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

基本信息

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
项目类别：
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

项目摘要

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).

概括人基因组中约有3000种蛋白质编码基因编码可与小的蛋白质相结合分子。这些“可毒基因”中的大多数编码仅属于三个蛋白质家族的蛋白质：蛋白质激酶，非野性G蛋白偶联受体（GPCR）和离子通道。毒品发现项目有专注于这些家庭的蛋白质良好的蛋白质。结果，FDA批准的药物仅针对这些约3000个可吸毒基因的部分。迫切需要填补这一空白并告知新颖疗法人类疾病的策略。为了满足这一需求，NIH的启发性可毒基因组（IDG）计划优先考虑了300多个理解的蛋白激酶，GPCR和离子通道的列表。高吞吐量屏幕在潜水员细胞类型和模型中，生物是药物发现的主要主体。斑马鱼很高吞吐量筛查和脊椎动物可以密切概括人类发展的复杂性，稳态和疾病。这些优势使斑马鱼独特地适合于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，以筛选通过产生双重突变，F0代的表型。这一突破加速了我们的能力从几个月到几天开始揭示数百个突变体的功能后果。激酶中的突变可以导致人类的潜水员功能障碍，包括癌症，异常发育和代谢障碍。我们分析了RFA-RM-20-109中列出的激酶基因的150个，并确定了126个斑马鱼直系同源物。在这126个基因中，已知21种突变会引起人类的发育障碍。目的该建议是为这21种编码可吸毒激酶的基因建立斑马鱼模型。我们将实现我们的目标，并通过以下目的检验我们的假设：1）生成斑马鱼库对应于21种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种吸毒中的8-10 基因将在斑马鱼中具有早期发育表型，并生成疾病模型以进一步的功能研究。我们的图书馆不仅可用于研究疾病病理学，还可用于激酶的功能测试抑制剂以阐明其目标。我们的突变收集将促进药物筛查以识别新疗法对于许多人类疾病，将通过斑马鱼国际资源中心广泛使用（锆）。