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：
10597161
负责人：
Jan M Skotheim
金额：
$ 23.44万
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/10597161
关键词：
Acute Lymphocytic Leukemia Address Alanine Alleles Binding Biochemistry Biological Assay Biological Sciences C-terminal Cancer Control Cancer Model Cell Cycle Arrest Cell Line Cell Proliferation Cell division Cells Clinical Trials Complex 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 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.

项目概要人类细胞分裂在 DNA 复制之前的 G1/S 转变中受到调节。通过 G1/S 转换的是细胞周期蛋白依赖性激酶 Cdk4 和 Cdk6 与 D 型细胞周期蛋白的复合物。该项目的目标是确定 Cyclin D-Cdk4/6 复合物用于驱动细胞分裂的机制。这对于癌症很重要，因为 Cdk4/6 活性在许多癌症中升高，包括乳腺癌、脑癌因此，抑制 Cyclin D-Cdk4/6 是癌症、急性淋巴细胞白血病和神经母细胞瘤的一种治疗方法。在此，我们建议确定 Cyclin D-Cdk4/6 如何发挥作用。首先，我们将通过识别细胞中 Cyclin D-Cdk4/6 激酶的所有靶标来实现这一点。其次，我们将确定 Cyclin D-Cdk4/6 使用的分子机制。在初步数据中，我们发现了其主要靶标视网膜母细胞瘤蛋白 Rb 的对接和磷酸化。 Cyclin D 在 Rb C 末端结合 α 螺旋我们现在的目标是确定 Cyclin D 上的位置。结合体外生物化学和结构生物学方法，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我们将追求破坏这种相互作用可用于靶向癌症治疗的原理证明。确定工具化合物的两个方向首先，我们将开发基于 Rb C 端的肽抑制剂。其次，我们将使用 FRET 分析筛选小分子文库。实验将深入分析 Cyclin D-Rb 相互作用，并确定它如何驱动细胞这项研究对Cyclin D分子对接机制和鉴定产生了更广泛的影响。 Cyclin D-Cdk4/6 底物可能是改善癌症治疗的新小分子的鉴定。