Functional characterization of understudied protein kinases implicated in developmental disorders using zebrafish

使用斑马鱼研究与发育障碍有关的蛋白激酶的功能特征

• 批准号: 10217780
• 负责人: Gaurav K Varshney
• 金额: $ 17.48万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217780
• 关键词: Address; Animal Model; Behavior; Binding; Biological Models; CRISPR/Cas technology; Candidate Disease Gene; Cell model; Code; Collection; Custom; Cyclic GMP-Dependent Protein Kinases; Data; Development; Disease; Disease model; Drug Screening; Drug Targeting; Embryo; Embryonic Development; Ensure; Evaluation; FDA approved; Fertilization; Functional disorder; G-Protein-Coupled Receptors; Gene Mutation; Generations; Genes; Genome; Homeostasis; Human; Human Development; Human Genome; Image; In Situ Hybridization; International; Investments; Ion Channel; Ion Channel Protein; Knock-out; Knowledge; Lead; Libraries; Light; Malignant Neoplasms; Mediating; Metabolic Diseases; Methods; Modeling; Morphology; Mutagenesis; Mutation; Nervous system structure; Orthologous Gene; Pathology; Pattern; Pharmaceutical Preparations; Phenotype; Phosphotransferases; Protein Family; Protein Kinase; Proteins; Published Comment; Reproducibility; Research Personnel; Resources; Role; System; Testing; Tissues; United States National Institutes of Health; Vertebrates; Zebrafish; base; behavioral phenotyping; cell type; clinically relevant; developmental disease; disease phenotype; drug discovery; high throughput screening; human disease; imaging system; insight; kinase inhibitor; knock-down; knockout gene; loss of function; mRNA Expression; mutant; new therapeutic target; novel therapeutic intervention; novel therapeutics; programs; protein complex; screening; small molecule; spatiotemporal

Summary About 3000 protein-coding genes in the human genome encode druggable proteins that can bind to small molecules. Most of these “druggable genes” encode proteins that belong to just three protein families: protein kinases, non-olfactory G-protein-coupled receptors (GPCRs), and ion channels. Drug discovery projects have focused on well-studied proteins from these families. As a consequence, FDA-approved drugs target only a fraction of these ~3000 druggable genes. There is an urgent need to fill this gap and inform novel therapeutic strategies for human disease. To meet this need, the NIH’s Illuminating Druggable Genome (IDG) program has prioritized a list of over 300 understudied protein kinases, GPCRs, and ion channels. High throughput screens in diverse cell types and model organisms are a mainstay of drug discovery. Zebrafish are amenable to high throughput screening and, as vertebrates, can closely recapitulate the complexity of human development, homeostasis, and disease. These advantages make zebrafish uniquely well suited to uncover clinically relevant drugs. We and others have optimized the CRISPR/Cas9 mediated mutagenesis method to screen for phenotypes in the F0 generation by generating biallelic mutations. This breakthrough accelerated our ability to uncover the functional consequences of hundreds of mutants from months to days. Mutations in kinases can lead to diverse dysfunctions in humans, including cancer, aberrant development, and metabolic disorders. We analyzed 150 of the understudied kinase genes listed in RFA-RM-20-109 and identified 126 zebrafish orthologs. Of these 126 genes, mutations in 21 are known to cause developmental disorders in humans. The objective of this proposal is to establish zebrafish models for these 21 understudied genes that encode druggable kinases. We will achieve our objective and test our hypothesis via the following aims: 1) Generate a Library of Zebrafish Mutants Corresponding to 21 Candidate Developmental Disorder Genes. 2) Elucidate the functional consequences of gene knockouts during embryonic development. We expect that 8-10 of the 21 druggable genes will have early development phenotypes in zebrafish and generate disease models for further functional studies. Our library will be useful not only for studying disease pathology but also for functional testing of kinase inhibitors to elucidate their targets. Our mutant collection will facilitate drug screening to identify new therapeutics for many human diseases and will be widely available through the Zebrafish International Resource Center (ZIRC).

总结 人类基因组中大约有3000个蛋白质编码基因编码可药用蛋白质， 分子。大多数这些“可药物化的基因”编码的蛋白质只属于三个蛋白质家族： 激酶、非嗅觉G蛋白偶联受体（GPCR）和离子通道。药物发现项目有 专注于这些家族中经过充分研究的蛋白质。因此，FDA批准的药物仅针对 这大约3000个可药用基因的一部分。迫切需要填补这一空白，并告知新的治疗方法。 人类疾病的策略。为了满足这一需求，美国国立卫生研究院的照明可药用基因组（IDG）计划， 优先考虑了300多种未充分研究的蛋白激酶、GPCR和离子通道。高通量筛选 在不同的细胞类型和模式生物中的应用是药物发现的主要支柱。斑马鱼能够适应高 作为脊椎动物，可以紧密地概括人类发育的复杂性， 稳态和疾病。这些优势使斑马鱼非常适合发现临床相关的 毒品我们和其他人已经优化了CRISPR/Cas9介导的诱变方法，以筛选 通过产生双等位基因突变在F0代中改变表型。这一突破加快了我们的能力， 从几个月到几天揭示数百种突变体的功能后果。激酶的突变可以 导致人类多种功能障碍，包括癌症、异常发育和代谢紊乱。我们 分析了RFA-RM-20-109中列出的150个未充分研究的激酶基因，并鉴定了126个斑马鱼直系同源物。 在这126个基因中，已知21个基因的突变会导致人类发育障碍。的目标 这项提议是为这21个未充分研究的编码可药用激酶的基因建立斑马鱼模型。 我们将通过以下目标实现我们的目标并验证我们的假设：1）生成斑马鱼文库 对应于21个候选发育障碍基因的突变体。2)阐明功能 胚胎发育过程中基因敲除的后果。我们预计21种药物中的8-10种 基因将在斑马鱼中具有早期发育表型，并产生疾病模型，用于进一步的功能研究。 问题研究我们的库不仅对疾病病理学的研究有用，而且对激酶的功能测试也有用 抑制剂，以阐明其目标。我们的突变体收集将促进药物筛选，以确定新的治疗方法 许多人类疾病，并将通过斑马鱼国际资源中心广泛提供 （ZIRC）。