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Project 1: Determine the mechanisms Cyclin D-Cdk4/6 uses to drive cell proliferation

项目 1：确定 Cyclin D-Cdk4/6 驱动细胞增殖的机制

基本信息

批准号：
10332380
负责人：
Jan M Skotheim
金额：
$ 27.65万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-03-25 至 2027-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10332380
关键词：
Acute Lymphocytic Leukemia Address Alanine Alleles Binding Biochemistry Biological Assay Biological Sciences C-terminal Cancer Model Cell Cycle Arrest Cell Line Cell Proliferation Cell division Cells Clinical Trials Complex Crystallization Cyclin D1 Cyclin-Dependent Kinases Cyclins DNA Data Docking Engineering Enzymes Fluorescence Polarization Fluorescence Resonance Energy Transfer G1/S Transition Goals Human In Vitro Knowledge Libraries Location Malignant Neoplasms Malignant neoplasm of brain Molecular Mus Mutation Neuroblastoma Peptides Pharmaceutical Preparations Phenotype Phosphorylation Phosphotransferases Property Protein Kinase Proteomics Reagent Regulation Retinoblastoma Protein Role Site Structure Testing Therapeutic Xenograft procedure alpha helix analog base cancer cell cancer therapy chemical genetics delivery vehicle design experimental study genetic approach improved in vitro testing in vivo inhibitor malignant breast neoplasm molecular imaging mouse model phosphoproteomics small molecule small molecule libraries structural biology targeted cancer therapy targeted treatment therapeutic target tool tumor tumor growth

项目摘要

PROJECT SUMMARY Human cell division is regulated at the G1/S transition before DNA is replicated. The primary drivers of cells through the G1/S transition are the cyclin-dependent kinases Cdk4 and Cdk6 in complex with D-type cyclins. The goal of this project is to determine the mechanisms that Cyclin D-Cdk4/6 complexes use to drive cell division. This is important for cancer because Cdk4/6 activity is elevated in many cancers including breast cancers, brain cancers, acute lymphoblastic leukemia, and neuroblastoma tumors. Inhibiting Cyclin D-Cdk4/6 is therefore a promising avenue for therapies targeting these cancers. Here, we propose to determine how Cyclin D-Cdk4/6 drives the G1/S transition. First, we will do this by identifying all the targets of Cyclin D-Cdk4/6 kinases in cells using a chemical genetic approach. Second, we will determine the molecular mechanism Cyclin D-Cdk4/6 uses to dock and phosphorylate its primary target, the retinoblastoma protein Rb. In preliminary data, we discovered that Cyclin D binds an alpha helix at the C-terminus of Rb. We now aim to determine the location on Cyclin D that docks Rb’s C-terminal helix using a combination of in vitro biochemistry and structural biology approaches. We propose to use knowledge of this docking interaction to develop a tool compound that disrupts the interaction and arrests the cell cycle. This will allow testing of the importance of the interactions in cells and provides a proof-of-principle that disrupting this interaction could be used in targeted cancer therapeutics. We will pursue two directions to identify a tool compound. First, we will develop a peptide inhibitor based on Rb’s C-terminal helix. Second, we will screen a small molecule library using a FRET assay. Taken together, the proposed experiments will provide an in-depth analysis of the Cyclin D-Rb interaction and will determine how it drives cell division. The broader impact of this study of the Cyclin D molecular docking mechanism and identification of Cyclin D-Cdk4/6 substrates may be the identification of new small molecules that improve cancer therapy.

项目摘要人类细胞分裂在DNA复制之前的G1/S转换时受到调节。细胞的主要驱动力通过G1/S转变的是与D型细胞周期蛋白复合的细胞周期蛋白依赖性激酶Cdk 4和Cdk 6。该项目的目标是确定细胞周期蛋白D-Cdk 4/6复合物用于驱动细胞分裂的机制。这对癌症很重要，因为Cdk 4/6活性在许多癌症中升高，包括乳腺癌、脑癌、乳腺癌和乳腺癌。癌症、急性淋巴细胞白血病和神经母细胞瘤肿瘤。因此，抑制细胞周期蛋白D-Cdk 4/6是一种有效的方法。有希望的针对这些癌症的治疗方法。在这里，我们建议确定细胞周期蛋白D-Cdk 4/6 推动G1/S过渡。首先，我们将通过鉴定细胞中细胞周期蛋白D-Cdk 4/6激酶的所有靶点来实现这一点使用化学遗传学方法。其次，我们将确定Cyclin D-Cdk 4/6的分子机制对接并磷酸化它的主要目标，视网膜母细胞瘤蛋白Rb。在初步数据中，我们发现细胞周期蛋白D在Rb的C末端结合α螺旋。我们现在的目标是确定细胞周期蛋白D的位置利用体外生物化学和结构生物学方法的结合，对接Rb的C-末端螺旋。我们建议利用这种对接相互作用的知识来开发一种破坏这种相互作用的工具化合物并阻止细胞周期这将允许测试细胞中相互作用的重要性，并提供了一种新的方法。原理证明，破坏这种相互作用可以用于靶向癌症治疗。我们将奉行两个方向来识别工具化合物。首先，我们将开发一种基于Rb的C-末端的肽抑制剂，螺旋。其次，我们将使用FRET测定筛选小分子文库。综合考虑，实验将提供细胞周期蛋白D-Rb相互作用的深入分析，并将确定它如何驱动细胞师.本研究对细胞周期蛋白D的分子对接机制和鉴定具有更广泛的影响，细胞周期蛋白D-Cdk 4/6底物可能是识别新的小分子，改善癌症治疗。