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，并且在转录调控中具有确定的作用。 延伸和起始、microRNA调节、凋亡和多能性。更重要的是， 直接涉及超过50%的人类癌症，并被认为是癌症起始的一般标志 和维护尽管如此，MYC的功能性化学探针很少， 我们已经发现了一种化合物KJ-Pyr-9，它能高效结合MYC， 特异性，下调MYC的转录活性，最重要的是，是第一个MYC配体， 在体内显示功效。然而，虽然SAR的努力能够改善药代动力学， 尽管支架的药效学性质不充分，但其仍然不足以用于治疗用途。这种奋进确实产生了 几种有价值的探针，其中之一，RSH 470，在MYC的存在下表现出荧光的增加。 HDX-MS实验显示RSH 470结合MYC关键bHLH-LZ基序中的一个新位点。令人兴奋的是， 该位点的建模和单氨基酸突变验证了这一发现，并提供了一个结构 抑制剂机制的解释。 利用RSH 470，我们开发了第一个基于荧光的HTS筛选竞争试验， 特异性鉴定MYC抑制剂，并且不需要蛋白质修饰、DNA结合或 互补二聚体伴侣MAX。此外，它简单、便宜，并且没有专有限制， 限制MYC的可用HTS检测。该方法的有效性已通过建立的正交试验进行了验证 方法（BLI、Bio-FET、SPR）和细胞致癌转化实验。此外，在结构上 通过对两者进行中试筛选，发现了具有特定细胞活性的不同化合物 斯克里普斯研究所校园。在这个建议中，我们提出了一个战略，以筛选整个> 665，000斯克里普斯 Drug Discovery Library（SDDL）用于鉴定新型MYC抑制剂支架。有和无MYC的二级HTS 将确定命中活动是否依赖于MYC。药物化学家选择的命中将被验证 通过生物层干涉法（BLI）、表面等离子体共振（SPR）和场效应晶体管分析（Bio-Effect Transistor Analysis）， FET）。细胞效力和MYC特异性将通过致癌转化试验确定， 正交癌基因以及抑制剂抗性MYC突变体细胞系。药代动力学特性 在建立的异种移植物模型中进行最终评价之前，将在体外评估先导化合物。这 一项研究计划将产生一组精确表征的化学线索， 体外和体内功效。这些化合物不仅有益于研究MYC功能， 可能导致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.