High-Throughput Screen for the Oncoprotein MYC

癌蛋白 MYC 的高通量筛选

• 批准号: 10276232
• 负责人: Kim Janda
• 金额: $ 43.29万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10276232
• 关键词: Affect; Affinity; Amino Acids; Animal Model; Apoptosis; Attenuated; Binding; Binding Proteins; Binding Sites; Biological Assay; Biology; Breast Cancer Model; California; Cell Line; Cell Proliferation; Cell division; Cell model; Cells; Cellular Assay; ChIP-seq; Chemicals; Chick Embryo; Competitive Binding; Complex; DNA Binding; Development; Dimerization; Disease; Dose; Drug Kinetics; E-Box Elements; Effectiveness; Ensure; Evaluation; Exhibits; Fibroblasts; Florida; Fluorescence; Fluorescent Probes; Follow-Up Studies; Gatekeeping; Genetic Transcription; Histologic; Human; Human Cell Line; In Vitro; Interferometry; Label; Lead; Libraries; Ligands; Light; MYC gene; Maintenance; Malignant Neoplasms; Methods; MicroRNAs; Modeling; Mutation; Nature; Oncogenes; Oncogenic; Oncoproteins; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phenotype; Plasma; Plasma Proteins; Post-Translational Protein Processing; Prevalence; Process; Property; Proteins; Regulation; Research; Resistance; Role; Running; Series; Site; Specificity; Structure; Surface Plasmon Resonance; Testing; Therapeutic; Therapeutic Uses; Tissues; Toxic effect; Transcription Elongation; Transcription Initiation; Transistors; United States National Institutes of Health; Validation; Xenograft Model; acute toxicity; base; cancer initiation; cell growth; dimer; drug discovery; efficacy evaluation; experimental study; high throughput screening; improved; in vivo; inhibitor/antagonist; mutant; non-Native; novel; pharmacokinetics and pharmacodynamics; pluripotency; programs; scaffold; screening; therapeutic target; transcription factor; tumor; tumor growth

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.

抽象的 MYC 是参与细胞增殖的关键转录调节因子，并已在转录中发挥作用。 延伸和起始、microRNA 调节、细胞凋亡和多能性。更重要的是，MYC已经 直接涉及超过 50% 的人类癌症，并被认为是癌症发生的一般标志 和维护。尽管如此普遍，但针对 MYC 的功能性化学探针却很少，而且还没有 针对它的治疗方法。我们发现了一种化合物 KJ-Pyr-9，它可以与 MYC 高效结合，并且 特异性，下调 MYC 的转录活性，最重要的是，它是第一个 显示体内功效。然而，虽然 SAR 的努力能够改善药代动力学和 支架的药效学特性，但其仍然不足以用于治疗用途。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 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结构 抑制剂机制的解释。 利用 RSH470，我们开发了第一个基于荧光的 HTS 筛选竞争测定法， 特异性识别 MYC 抑制剂，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、便宜且不受专有限制， 限制 MYC 可用的 HTS 检测。该测定的有效性已通过建立的正交试验得到验证 方法（BLI、Bio-FET、SPR）和细胞致癌转化实验。此外，从结构上 通过对两者进行的中试筛选，发现了具有特定细胞活性的不同化合物 斯克里普斯研究园区。在本提案中，我们提出了一项筛选整个 >665,000 斯克里普斯的策略 用于识别新型 MYC 抑制剂支架的药物发现库 (SDDL)。有或没有 MYC 的二级 HTS 将确定命中活动是否依赖于 MYC。然后将验证药物化学家选择的命中 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- 场效应管）。细胞效力和 MYC 特异性将通过致癌转化测定来确定 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 先导化合物将在体外进行评估，然后再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先导化合物，这些先导化合物之间具有很强的相关性 体外和体内功效。这些化合物不仅有利于 MYC 功能的研究，而且 可能会导致针对 MYC 驱动的癌症的治疗策略。