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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：
9189594
负责人：
Mark G. Alexandrow
金额：
$ 18.71万
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/9189594
关键词：
ATP Hydrolysis ATP phosphohydrolase Acute Alanine Antineoplastic Agents Appearance Baculoviridae Baculoviruses Binding Biochemical Biological Assay Cancer Intervention Cell Cycle Progression Cells Chemicals Clinical Complex DNA DNA biosynthesis Enzymes Exons Fluorescence Fluorescence Polarization Future Goals Growth Growth Factor Human In Vitro Insecta Libraries Malignant Neoplasms Measurement Measures Mediating Mediator of activation protein Methods Molecular Monitor Mutagenesis N-terminal Normal Cell Nucleic Acid Binding Peptides Pharmaceutical Preparations Preclinical Drug Evaluation Process Property Reader Research Retinoblastoma Protein S Phase Scanning Structure System Testing Therapeutic Index Validation Xenopus base cancer cell cell growth drug development drug discovery experimental study helicase high throughput screening inhibitor/antagonist innovation insight loss of function mutant neoplastic cell novel novel anticancer drug public health relevance 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（由申请人提供）：TGF β 1是细胞周期进程的有效抑制剂，并且不仅在早期G1中，而且在S期开始之前加入细胞中时，可以引起生长停滞。我们研究的目标是在机制水平上了解这种G1晚期TGF β 1抑制过程，并确定TGF β 1急性靶向哪些酶以及如何靶向。然后，我们使用这些信息来鉴定模拟抑制TGF β 1靶点的小分子，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目标，CMG复制解旋酶，和一个潜在的手段来抑制解旋酶。TGF β 1急性阻断CMG解旋酶的激活，CMG解旋酶完全形成并准备在促进G1-S转运中起作用。在TGF β 1停滞的条件下，CMG与Rb蛋白形成物理复合物，这是解旋酶保持失活所必需的。Rb与CMG的至少一个亚基Mcm 7直接相互作用，并且这种相互作用经由Rb的N-末端（RbN）和Mcm 7的C-末端（Mcm 7 CT）发生。我们的研究结果表明，Rb是CMG解旋酶的抑制剂，我们进一步证明，RbN可以抑制解旋酶在非洲爪蟾无细胞生化系统。使用这个系统，我们还表明，这是来自外显子7的RbN，这是在人类癌症中丢失。抑制CMG解旋酶为药物开发和癌症干预提供了创新机会。癌细胞比正常细胞对CMG功能的抑制更敏感。抑制CMG功能也增加了肿瘤细胞对DNA复制抑制药物的敏感性，临床上有很多这种药物。因此，化学抑制CMG的手段具有增加现有抗肿瘤药物的治疗指数的倾向，并且单独地可以提供抑制肿瘤生长的有效和创新的手段。本提案的具体目的将通过严格测试RbN和外显子7直接抑制纯化CMG解旋酶的延伸和/或ATP酶活性的能力和方法来扩展这些重要的观察结果。将检测缺乏关键结构域的RbN突变体的功能丧失，并进行丙氨酸扫描诱变，以鉴定外显子7内介导CMG抑制的重要残基。这些实验目标将有助于理解CMG是如何调节的，特别是外显子7如何实现CMG的抑制。我们还将建立有效的CMG荧光为基础的测定用于靶向文库筛选的化学抑制剂的解旋酶，这是兼容的HT方法。NCI Diversity Set IV文库将用于鉴定CMG解旋酶的少量抑制剂/探针，用于未来扩展到涉及HT分析的更大药物发现项目。