Inhibition of the CMG Helicase as Novel Anti-Neoplastic Approach

 描述(由申请人提供)：转化生长因子1是一种有效的细胞周期进程抑制剂，不仅可以在G1期早期诱导生长停滞，而且当加入细胞中时正好在S期开始之前。我们研究的目的是在机制水平上了解这种G1晚期的转化生长因子1抑制过程，并确定转化生长因子1作用于哪些酶以及如何作用。然后，我们使用这些信息来识别模拟抑制转化生长因子1靶标的小分子，并在抑制癌症生长方面提供临床应用。我们的团队已经确定了一个这样的靶点，CMG复制解旋酶，以及一个潜在的抑制解旋酶的方法。转化生长因子1能有效地阻断CMG解旋酶的激活，CMG解旋酶已经完全形成，可以促进G1-S转运。在转化生长因子1停滞的条件下，CMG与Rb蛋白处于物理复合体中，Rb蛋白是解旋酶保持不活跃所必需的。Rb直接与CMG的至少一个亚基MCM7相互作用，这种相互作用通过Rb的N端(RBN)和MCM7的C端(Mcm7CT)发生。我们的结果表明，Rb是CMG解旋酶的抑制剂，我们进一步证明了在非洲爪哇无细胞生化系统中，RBN可以抑制解旋酶。利用这个系统，我们还表明这是源于RBN的外显子7，该外显子在人类癌症中缺失。抑制CMG解旋酶为药物开发和癌症干预提供了一个创新的机会。癌细胞对CMG功能的抑制比正常细胞更敏感。抑制CMG功能还会增加肿瘤细胞对DNA复制抑制药物的敏感性，临床上有许多药物。因此，化学抑制CMG的方法具有增加现有抗肿瘤药物的治疗指数的倾向，并且仅此一项就可以提供一种有效和创新的方法来抑制肿瘤生长。该提案的具体目的将通过严格测试RBN和Exon7直接抑制纯化的CMG解旋酶的延伸和/或ATPase活性的能力和方法来扩展这些重要的观察结果。缺少关键结构域的RBN突变体将接受功能丧失测试，并将进行丙氨酸扫描突变，以确定外显子7中介导CMG抑制的重要残基。这些实验目标将有助于理解CMG是如何被调控的，特别是Exon7是如何实现对CMG的抑制的。我们还将建立有效的基于CMG荧光的分析方法，用于解旋酶化学抑制剂的靶向文库筛选，并与HT方法兼容。NCI多样性集IV文库将用于鉴定CMG解旋酶的少量抑制剂/探针，以便将来扩展到涉及HT分析的更大的药物发现项目。

 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.