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 信号传导酶，例如激酶和磷酸酶控制细胞分化的多个方面， 行为，并且在将信号从细胞表面受体转换为细胞命运变化方面尤为重要 或功能。使用经过验证的细胞可渗透药物激活感兴趣的信号蛋白的能力将是 对于研究这些蛋白质在细胞或动物中的功能非常有用，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激酶和磷酸酶的化学控制的新方法 药物诱导的束缚自身抑制域（AID）的位移。我们将测试和 使用磷酸酶钙调蛋白（CAN）和钙/钙调蛋白激酶IV（CAMKIV）验证该方法，两个 艾滋病被辅助抑制并被涉及辅助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元素将艾滋病定位在酶活性位置附近，然后使用 小分子药物破坏这种相互作用并将艾滋病从活跃部位移位。我们将执行 以下特定目的：（1）创建药物激活可以使用化学可解散的自身抑制性肽， （2）使用化学可解散的自身抑制性肽创建药物激活的CAMKIV，（3）检查 使用药物激活的蛋白质在T细胞中CAN和CAMKIV在IL-2转录中的作用。 我们的设计具有几个独特而创新的功能。单链设计应提高可靠性和 降低对多组分系统的复杂性。合理调节接头长度，异二聚体的能力 亲和力和辅助酶亲和力为构造优化提供了多种途径。化学上多重 可分离的相互作用是已知的，允许多发性药物控制蛋白。最后，鉴于这一点 分子内艾滋病应低而不是高亲和力，可以选择或设计用于 缺乏天然艾滋病的信号酶。我们通过药物引起的蛋白质控制方法 因此，自身抑制域应具有独特的有用，鲁棒性和可推广。