Chemogenetic control of kinase and phosphatase activity by modulating autoinhibition

摘要 激酶和磷酸酶等信号酶控制细胞分化的多个方面，并 行为，在从细胞表面受体向细胞命运的改变传递信号方面尤其重要 或功能。使用有效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 对基因和细胞疗法的控制。 在这里，我们提出了一种新的方法来赋予对激酶和磷酸酶的化学控制，基于 药物诱导的自抑制结构域(AID)从活性部位上移位。我们将测试和 使用磷酸酶钙调神经磷酸酶(CaN)和钙/钙调蛋白激酶IV(CaMKIV)验证该方法，两个 被AID天然抑制，并被AID解离机制激活的酶。在我们的 方法，我们将使用融合的异源二聚体元件将AIDS定位在酶活性部位附近，然后使用 小分子药物破坏这种相互作用，取代艾滋病的活性部位。我们将执行 具体目的如下：(1)使用化学上可解离的自身抑制肽来创造药物激活的CAN， (2)使用化学上可解离的自身抑制肽创建药物激活的CaMKIV，以及(3)检测 CaN和CaMKIV利用药物激活蛋白在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，而AID-酶亲和力为构建优化提供了多种途径。多种化学成分 解离的相互作用是已知的，允许多个药物控制的蛋白质。最后，鉴于 分子内艾滋病应该是低亲和力而不是高亲和力，可以选择或设计多肽抑制剂 缺乏天然艾滋病的信号酶。我们通过药物诱导的An置换来控制蛋白质的方法 因此，自抑制域应该是唯一有用的、健壮的和可推广的。

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.