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

描述（由申请人提供）：MYC是人类癌症中最常扩增的癌基因，发生在广泛的组织类型中，包括乳腺癌、肺癌和前列腺癌。MYC过表达发生在至少30%的人类癌症中，并且通常与不良临床结果和复发机会增加相关。据估计，每年有45万美国人被诊断患有MYC依赖性癌症。这些患者需要新的和有效的治疗策略。c-Myc是螺旋-环-螺旋转录因子，其通过与Max形成异二聚体、结合序列特异性DNA元件并刺激增殖相关基因的转录来驱动增殖细胞状态。转录因子是细胞状态的关键调节因子，因为它们控制驱动细胞类型特化的基因表达程序，并且通常是信号级联的末端组分。这些基因表达程序通常在疾病状态下去调节，使得转录因子成为用于治疗靶向的理想蛋白质类别。然而，大多数转录因子缺乏用于小分子结合的明确口袋，因此在很大程度上被认为是当前技术不可用的。化学生物学领域的一个主要挑战是开发有效的小分子转录因子抑制剂。虽然先前发表的工作已经确定了小分子抑制剂的c-Myc/Max异源二聚化使用截短的蛋白质在FRET和酵母双杂交测定，只有几千种化合物进行了筛选，在每种情况下，这些抑制剂的体外效力是有限的。事实上，在动物模型中，该效力未能转化为体内活性。一般来说，这些化合物不能满足可接受的化学探针的普遍认可的标准。迫切需要能够成功开发成化学探针的新化学型。）该研究旨在使用新技术鉴定c-Myc/Max二聚化和DNA结合的抑制剂。已经开发了一种稳健的高通量体外测定来筛选c-Myc/Max二聚化和随后与其DNA结合位点结合的抑制剂。已经开发了二级生物化学和细胞测定来验证来自初级筛选的命中并更详细地研究它们。通过MLPCN使用这些稳健的检测方法进行的大规模高通量筛选应可为生物学效应的开发和验证提供易于处理的结果。通过反复使用本文概述的下游测定来优化这些先导分子的药物化学提供了产生有用的化学探针以研究c-Myc功能的机会。这样的探针将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见解。

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.