Modulating c-Myc transcription by G-quadruplex-interactive small molecules

描述（由申请人提供）：通过g -四重体相互作用小分子DNA调节c-MYC转录最近发现g -四重体二级结构作为转录调节剂在近端启动子区域形成，被认为是抗癌药物的一类新的分子靶点。具体来说，c-MYC是人类癌症中最常见的失调基因之一，在启动子核酸酶超敏感元件（NHE） III1中具有DNA g -四重基序，该基序调节其总转录的80-95%。在c-MYC NHE III1中形成的DNA g -四重体已被证明是转录沉默元件；结合并稳定g -四重构象的化合物可以减少c-MYC的表达并具有抗肿瘤作用。我们最近发现NM23-H2蛋白展开c-MYC启动子g -四重体来激活基因转录。然而，尽管c-MYC启动子g -四重体是第一个也是研究最广泛的系统，但对其与小分子和蛋白质的分子相互作用知之甚少。要验证的假设是，c-MYC g -四重体相互作用化合物的生理功能不仅通过g -四重体介导，而且通过g -四重体相互作用蛋白介导。我们已经确定了一个椭圆类似物作为我们的先导化合物，以进一步优化c-MYC启动子G-四重体。Ellipticine具有良好的“药物样”性质，并已被证明可以选择性地结合c-MYC g-四重体。我们将使用核磁共振来了解与c- MYC g -四重体的分子相互作用，并使用ITC来表征药物结合的热力学贡献（目的1）。基于这些信息，我们将合理设计和合成新的具有不同取代基的C9， N2， N6和C3位置的椭圆类似物（Aim 2）。我们将使用生化、生物物理和生物分析来检验椭圆素对抑制NM23-H2结合和c-MYC g -四重体展开的影响，以及它们在c-MYC转录抑制中的有效性。结构和生物学研究的结合将使我们能够了解Ellipticine导致NM23-H2蛋白抑制和c-MYC转录的特定g -四重体相互作用。本研究的总体目标是建立椭圆类药物抑制c-MYC的构效关系和潜在的分子机制，并为进一步的药物开发设计/合成新的类似物。具体目的是：1)确定椭圆和相关分子与c-MYC G-四重体相互作用的结构和热力学细节。2)设计和合成新的C9-、N2-、N6-和c3取代的椭圆类似物，并研究针对c-MYC G-四联体的椭圆类似物的构效关系（SAR）。3)确定Ellipticine类似物如何调节NM23-H2结合和c-MYC g -四重体的展开，以及这与c-MYC转录抑制的关系。

DESCRIPTION (provided by applicant): Modulating c-MYC Transcription by G-quadruplex-interactive Small Molecules DNA G-quadruplex secondary structures have recently been found to form in proximal promoter regions as transcriptional regulators, and are considered as a new class of molecular targets for anticancer drugs. Specifically, c-MYC, one of the most commonly deregulated genes in human cancers, has a DNA G-quadruplex motif in the promoter Nuclease Hypersensitive Element (NHE) III1 which regulates 80-95% of its total transcription. The DNA G-quadruplex formed in the c-MYC NHE III1 has been shown to be a transcriptional silencer element; compounds that bind to and stabilize the G-quadruplex conformation can reduce c-MYC expression and are anti- tumorigenic. We have recently discovered that the NM23-H2 protein unfolds the c-MYC promoter G-quadruplex to activate gene transcription. However, although the c-MYC promoter G-quadruplex is the first and most extensively studied system, little is known about its molecular interactions with small molecules and proteins. The hypothesis to be tested is that the physiological functions of c-MYC G-quadruplex-interactive compounds are mediated through not only the G-quadruplex but also the G-quadruplex-interactive protein. We have identified an Ellipticine analog as our lead compound for further optimization to target the c-MYC promoter G- quadruplex. Ellipticine has good "drug-like" properties and has been shown to selectively bind the c-MYC G-quadruplex. We will use NMR to understand the molecular interactions with the c- MYC G-quadruplex and ITC to characterize the thermodynamic contributions of drug binding (Aim 1). Based on this information, we will rationally design and synthesize new Ellipticine analogs with various substituents at C9, N2, N6, and C3 positions (Aim 2). We will use biochemical, biophysical, and biological assays to examine the effects of the Ellipticines on inhibiting NM23-H2 binding and unfolding of the c-MYC G-quadruplex and their effectiveness in c-MYC transcriptional suppression. A combination of structural and biological studies will allow us to understand the specific G-quadruplex interactions of Ellipticine that lead to inhibition of the NM23-H2 protein and suppression of c-MYC transcription. The overall objectives of this research are to establish the structure-activity relationship and underlying molecular mechanism of Ellipticines for c-MYC suppression and to design/synthesize new analogs for further drug development. The specific aims are: 1) To determine structural and thermodynamic details of molecular interactions of Ellipticines and related molecules with the c-MYC G- quadruplex. 2) To design and synthesize new C9-, N2-, N6-, and C3-substituted Ellipticine analogs and to study structure-activity relationship (SAR) of Ellipticines targeting the c-MYC G- quadruplex. 3) To determine how Ellipticine analogs modulate NM23-H2 binding and unfolding of the c-MYC G-quadruplex, and how this correlates with c-MYC transcriptional suppression.