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.

摘要