High-Throughput Screen for the Oncoprotein MYC

癌蛋白 MYC 的高通量筛选

• 批准号: 10657663
• 负责人: Kim Janda
• 金额: $ 40.58万
• 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/10657663
• 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; Libraries; Ligands; Light; MYC gene; Maintenance; Malignant Neoplasms; Medicine; Methods; MicroRNAs; Microsomes; Modeling; Mutation; Nature; Oncogenes; Oncogenic; Oncoproteins; Pharmaceutical Chemistry; Phenotype; Plasma; Plasma Proteins; Post-Translational Protein Processing; Prevalence; Process; Property; Proteins; Regulation; Research; Resistance; Role; Running; Series; Site; Specificity; 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; cancer initiation; cell growth; dimer; drug discovery; drug-like compound; efficacy evaluation; experimental study; high throughput screening; improved; in vivo; inhibitor; 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剂，并在转录中确立了作用 伸长和启动，microRNA调节，凋亡和多能性。更重要的是，MYC一直是 直接与超过50％的人类癌症有关，被认为是癌症启动的一般标志 和维护。尽管存在这种流行，但MYC几乎没有功能化学探针 针对它的治疗剂。我们发现了一个化合物KJ-PYR-9，该化合物以高效力与MYC结合，并且 特异性，下调了MYC的转录活动，最重要的是，是第一个MYC配体 在体内显示功效。但是，尽管SAR努力能够改善药代动力学和 脚手架的药效学特性，不足以治疗。这项工作确实屈服了 几种有价值的探针，其中一种RSH470在MYC存在下表现出荧光的增加。 HDX-MS实验表明，RSH470结合了MYC临界BHLH-Lz基序中的一个新位点。令人兴奋的是 现场的建模和单氨基酸突变已经验证了这一发现并提供了结构性 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筛选竞赛测定法 明确识别MYC抑制剂，不需要蛋白质修饰，DNA结合或 免费二聚体合作伙伴Max。此外，它是简单，便宜的，并且没有专有限制 限制MYC的可用HTS分析。该测定的有效性已通过已建立的正交验证 方法（BLI，Bio-Fet，SPR）和细胞致癌转化实验。此外，在结构上 通过对两者进行的试验屏幕发现了具有特定细胞活性的不同化合物 Scripps研究校园。在此提案中，我们提出了筛选整个> 665,000 scripps的策略 药物发现文库（SDDL）识别新型MYC抑制剂支架。有和没有MYC的次要HTS 将确定命中活动是否取决于MYC。然后，由药剂师选择的命中将得到验证 通过生物层干涉法（BLI），表面等离子体共振（SPR）和现场效应晶体管分析（Bio-- Fet）。细胞效力和MYC特异性将通过与致癌转化测定法建立 正交癌基因以及抑制剂抗体MYC突变体细胞系。药代动力学特性 在已建立的异种移植模型中最终评估之前，将在体外评估铅化合物的。这 研究计划将产生一组精确特征的化学导管，并在 体外和体内功效。这些化合物不仅在研究MYC功能中有益，而且它们会 可能会导致MYC驱动的癌症的治疗策略。