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.

项目摘要在复制DNA之前，在G1/s转变处调节人细胞分裂。细胞的主要驱动因素通过G1/s的转变是与D型细胞周期蛋白复合物中的细胞周期蛋白依赖性激酶CDK4和CDK6。该项目的目的是确定Cyclin D-CDK4/6复合物用于驱动细胞分裂的机制。这对癌症很重要，因为在包括乳腺癌，大脑在内的许多癌症中CDK4/6的活性升高癌症，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-CDK4/6是针对这些癌症的疗法有希望的途径。在这里，我们建议确定细胞周期蛋白D-CDK4/6 驱动G1/S过渡。首先，我们将通过识别细胞中细胞周期蛋白D-CDK4/6激酶的所有靶标来做到这一点使用化学遗传方法。其次，我们将确定分子机制细胞周期蛋白D-CDK4/6使用对接并磷酸化其主要靶标，即视网膜细胞瘤蛋白RB。在初步数据中，我们发现了该细胞周期蛋白D在RB的C端结合α螺旋。我们现在旨在确定细胞周期蛋白D的位置这是使用体外生物化学和结构生物学方法组合的RB RB的C末端螺旋。我们建议使用这种对接交互的知识来开发一种工具化合物，以破坏交互作用并阻止细胞周期。这将允许测试细胞中相互作用的重要性，并提供破坏这种相互作用的原理可以用于靶向癌症治疗。我们将追求两个方向以识别工具化合物。首先，我们将根据RB的C端开发肽抑制剂螺旋。其次，我们将使用FRET分析筛选一个小分子库。两者一起，提议实验将对细胞周期蛋白D-RB相互作用提供深入的分析，并确定如何驱动细胞分配。这项研究对细胞周期蛋白D分子对接机制的更广泛影响和鉴定细胞周期蛋白D-CDK4/6底物可以是改善癌症治疗的新小分子的鉴定。