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.

项目摘要 靶向失调蛋白激酶的小分子的发展导致范式转移 癌症的治疗。然而，选择性证明了对激酶抑制的新方法的需求 挑战和耐药性的出现。该建议探讨了腺苷的RNA编辑如何 作用于RNA（ADAR）酶的脱氨酶可用于调节癌症中的靶蛋白活性， 在特定序列上下文中促进编辑的指导寡核苷酸的优化。 Adar家族 酶的酶将腺苷转化为RNA中的肌苷，可以通过A将腺苷转化为靶腺苷 完全引导寡核苷酸。远处，ADAR酶的RNA编辑领域已重点放在 由于ADAR偏爱编辑腺丝 在终止密码子内。但是，已知赖氨酸密码子被ADAR脱离，并且特别是 由于蛋白激酶中存在保守的催化赖氨酸居住而引起的兴趣。赖氨酸的反应 带有ADAR2的密码子会产生精氨酸密码子：蛋白激酶中已知的灭活突变。开车 在激酶mRNA中的催化赖氨酸的编辑，这是一种已建立的理性设计方法 将使用寡核苷酸化学修饰。我们针对现场RNA编辑的策略具有较高的 治疗潜力是因为它利用内源性ADAR酶，因此仅需要递送 指南寡核苷酸。酶递送面临效率和免疫刺激的障碍，但 寡核苷酸疗法最近成功地递送了。该项目的中心假设 是指导寡核苷酸指导的蛋白激酶mRNA的编辑将导致下游细胞的减少 癌症中的信号传导。 AIM 1将定义指导RNA修改如何影响目标腺苷的编辑 通过寡核苷酸化学合成和体外活性评估的密码子。 AIM 2将评估蛋白质的效果 胶质母细胞瘤细胞中蛋白质磷酸化和凋亡的激酶mRNA编辑。细胞培养的培训， 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。 这种原则实验证明将通过建立有效性对癌症治疗具有广泛的影响 RNA编辑以调节无序蛋白活性。更广泛地这将扩大潜在疗法 通过定义如何设计指南RNA以非偏见的序列作用，ADAR编辑的应用 上下文。