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

描述（由申请人提供）：通过G-四链体相互作用的小分子DNA调节c-MYC转录最近发现G-四链体二级结构作为转录调节因子形成于近端启动子区域，并被认为是抗癌药物的一类新的分子靶标。具体而言，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元件（NHE）III 1中具有DNA G-四链体基序，其调节其总转录的80-95%。在c-MYC NHE III 1中形成的DNA G-四链体已被证明是转录沉默元件;结合并稳定G-四链体构象的化合物可降低c-MYC表达并具有抗肿瘤性。我们最近发现，NM 23-H2蛋白解折叠c-MYC启动子G-四链体以激活基因转录。然而，尽管c-MYC启动子G-四链体是第一个也是最广泛研究的系统，但对其与小分子和蛋白质的分子相互作用知之甚少。待检验的假设是c-MYC G-四链体相互作用化合物的生理功能不仅通过G-四链体而且通过G-四链体相互作用蛋白介导。我们已经鉴定了椭圆藤碱类似物作为我们的先导化合物用于进一步优化以靶向c-MYC启动子G-四链体。椭圆藤碱具有良好的“药物样”性质，并已显示出选择性结合c-MYC G-四链体。我们将使用NMR来理解与c-MYC G-四链体和ITC的分子相互作用，以表征药物结合的热力学贡献（目的1）。基于这些信息，我们将合理地设计和合成在C9，N2，N6和C3位置具有各种取代基的新的椭圆形碱类似物（目标2）。我们将使用生物化学、生物物理学和生物学测定来检查椭圆霉素对抑制NM 23-H2结合和c-MYC G-四链体的解折叠的影响以及它们在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椭圆果碱的特定G-四链体相互作用，从而抑制NM 23-H2蛋白和c-MYC转录。本研究的总体目标是建立椭圆霉素抑制c-MYC的构效关系和潜在的分子机制，并设计/合成新的类似物用于进一步的药物开发。具体目标是：1）确定椭圆霉素和相关分子与c-MYC G-四链体的分子相互作用的结构和热力学细节。2)设计并合成C9、N2、N6和C3取代的椭圆霉素类似物，并研究以c-MYC G-四链体为靶点的椭圆霉素类化合物的构效关系。3)确定椭圆形碱类似物如何调节NM 23-H2结合和c-MYC G-四链体的解折叠，以及这如何与c-MYC转录抑制相关。

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.