Inhibition of the CMG Helicase as Novel Anti-Neoplastic Approach

抑制 CMG 解旋酶作为新型抗肿瘤方法

基本信息

批准号：
8993839
负责人：
Mark G. Alexandrow
金额：
$ 21.99万
依托单位：
H. LEE MOFFITT CANCER CTR & RES INST
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-01-01 至 2017-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8993839
关键词：
ATP Hydrolysis ATP phosphohydrolase Alanine Appearance Baculoviruses Binding Biochemical Biological Assay Cancer Intervention Cell Cycle Progression Cells Chemicals Clinical Complex Cytolysis DNA DNA biosynthesis Enzymes Exons Fluorescence Fluorescence Polarization Future Goals Growth Human In Vitro Insecta Libraries Malignant Neoplasms Measurement Measures Mediating Mediator of activation protein Methods Molecular Monitor Mutagenesis N-terminal Normal Cell Peptides Pharmaceutical Preparations Preclinical Drug Evaluation Process Property Reader Research Retinoblastoma Protein S Phase Scanning System Testing Therapeutic Index Validation Xenopus base cancer cell cell growth drug development drug discovery helicase high throughput screening inhibitor/antagonist innovation insight loss of function mutant neoplastic neoplastic cell novel nucleic acid structure public health relevance research study screening small molecule small molecule inhibitor tumor growth

项目摘要

DESCRIPTION (provided by applicant): TGFß1 is a potent inhibitor of cell cycle progression and can elicit a growth arrest not only in early-G1, but also when added to cells just before S-phase begins. The goal of our research is to understand this late-G1 TGFß1- inhibitory process at the mechanistic level, and determine which enzymes TGFß1 targets acutely and how. We then use this information to identify small molecules that mimic inhibition of TGFß1 targets and offer clinical utility in suppressing cancer growth. Our group has identified one such target, the CMG replicative helicase, and a potential means to inhibit the helicase. TGFß1 acutely blocks activation of the CMG helicase, which is fully formed and ready to function in promoting G1-S transit. Under conditions of TGFß1 arrest, the CMG is in a physical complex with the Rb protein, which is required for the helicase to remain inactive. Rb directly interacts with at least one subunit of the CMG, Mcm7, and this interaction occurs via the N-terminus of Rb (RbN) and the C- terminus of Mcm7 (Mcm7CT). Our results indicate that Rb is an inhibitor of the CMG helicase, and we further demonstrate that RbN can inhibit the helicase in the Xenopus cell-free biochemical system. Using this system, we also show that this is derived from Exon7 of RbN, which is lost in human cancers. Inhibiting the CMG helicase presents an innovative opportunity for drug development and cancer intervention. Cancer cells are more sensitive than normal cells to inhibition of CMG function. Inhibiting CMG function also increases the sensitivity of tumor cells to DNA replication-suppressing drugs, of which there are many in the clinical arsenal. Thus, a means to chemically inhibit the CMG has the propensity to increase the therapeutic index of existing anti-neoplastic drugs, and alone can provide for an effective and innovative means to suppress tumor growth. The Specific Aims of this proposal will extend these important observations by rigorously testing the ability and means by which RbN and Exon7 directly inhibit elongation and/or ATPase activities of the purified CMG helicase. RbN mutants lacking critical domains will be tested for loss of function, and alanine-scanning mutagenesis will be performed to identify important residues within Exon7 that mediate CMG inhibition. These experimental goals will aid in understanding how the CMG is regulated, and specifically how Exon7 achieves inhibition of the CMG. We will also establish effective CMG fluorescence-based assays to be used in targeted library screening for chemical inhibitors of the helicase, and which are compatible with HT approaches. The NCI Diversity Set IV library will be used to identify a small number of inhibitors/probes of the CMG helicase, for future expansion into larger drug discovery projects involving HT analysis.

描述（由适用提供）：TGFß1是细胞周期进展的潜在抑制剂，不仅可以在早期G1中引起生长停滞，而且还可以在S相开始之前添加到细胞中时。我们研究的目的是在机械水平上了解G1TGFß1后期的抑制过程，并确定哪种酶TGFß1敏锐地靶向。然后，我们使用这些信息来识别模仿TGFß1靶标的小分子，并提供抑制癌症生长的临床实用性。我们的小组已经确定了一个这样的靶标，即CMG复制性解旋酶以及抑制解旋酶的潜在手段。 TGFß1急性阻断CMG解旋酶的激活，该酶完全形成并准备在促进G1-S传输中发挥作用。在TGFß1停滞的条件下，CMG与RB蛋白的物理复合物处于物理复合物中，这是解旋酶保持不活跃所必需的。 RB直接与CMG，MCM7的至少一个亚基相互作用，并且这种相互作用是通过RB（RBN）的N端和MCM7（MCM7CT）的C端进行的。我们的结果表明，RB是CMG解旋酶的抑制剂，我们进一步证明RBN可以抑制无爪蟾细胞中无细胞的生化系统中的解旋酶。使用此系统，我们还表明，这是从RBN的Exon7衍生而来的，RBN在人类癌症中丢失。抑制CMG解旋酶为药物开发和癌症干预提供了创新的机会。癌细胞比正常细胞对CMG功能更敏感。抑制CMG功能还增加了肿瘤细胞对DNA复制抑制药物的敏感性，其中临床库中有许多。这是化学抑制CMG的一种手段有望增加现有抗肿瘤药物的治疗指数，并且单独可以提供有效且创新的手段来抑制肿瘤生长。该提案的具体目的将通过严格测试RBN和EXON7直接抑制纯化CMG解旋酶的伸长伸长和/或ATPase活性的能力和手段来扩展这些重要观察结果。缺乏关键结构域的RBN突变体将测试功能丧失，并将执行丙氨酸扫描诱变，以识别Exon7中介导CMG抑制的重要残差。这些实验目标将有助于了解CMG的调节方式，特别是外部7如何实现CMG的抑制作用。我们还将建立有效的基于CMG荧光的测定法，以用于靶向图书馆筛选的解旋酶的化学抑制剂，并与HT方法兼容。 NCI多样性集IV库将用于识别CMG解旋酶的少量抑制剂/探针，以将来扩展到涉及HT分析的大型药物发现项目中。