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

描述(申请人提供)：最近发现G-四链相互作用的小分子DNA G-四链二级结构作为转录调节形成在近端的启动子区域，被认为是一类新的抗癌药物的分子靶点。具体地说，c-myc是人类癌症中最常见的非调控基因之一，它在启动子核酸酶超敏元件(NHE)III1中有一个DNA G-四链基序，调控其总转录的80%-95%。在c-myc NHE III1中形成的DNA G-四链被证明是一个转录沉默元件；与G-四链构象结合并稳定的化合物可以减少c-myc的表达，并具有抗肿瘤作用。我们最近发现，NM23-H2蛋白通过激活c-myc启动子G-四链来激活基因转录。然而，尽管c-myc启动子G-四链是第一个也是研究最广泛的系统，但人们对其与小分子和蛋白质的分子相互作用知之甚少。需要检验的假设是，c-myc G-四链相互作用化合物的生理功能不仅通过G-四链，而且还通过G-四链相互作用蛋白来调节。我们已经确定了一种椭圆碱类似物作为我们的先导化合物，用于进一步优化靶向c-myc启动子G-四链的目标。椭圆素具有良好的“类药物”性质，已被证明能选择性地结合c-myc G-四链。我们将使用核磁共振来了解与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(目标1)。根据这些信息，我们将合理地设计和合成具有不同取代基的新的椭圆碱类似物，这些取代基分别位于C9、N2、N6和C3位置(目标2)。我们将使用生化、生物物理和生物学方法来检测椭圆素对c-myc G-四链的NM23-H2结合和展开的抑制作用以及它们在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碱特定的G-四链相互作用，这些相互作用导致抑制NM23-H2蛋白和抑制c-myc转录。本研究的总体目标是建立椭圆素类化合物抑制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)确定椭圆碱及其相关分子与c-myc G-四链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取代的椭圆碱类似物，并研究以c-myc G-四链体为靶点的椭圆碱的构效关系(SAR)。3)确定椭圆碱类似物如何调节c-myc G-四链的NM23-H2结合和解折叠，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.