Discovery of small molecule inhibitors of c-Myc/Mac dimerization and DNA binding

描述(申请人提供)：MYC是人类癌症中最常被扩增的癌基因，发生在包括乳腺癌、肺癌和前列腺癌在内的多种组织类型中。MYC的过度表达发生在至少30%的人类癌症中，并且经常与不良的临床结果和增加复发的机会相关。据估计，每年有45万美国人被诊断出患有MYC依赖型癌症。这些患者需要新的有效的治疗策略。C-Myc是一种螺旋-环-螺旋转录因子，通过与MAX形成异源二聚体，结合序列特异性DNA元件，刺激增殖相关基因的转录，从而驱动细胞增殖状态。转录因子是细胞状态的关键调节因子，因为它们控制着驱动细胞类型规范的基因表达程序，通常是信号级联的末端组件。这些基因表达程序通常在疾病状态下被解除调控，使转录因子成为治疗靶向的理想蛋白质类别。然而，大多数转录因子缺乏明确的小分子结合口袋，因此在很大程度上被认为是当前技术无法治愈的。化学生物学领域的一个主要挑战是开发有效的转录因子小分子抑制剂。虽然以前发表的工作已经在FRET和酵母双杂交试验中使用截断蛋白确定了c-Myc/Max异源二聚的小分子抑制剂，但在每种情况下只筛选了数千个化合物，这些抑制剂的体外效力有限。事实上，在动物模型中，这种效力未能转化为体内的活动。一般来说，这些化合物不符合公认的可接受的化学探针标准。迫切需要新的化学类型，可以成功地开发成化学探针。)拟议的研究旨在利用新技术识别c-Myc/Max二聚化和DNA结合的抑制剂。建立了一种可靠的高通量体外试验来筛选c-Myc/Max二聚化的抑制剂以及随后与其DNA结合位点的结合。已经开发了二级生化和细胞分析来验证来自初级筛查的命中并对它们进行更详细的研究。可以通过MLPCN使用这些稳健的分析方法提供的大的高通量筛查应该为生物效应的开发和验证提供易于处理的点击。药物化学通过迭代使用这里概述的下游分析来优化这些先导分子，然后提供了产生有用的化学探针来研究c-Myc功能的机会。这些探针有望引领针对这种典型癌症靶点的新疗法，并为直接抑制转录因子的机制提供洞察力。 公共卫生相关性：c-Myc功能失控导致了至少30%的人类癌症，并经常与不良的临床结果和复发机会增加相关。然而，临床上有用的c-Myc功能抑制剂还没有被开发出来治疗这些患者。这项拟议的研究旨在开发c-Myc功能的直接抑制剂，这可能会引领针对这种典型致癌蛋白的新疗法。))

DESCRIPTION (provided by applicant): MYC is the most frequently amplified oncogene in human cancers occurring in a wide range of tissue types including breast, lung, and prostate. MYC overexpression occurs in at least 30% of all human cancers and frequently correlates with poor clinical outcome and increased chance of relapse. An estimated 450,000 Americans are diagnosed with a MYC-dependent cancer each year. These patients are in need of novel and effective treatment strategies. c-Myc is a helix-loop-helix transcription factor that drives a proliferative cell state by forming a heterodimer with Max, binding sequence-specific DNA elements and stimulating transcription of proliferation-associated genes. Transcription factors are key regulators of cell state as they control the gene expression programs that drive cell type specification and commonly are terminal components of a signaling cascade. These gene expression programs are often deregulated in disease states making transcription factors an ideal class of proteins for therapeutic targeting. However, most transcription factors lack clear pockets for small molecule binding and therefore have been largely considered undruggable with current technologies. A major challenge in the chemical biology field has been to develop potent small molecule inhibitors of transcription factors. While previously published work has identified small molecule inhibitors of c-Myc/Max heterodimerization using truncated proteins in FRET and yeast two-hybrid assays, only a few thousand compounds were screened in each case and the in vitro potency of these inhibitors is limited. Indeed, the potency fails to translate to in vivo activity in animal models. In general, these compounds fail to meet the generally agreed upon criteria for acceptable chemical probes. New chemotypes, which can be successfully developed into chemical probes, are desperately needed.)The proposed research aims to identify inhibitors of c-Myc/Max dimerization and DNA binding using novel technology. A robust high-throughput in vitro assay has been developed to screen for inhibitors of c-Myc/Max dimerization and subsequent binding to its DNA binding site. Secondary biochemical and cellular assays have been developed to validate hits from the primary screen and study them in greater detail. A large high-throughput screen as could be provided through the MLPCN using these robust assays should provide tractable hits for development and validation of biological effect. Medicinal chemistry optimizing these lead molecules through iterative use of downstream assays outlined herein then provides the opportunity to generate useful chemical probes to study c-Myc function. Such probes will hopefully lead the way to new therapeutics against this quintessential cancer target and offer insights into mechanisms for directly inhibiting transcription factors. PUBLIC HEALTH RELEVANCE: Deregulated c-Myc function drives at least 30% of all human cancers and frequently correlates with poor clinical outcome and increased chance of relapse. However, clinically useful inhibitors of c-Myc function have not been developed to treat these patients. The proposed research aims to develop direct inhibitors of c-Myc function, which could lead the way to new therapeutics against this quintessential oncogenic protein. ) )