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

摘要 人类基因组中约3000个蛋白质编码基因编码可与小分子药物结合的蛋白质。 分子。这些“可药物基因”中的大多数编码的蛋白质只属于三个蛋白质家族：蛋白质 激酶、非嗅觉G蛋白偶联受体(GPCRs)和离子通道。药物发现项目已经 重点研究了这些家族中研究得很好的蛋白质。因此，FDA批准的药物只针对 这些约3000个可用药基因的一部分。迫切需要填补这一空白，并提供新的治疗方法 人类疾病的战略。为了满足这一需求，美国国立卫生研究院的启发性可药物基因组(IDG)计划 对300多个未被研究的蛋白激酶、GPCRs和离子通道进行了优先排序。高通量筛网 在不同的细胞类型和模型中，生物是药物发现的支柱。斑马鱼容易兴奋 作为脊椎动物，吞吐量筛选可以很好地概括人类发育的复杂性， 动态平衡和疾病。这些优势使斑马鱼独一无二地非常适合揭示临床相关 毒品。我们和其他人已经优化了CRISPR/Cas9介导的突变方法来筛选 通过产生双等位基因突变在F0代中的表型。这一突破加速了我们的能力 从几个月到几天揭示数百个突变体的功能后果。激酶的突变可以 导致人类多种功能障碍，包括癌症、发育异常和代谢紊乱。我们 分析了RFA-RM-20-109中列出的150个未被研究的激酶基因，并鉴定了126个斑马鱼同源基因。 在这126个基因中，已知有21个基因的突变会导致人类发育障碍。的目标是 这项提议是为这21个未被研究的编码可药物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)建立斑马鱼库 与21个候选发育障碍基因对应的突变体。2)阐明功能 胚胎发育过程中基因敲除的后果。我们预计21个可用药中有8-10个 基因将在斑马鱼中具有早期发育表型，并为进一步的功能产生疾病模型 学习。我们的文库将不仅用于研究疾病病理学，而且还将用于激酶的功能测试 抑制剂来阐明它们的靶标。我们的突变体收集将促进药物筛选，以确定新的治疗方法 针对许多人类疾病，将通过斑马鱼国际资源中心广泛提供 (Zirc)。

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).