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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.

项目总结人类细胞分裂在DNA复制之前的G1/S过渡期受到调控。细胞的主要驱动力在G1/S转变过程中，细胞周期蛋白依赖性蛋白激酶CDK4和CDK6与D型细胞周期蛋白形成复合体。该项目的目标是确定Cyclin D-CDK4/6复合体驱动细胞分裂的机制。这对癌症很重要，因为CDK4/6活性在许多癌症中都升高，包括乳腺癌、脑癌癌症、急性淋巴细胞性白血病和神经母细胞瘤。因此抑制Cyclin D-CDK4/6是一种针对这些癌症的治疗方法前景看好。在这里，我们建议确定Cyclin D-CDK4/6是如何驱动G1/S过渡。首先，我们将通过确定细胞中Cyclin D-CDK4/6激酶的所有靶点来完成这项工作使用化学遗传学方法。第二，我们将确定Cyclin D-CDK4/6使用的分子机制对接和磷酸化其主要靶点，视网膜母细胞瘤蛋白Rb。在初步数据中，我们发现 Cyclin D在Rb的C末端结合了一个α螺旋。我们现在的目标是确定Cyclin D的位置它结合了体外生物化学和结构生物学的方法，对接了RB的C-末端螺旋。我们建议利用对接相互作用的知识来开发一种扰乱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，并提供破坏这种相互作用的原则证明可以用于靶向癌症治疗。我们将继续追查识别工具化合物的两个方向。首先，我们将开发一种基于Rb的C-末端的多肽抑制剂螺旋。其次，我们将使用FRET实验筛选一个小分子文库。总而言之，拟议的实验将提供对Cyclin D-RB相互作用的深入分析，并将确定其如何驱动细胞组织。这项研究对Cyclin D分子对接机制的更广泛影响和对细胞周期蛋白D-CDK4/6底物可能是识别新的小分子来改善癌症治疗的方法。