High-Throughput Screen for the Oncoprotein MYC

摘要 MYC是参与细胞增殖的关键转录调节因子，已在转录调控中发挥作用。 伸长和起始、microRNA调节、细胞凋亡和多能性。更重要的是，MYC一直在 直接与50%以上的人类癌症有关，被认为是癌症发生的一般标志 和维护。尽管这种情况很普遍，但针对MYC的功能性化学探针很少，而且没有 以此为目标的治疗学。我们发现了一种化合物KJ-PYR-9，它能高效地与MYC结合，并 特异性，下调MYC的转录活性，最重要的是，它是第一个 在活体内显示疗效。然而，虽然SAR的努力能够改善药物动力学和 尽管支架具有药效学特性，但仍不足以用于治疗。这一努力确实取得了成果。 几种有价值的探针，其中之一，RSH470，在MYC存在下表现出荧光增强。 HDX-MS实验表明，RSH470在MYC的关键bHLH-LZ基序中结合了一个新的位点。令人兴奋的是， 该位点的模拟和单一氨基酸突变证实了这一发现，并提供了一种结构 对缓蚀剂作用机理的解释。 利用RSH470，我们开发了第一个基于荧光的HTS筛选竞争分析方法 专门识别MYC抑制剂，不需要蛋白质修饰、DNA结合或 免费赠送二聚体搭档麦克斯。此外，它简单、廉价且不受专有限制 限制可用于MYC的HTS检测。通过建立的正交法验证了该方法的有效性。 方法(BLI、Bio-FET、SPR)和细胞致癌实验。此外，在结构上， 通过对这两种化合物进行试点筛选，已经发现了具有特定细胞活性的不同化合物 斯克里普斯研究校园。在这个提案中，我们提出了一种策略，以筛选整个&>665,000个Scripps 药物发现文库(SDDL)，以确定新的MYC抑制剂支架。有MYC和没有MYC的二级HTS 将确定HIT活动是否依赖于MYC。然后，由药物化学家选择的匹配将被验证 通过生物层干涉测量(BLI)、表面等离子体共振(SPR)和场效应晶体管分析(Bio- Fet)。细胞潜能和MYC特异性将通过致癌转化分析确定 正交癌基因以及与耐药MYC突变细胞系。其药代动力学特性 的先导化合物将在体外进行评估，然后在已建立的异种移植模型中进行最终评估。这 研究计划将产生一组精确表征的化学铅，它们之间具有很强的相关性 体外和体内疗效。这些化合物不仅对研究MYC的功能有好处，而且还会 可能导致针对MYC驱动的癌症的治疗策略。

ABSTRACT MYC is a key transcriptional regulator involved in cellular proliferation and has established roles in transcriptional elongation and initiation, microRNA regulation, apoptosis, and pluripotency. More importantly, MYC has been directly implicated in over 50% of human cancers and is recognized as a general hallmark of cancer initiation and maintenance. Despite this prevalence, there are few functional chemical probes for MYC and no therapeutics that target it. We have discovered a compound, KJ-Pyr-9, that binds to MYC with high potency and specificity, downregulates the transcriptional activities of MYC and, most importantly, is the first MYC ligand that shows efficacy in vivo. However, while SAR efforts were able to improve the pharmacokinetic and pharmacodynamic properties of the scaffold, it remains insufficient for therapeutic use. This endeavor did yield several valuable probes, one of which, RSH470, exhibits an increase in fluorescence in the presence of MYC. HDX-MS experiment revealed that RSH470 binds a novel site in the critical bHLH-LZ motif of MYC. Excitingly, modeling and single amino acids mutations of the site have validated this finding and provided a structural explanation of the inhibitor mechanism. Utilizing RSH470, we have developed the first fluorescence-based HTS screening competition assay that specifically identifies MYC inhibitors and does not require protein modification, DNA binding, or the complimentary dimer partner MAX. Furthermore, it is simple, inexpensive, and free of proprietary restrictions that limit available HTS assays for MYC. The effectiveness of this assay has been validated by established orthogonal methods (BLI, Bio-FET, SPR) and cellular oncogenic transformation experiments. Furthermore, structurally distinct compounds, with specific cellular activity, have been discovered by pilot screens performed on both Scripps Research campuses. In this proposal, we present a strategy to screen of the entire >665,000 Scripps Drug Discovery Library (SDDL) to identify novel MYC inhibitor scaffolds. A secondary HTS with and without MYC will determine whether hit activity is MYC dependent. Hits selected by a medicinal chemist will then be validated by bio-layer interferometry (BLI), surface plasmon resonance (SPR), and field-effect transistor analysis (Bio- FET). Cellular potency and MYC specificity will be established through oncogenic transformation assays with orthogonal oncogenes as well as with inhibitor resistant MYC mutants cell lines. The pharmacokinetics properties of leads compounds will be assessed in vitro before their final evaluation in an established xenograft model. This research program will produce a set of precisely characterized chemical leads with a strong correlation between in vitro and in vivo efficacy. Not only will these compounds be beneficial in the study MYC functions, but they will may lead to a therapeutic strategy for MYC driven cancers.