Inhibition of the CMG Helicase as Novel Anti-Neoplastic Approach

 描述（由申请人提供）：TGFβ1 是细胞周期进程的有效抑制剂，不仅可以在 G1 早期引起生长停滞，而且在 S 期开始之前添加到细胞中时也可以引起生长停滞。我们研究的目标是在机制水平上了解这种晚期 G1 TGFß1 抑制过程，并确定 TGFß1 敏锐地靶向哪些酶以及如何靶向。然后，我们利用这些信息来识别模拟抑制 TGFβ1 靶标的小分子，并在抑制癌症生长方面提供临床实用性。我们的小组已经确定了一个这样的目标，即 CMG 复制解旋酶，以及抑制解旋酶的潜在方法。 TGFβ1 急剧阻断 CMG 解旋酶的激活，该解旋酶已完全形成并准备好在促进 G1-S 转运中发挥作用。在 TGFβ1 停滞的情况下，CMG 与 Rb 蛋白形成物理复合物，这是解旋酶保持非活性所必需的。 Rb 直接与 CMG 的至少一个亚基 Mcm7 相互作用，并且这种相互作用通过 Rb 的 N 末端 (RbN) 和 Mcm7 的 C 末端 (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 RbN 可以抑制非洲爪蟾无细胞生化系统中的解旋酶。使用该系统，我们还表明它源自 RbN 的外显子 7，该外显子在人类癌症中丢失。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 功能还会增加肿瘤细胞对 DNA 复制抑制药物的敏感性，临床上有很多此类药物。因此，化学抑制CMG的方法有可能提高现有抗肿瘤药物的治疗指数，并且单独使用可以提供抑制肿瘤生长的有效且创新的方法。该提案的具体目标将通过严格测试 RbN 和 Exon7 直接抑制纯化的 CMG 解旋酶的延伸和/或 ATP 酶活性的能力和手段来扩展这些重要的观察结果。将测试缺乏关键结构域的 RbN 突变体的功能丧失，并进行丙氨酸扫描诱变以鉴定 Exon7 内介导 CMG 抑制的重要残基。这些实验目标将有助于了解 CMG 是如何调节的，特别是 Exon7 如何实现对 CMG 的抑制。我们还将建立有效的基于 CMG 荧光的检测方法，用于解旋酶化学抑制剂的靶向文库筛选，并且与 HT 方法兼容。 NCI Diversity Set IV 库将用于鉴定少量 CMG 解旋酶抑制剂/探针，以便将来扩展到涉及 HT 分析的更大药物发现项目。

 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.