Site-directed RNA Editing to Modulate Kinase Activity as a Chemotherapeutic

通过定点 RNA 编辑调节激酶活性作为化疗药物

• 批准号: 10313239
• 负责人: Erin E Doherty
• 金额: $ 3.82万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10313239
• 关键词: Adenosine; Affect; Antineoplastic Agents; Apoptosis; Arginine; Base Pairing; Biological Assay; Cell Culture Techniques; Cell Death; Cells; Chemicals; Clinical; Codon Nucleotides; Complementary DNA; Crystallization; DNA; DRADA2b protein; Development; Dideoxy Chain Termination DNA Sequencing; Disease; Dose; Double-Stranded RNA; Drug Targeting; Drug resistance; Engineering; Enzymes; Face; Family; Fluorescence; Fluorescence Microscopy; Genetic Diseases; Glioblastoma; Guanosine; Guide RNA; Immunization; In Vitro; Inosine; Lead; Lysine; Malignant Neoplasms; Measures; Mediating; Messenger RNA; Methods; Modification; Mutation; Oligonucleotides; Oncology; Pathway interactions; Phosphoproteins; Phosphorylation; Phosphotransferases; Prevalence; Protein Analysis; Protein Kinase; Proteins; RNA; RNA Editing; Reaction; Research; Resolution; Roentgen Rays; Signal Transduction; Signal Transduction Pathway; Site; Structure; Techniques; Terminator Codon; Therapeutic; Therapeutic Uses; Time; Training; Tryptophan; Western Blotting; Work; adenosine deaminase; anti-cancer; base; cancer therapy; cell growth; chemical synthesis; design; experience; experimental study; in vitro activity; inhibitor/antagonist; interest; kinase inhibitor; mutant; novel; overexpression; preference; premature; programs; response; single molecule; small molecule; success; targeted delivery

PROJECT SUMMARY The development of small molecules that target dysregulated protein kinases led to a paradigm shift in the treatment of cancer. However, the need for novel methods of kinase inhibition is demonstrated by selectivity challenges and the emergence of drug resistance. This proposal explores how RNA editing by Adenosine Deaminase Acting on RNA (ADAR) enzymes can be used to modulate target protein activity in cancers, through the optimization of guide oligonucleotides that promote editing in specific sequence contexts. The ADAR family of enzymes converts adenosine to inosine in RNA, and can be directed to a target adenosine by a complementary guide oligonucleotide. Thus far, the field of RNA editing by ADAR enzymes has focused on correcting disease-causative premature termination codons, due to ADAR’s preference for editing adenosines within a termination codon. However, lysine codons are known to be deaminated by ADARs, and are of particular interest due to the presence of a conserved catalytic lysine residue in protein kinases. Reaction of the lysine codon with ADAR2 would produce an arginine codon: a known inactivating mutation in protein kinases. To drive editing at the catalytic lysine within kinase mRNA, an established rational design approach to guide oligonucleotide chemical modifications will be used. Our strategy for site-directed RNA editing has high therapeutic potential because it utilizes endogenous ADAR enzymes, and therefore only requires the delivery of a guide oligonucleotide. While enzyme delivery faces barriers in efficiency and immune stimulation, oligonucleotide therapies have had recent successes in targeted delivery. The central hypothesis of this project is that guide oligonucleotide-directed editing of protein kinase mRNA will lead to a reduction in downstream cell signaling in cancers. Aim 1 will define how guide RNA modifications affect editing of target adenosines in lysine codons via oligonucleotide chemical synthesis, and in vitro activity assays. Aim 2 will evaluate the effect of protein kinase mRNA editing on protein phosphorylation and apoptosis in glioblastoma cells. Training in cell culture, Western blotting, and fluorescence microscopy will enable the cellular analysis of protein kinase mRNA editing. This proof of principle experiment will have broad implications in cancer therapeutics by establishing the efficacy of RNA editing to modulate disordered protein activity. More broadly this will expand the potential therapeutic applications of ADAR editing by defining how guide RNAs can be engineered to act in non-preferred sequence contexts.

项目概要 针对失调蛋白激酶的小分子的开发导致了范式转变 癌症的治疗。然而，选择性证明了对激酶抑制新方法的需求 挑战和耐药性的出现。该提案探讨了腺苷如何编辑 RNA 作用于 RNA 的脱氨酶 (ADAR) 可用于调节癌症中的靶蛋白活性，通过 优化引导寡核苷酸，促进特定序列环境中的编辑。 ADAR 家族 酶将 RNA 中的腺苷转化为肌苷，并且可以通过 互补引导寡核苷酸。迄今为止，ADAR 酶的 RNA 编辑领域主要集中在 由于 ADAR 偏好编辑腺苷，因此纠正导致疾病的过早终止密码子 在终止密码子内。然而，已知赖氨酸密码子会被 ADAR 脱氨基，并且具有特殊的性质。 由于蛋白激酶中存在保守的催化赖氨酸残基而引起人们的兴趣。赖氨酸的反应 ADAR2 的密码子会产生精氨酸密码子：一种已知的蛋白激酶失活突变。开车 编辑激酶 mRNA 内的催化赖氨酸，这是一种既定的合理设计方法来指导 将使用寡核苷酸化学修饰。我们的定点 RNA 编辑策略具有很高的 治疗潜力，因为它利用内源性 ADAR 酶，因此只需要输送 引导寡核苷酸。虽然酶递送在效率和免疫刺激方面面临障碍， 寡核苷酸疗法最近在靶向递送方面取得了成功。本项目的中心假设 是引导寡核苷酸定向编辑蛋白激酶 mRNA 将导致下游细胞减少 癌症中的信号传导。目标 1 将定义引导 RNA 修饰如何影响赖氨酸中目标腺苷的编辑 通过寡核苷酸化学合成和体外活性测定获得密码子。目标 2 将评估蛋白质的效果 激酶 mRNA 编辑对胶质母细胞瘤细胞中蛋白质磷酸化和细胞凋亡的影响。细胞培养培训， 蛋白质印迹和荧光显微镜将使蛋白激酶 mRNA 编辑的细胞分析成为可能。 这项原理证明实验将通过确定疗效来对癌症治疗产生广泛的影响 RNA编辑来调节无序蛋白质活性。更广泛地说，这将扩大潜在的治疗 ADAR 编辑的应用，定义如何设计指导 RNA 以在非首选序列中发挥作用 上下文。