Structure-based design of novel low molecular weight c-Myc inhibitors

描述(由申请人提供)：C-myc癌基因失控是人类癌症中最常见的分子异常之一。C-Myc是一种bHLH-ZIP转录因子，它与另一种bHLH-ZIP蛋白MAX的二聚化是转化和所有其他生物学活性所必需的。C-Myc对细胞增殖是必不可少的，大多数正常细胞是非周期的，因此不表达c-Myc，而c-Myc的短暂抑制可能足以实现永久的肿瘤消退，这使得以癌蛋白为靶点的治疗非常有吸引力。我们以前已经鉴定了7个低分子量化合物(“Myc化合物”)，它们可以消除/阻止bHLH-ZIP介导的c-Myc-Max异二聚化。其中一种化合物的非结构导向(即“无偏”)修饰导致了“第二代”类似物的产生，其效价提高了8倍，从而表明，即使在没有与其目标部位相关的化合物模型的情况下，化合物功效也可以获得显著的改善。为了以更合理、更有意义的方式鉴定更好的类似物，我们已经证明了我们原来的3个Myc化合物与固有无序的c-Myc bHLH-ZIP单体上的不同位点结合，改变了它的二级结构，并阻止了它与MAX的结合。剩下的4个化合物是多余的，并与其中一个位点结合。利用核磁共振波谱，我们已经生成了前三个化合物的结构模型，这些结构模型与含有同源结合位点的合成肽有关。因此，在具体目标1中，我们将利用计算方法和硅胶筛选中的高吞吐量来确定新的、更有效的“第三代”类似物，其结构是基于最初的3个创始成员的结构合理预测的。在具体目标2中，我们将比较这些新化合物抑制c-Myc-Max结合和c-Myc介导的肿瘤生长的能力。在具体目标3中，我们将对最有希望的类似物进行体内药理学测试，根据它们的“admet”特征优先考虑。最后，在特定目标4中，在特定目标2和3中确定的选定类似物将被化学连接，以生成能够同时与c-Myc bHLH-ZIP结构域上的两个不同位点结合的“第四代”化合物。这一建议具有高度的跨学科和互动性，因为它利用了儿科肿瘤学家/分子生物学家(Prochownik)、有机化学家(Metallo)、计算生物学家(Behar和Mustata)以及药理学家(Lazo、Eiseman和Egorin)的专业知识。来自两个机构的这个多样化、互补和协同的研究小组共同提出了关于Myc化合物的合理设计以及它们使这一关键癌蛋白失效的机制的新见解。与公共健康相关：这一高度集中的应用是来自两个机构的七名研究人员的多学科转化努力，目的是开发以c-Myc癌蛋白为靶点的合理设计的小分子。我们已经确定了三个直接与c-Myc单体上的不同位置结合并抑制其活性的化合物的三维核磁共振结构。以此为起点，我们建议设计和表征一系列更有效的类似物，其中一些将在化学上连接起来，以提供协同结合和生物活性。

DESCRIPTION (provided by applicant): De-regulation of the C-MYC oncogene is among the most frequent molecular abnormalities in human cancer. c-Myc is a bHLH-ZIP transcription factor whose dimerization with another bHLH-ZIP protein, Max, is necessary for transformation and all other biological activities. That c-Myc is indispensable for cell proliferation, that most normal cells are non-cycling and thus non-c-Myc-expressing, and that transient inhibition of c-Myc may be sufficient to achieve permanent tumor regression, makes therapeutic targeting of the oncoprotein highly appealing. We have previously identified 7 low molecular weight compounds that abrogate/prevent bHLH-ZIP-mediated c-Myc-Max heterodimerization ("Myc compounds"). Non structure- guided (i.e. "unbiased") modifications of one of these has resulted in the generation of "2nd generation" analogs with up to 8-fold enhanced potencies, thus demonstrating that significant improvements in compound efficacy can be obtained even in the absence of a model of the compound in association with its target site. In order to identify even better analogs in a more rational and meaningful way, we have demonstrated that 3 of our original Myc compounds bind to distinct sites on the intrinsically disordered c- Myc bHLH-ZIP monomer, alter its secondary structure, and prevent its association with Max. The remaining 4 compounds are redundant and bind to one of these sites. Using NMR spectroscopy, we have generated structural models of the first three compounds in association with synthetic peptides comprising their cognate binding sites. Thus, in Specific Aim 1, we will utilize computational methods and high throughput in silico screens to identify new and more potent "3rd generation" analogs whose structures are rationally predicated on those of the original 3 founding members. In Specific Aim 2, we will compare the abilities of these new compounds to inhibit c-Myc-Max association and c-Myc-mediated neoplastic growth. In Specific Aim 3, we will conduct in vivo pharmacologic testing of the most promising analogs prioritized by their "ADMET" profiles. Finally, in Specific Aim 4, selected analogs identified in Specific Aims 2 and 3 will be chemically linked in order to generate "4th generation" compounds capable of simultaneous binding to two distinct sites on the c-Myc bHLH-ZIP domain. This proposal is highly interdisciplinary and interactive in that it utilizes the expertise of a pediatric oncologist/molecular biologist (Prochownik), an organic chemist (Metallo), computational biologists (Behar and Mustata), and pharmacologists (Lazo, Eiseman and Egorin). Together, this diverse, complementary, and synergistic group of investigators, from two institutions, proposes studies that promise new insights regarding the rational design of Myc compounds and the mechanisms by which they disable this critical oncoprotein. PUBLIC HEALTH RELEVANCE: This highly focused application is a multidisciplinary translational effort by seven investigators from two institutions to develop rationally designed small molecules that target the c-Myc oncoprotein. We have already determined the 3D NMR structures of three compounds that bind directly to different sites on the c-Myc monomer and inhibit its activity. Using these as a starting point, we propose to design and characterize a series of more potent analogs, some of which will be chemically linked to provide synergistic binding and biological activities.