磷酸酶控制细胞分化的多个方面， 行为，并在从细胞表面受体到细胞命运变化的信号转导中特别重要 或功能。使用经验证的细胞可渗透药物激活感兴趣的信号传导蛋白的能力将是可行的。 对于研究这些蛋白质在细胞或动物中的功能非常有用，并可以提供急需的 控制基因和细胞疗法。 在这里，我们提出了一种新的方法，赋予化学控制激酶和磷酸酶的基础上， 药物诱导的栓系自抑制结构域（AID）从活性位点的置换。我们将测试和 使用磷酸酶钙调磷酸酶（CaN）和钙/钙调蛋白激酶IV（CaMKIV）验证该方法， 被AID天然抑制并被涉及AID解离的机制激活的酶。在我们 方法，我们将使用融合的异源二聚化元件将AIDS定位在酶活性位点附近，然后使用 小分子药物来破坏这种相互作用，并将艾滋病从活性部位置换出来。创新实施 具体目的如下：（1）使用化学可解离的自抑制肽产生药物活化的CaN， (2)使用化学可解离的自抑制肽产生药物活化的CaMKIV，以及（3）检查 CaN和CaMKIV在使用药物活化蛋白的T细胞中的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， 降低多组分系统的复杂性。合理调节接头长度、异二聚体 亲和性和AID-酶亲和性为构建体优化提供了多种途径。多化学 可解离的相互作用是已知的，允许多重药物可控的蛋白质。最后，鉴于 分子内AIDS应该是低亲和力而不是高亲和力的，可以选择或设计肽抑制剂用于 缺乏天然艾滋病的信号酶。我们的方法是通过药物诱导的蛋白质置换来控制蛋白质， 因此，自身抑制结构域应该是唯一有用的、稳健的和可推广的。