Chemical Probes and Drug Discovery

化学探针和药物发现

• 批准号: 10627694
• 负责人: Joseph M Salvino
• 金额: $ 34.3万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-11 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10627694
• 关键词: Antineoplastic Agents; Binding; Biochemical; Biological Assay; Biology; Cancer cell line; Carcinoma; Cell Cycle Progression; Cell model; Cells; Chemicals; Clinical; Combined Modality Therapy; CpG Island Methylator Phenotype; Cytosine; DNA; DNA Methylation; Data; Decitabine; Development; Diversity Library; Drug Combinations; Enzymes; Epigenetic Process; Epstein-Barr Virus Infections; Epstein-Barr Virus latency; Gene Expression Regulation; Human Herpesvirus 4; Lead; Libraries; Ligands; Malignant Neoplasms; Mammalian Cell; Measures; Metabolic Control; Mus; Oligonucleotides; PHD Finger; Pennsylvania; Pharmaceutical Chemistry; Pharmaceutical Preparations; Play; Poly(ADP-ribose) Polymerase Inhibitor; Proteins; Ring Finger Domain; Screening Result; Signal Pathway; Synthesis Chemistry; Ubiquitin; Ubiquitination; Universities; Virus Inhibitors; Western Blotting; Work; Xenograft Model; assay development; chemical synthesis; clinically relevant; demethylation; design; drug discovery; efficacy study; gastric cancer cell; high throughput screening; improved; in vivo; inhibitor; inhibitor therapy; innovation; latent infection; lead optimization; lead series; mouse model; novel; novel strategies; novel therapeutic intervention; protein function; prototype; recruit; scale up; screening; small hairpin RNA; small molecule inhibitor; synergism; tool; trimethyllysine; tumor growth; ubiquitin-protein ligase

CORE B – PROJECT SUMMARY The Chemical Probes and Drug Discovery (Core B) will provide state-of-the-art assay development, screening, and synthetic/medicinal chemistry expertise to support all three Projects. In Project 1, Core B will develop new EBNA1 Probes and DNA-Tacs targeting EBNA1 degradation. Degraders of EBNA1 are predicted to be more efficacious. Novel approaches for degraders will leverage shRNA PLOD1 silencing showing EBNA1 degradation and the use of novel EBNA1 targeted PLOD1 inhibition. We will use small DNA oligos as targeting ligands conjugated to E3 ligase recruiting molecules as chemical tool compounds for degradation of EBNA1. In Project 2, we will develop selective PARP1 degraders and compare these to known PARP inhibitors to compare the effect of degradation of PARP1 protein compared to inhibition of PARP1 enzymatic activity. We have shown that PARP inhibitors and importantly PARP PROTACs synergize with ATR inhibition, ATM inhibition, and decitabine. The observation that PARP1 selective PROTACs kill SNU719 cells equally or with greater potency as Olaparib suggests trapping may not be required for bioactivity. We have developed prototype PARP1 inhibitors using the clinically relevant PARP inhibitor Olaparib conjugated to E3 ligase recruiting ligands to recruit cereblon (CRBN) or Mdm2. We found that our prototype compounds selectively degrade PARP1 and have observed that degraders utilizing the Mdm2 recruiting ligand are effective in killing SNU719 EBV gastric cancer cells. We utilize an innovative plate based In-Cell Western assay to optimize the activity of the PROTACs to increase the assay through-put and accuracy, and to prioritize compounds for Western blot confirmation. In Project 3, we will develop novel inhibitors and degraders for UHRF1, an important protein that plays a key role in maintaining DNA methylation in mammalian cells. Core B has developed an HTRF assay for measuring UHRF1 binding to specific trimethyl-lysine ligand to further enable the development of UHRF1 degraders and inhibitors that block its mechanism in CIMP. Preliminary screening results have identified potent sub-micromolar UHRF1 ligands, including bi-functional molecules being evaluated as novel UHRF1 degraders. Core B will also provide assay development and screening for all the Projects. For example, synergy screening in the EBV positive SNU719 gastric cancer cell line typically using the NCI library of known pan-cancer drugs and epigenetic library of known clinical drugs. The specific aims for Core B are (1) to provide state-of-the- art Synthetic chemistry/ Medicinal chemistry capabilities to design and synthesize chemical probes for all the projects, and (2) to develop suitable biochemical and cell-based assays to support probe optimization efforts and to provide synergy screens to enhance the activity of our current probes.

核心B - 项目摘要 化学探针和药物发现（核心B）将提供最新的测定开发，筛查， 以及合成/药物化学专业知识，以支持这三个项目。在项目1中，核心B将开发新的 EBNA1探针和靶向EBNA1降解的DNA-TAC。 EBNA1的降级器预计会更 降级器的新方法将利用shrna plod1沉默显示EBNA1退化 以及新型EBNA1靶向PLOD1抑制的使用。我们将使用小的DNA寡聚作为靶向配体 将E3连接酶募集的分子作为化学工具化合物，以降解EBNA1。在项目中 2，我们将开发选择性PARP1降解器，并将其与已知的PARP抑制剂进行比较以比较 与抑制PARP1酶活性相比，PARP1蛋白降解的影响。我们已经表明 PARP抑制剂和重要的是PARP Protac随着ATR抑制，ATM抑制和Decideabine协同作用。 观察到PARP1选择性protac均等杀死SNU719细胞，或者像Olaparib一样具有更大的效力 暗示生物活性可能不需要捕获。我们已经使用了原型PARP1抑制剂 临床相关的PARP抑制剂Olaparib与E3连接酶招募配体以募集Cereblon（CRBN） 或MDM2。我们发现我们的原型化合物选择性降解PARP1，并观察到 利用MDM2募集配体的降级器可有效杀死SNU719 EBV胃癌细胞。我们利用 基于创新板的基于细胞内西部测定法，以优化Protac的活性以增加测定 贯穿和准确性，并优先考虑蛋白质印迹确认的化合物。在项目3中，我们将 为UHRF1开发新型抑制剂和降解器，这是一种重要的蛋白质，在维持DNA中起关键作用 哺乳动物细胞中的甲基化。核心B开发了用于测量UHRF1结合特定的HTRF测定法 三甲基 - 赖氨酸配体进一步使UHRF1降解器和抑制剂的发展能够阻止其 CIMP中的机制。初步筛选结果已经确定了潜在的亚微摩尔摩尔UHRF1配体， 包括被评估为新型UHRF1降解器的生物功能分子。 核心B还将为所有项目提供组装开发和筛选。例如，协同作用 在EBV阳性SNU719胃癌细胞系中筛选通常使用已知泛伴奏的NCI库 已知临床药物的药物和表观遗传文库。核心B的具体目的是（1）提供最新的 ART合成化学/药物化学能力，用于设计和合成化学问题的所有功能 项目，以及（2）开发合适的生化和基于细胞的测定法，以支持探针优化工作和 提供协同屏幕以增强我们当前问题的活性。