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 -项目总结 化学探针和药物发现（核心B）将提供最先进的检测开发，筛选， 和合成/药物化学专业知识，以支持所有三个项目。在项目1中，核心B将开发新的 靶向EBNA 1降解的EBNA 1探针和DNA-Tac。EBNA 1的降解物预计将更多 灵验。降解物的新方法将利用shRNA PLOD 1沉默，显示EBNA 1降解 以及新的EBNA 1靶向PLOD 1抑制的用途。我们将使用小的DNA寡核苷酸作为靶向配体 偶联至E3连接酶募集分子作为用于降解EBNA 1的化学工具化合物。在项目 2，我们将开发选择性PARP 1降解剂，并将其与已知的PARP抑制剂进行比较， 与抑制PARP 1酶活性相比，PARP 1蛋白降解的影响。我们已经证明 PARP抑制剂和重要的PARP PROTAC与ATR抑制、ATM抑制和地西他滨协同作用。 观察到PARP 1选择性PROTAC杀死SNU 719细胞的效力与奥拉帕尼相同或更高 表明捕获可能不是生物活性所必需的。我们已经开发了原型PARP 1抑制剂， 与E3连接酶募集配体偶联的临床相关PARP抑制剂奥拉帕尼以募集cereblon（CRBN） 或Mdm 2。我们发现，我们的原型化合物选择性地降解PARP 1，并观察到， 利用Mdm 2募集配体的降解剂有效杀死SNU 719 EBV胃癌细胞。我们利用 一种创新的基于平板的In-Cell Western分析，用于优化PROTAC的活性，以提高分析 生产量和准确性，并优先考虑用于Western印迹确认的化合物。在项目3中，我们 开发新的UHRF 1抑制剂和降解剂，UHRF 1是一种重要的蛋白质，在维持DNA中起着关键作用 哺乳动物细胞中的甲基化。核心B开发了一种HTRF测定法，用于测量UHRF 1与特异性 三甲基赖氨酸配体，以进一步使UHRF 1降解剂和抑制剂的发展，阻止其 CIMP中的机制。初步筛选结果已经鉴定出有效的亚微摩尔UHRF 1配体， 包括被评价为新型UHRF 1降解剂的双功能分子。 核心B还将为所有项目提供检测试剂盒开发和筛选。比如说协同 在EBV阳性SNU 719胃癌细胞系中的筛选，通常使用已知的泛癌症的NCI文库 药物和已知临床药物的表观遗传文库。核心B的具体目标是：（1）提供最先进的 art合成化学/药物化学能力，设计和合成化学探针， 项目，和（2）开发合适的生物化学和基于细胞的测定，以支持探针优化工作， 以提供协同筛选来增强我们当前探针的活性。