Chemical Probes and Drug Discovery

核心B--项目摘要 化学探针和药物发现(核心B)将提供最先进的分析开发、筛选、 以及合成/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 针对EBNA1降解的EBNA1探针和DNA-Tacs。EBNA1的降解者预计会更多 很有效。降解者的新方法将利用shRNA PLOD1沉默显示EBNA1降解 并利用新型EBNA1靶向抑制PLOD1。我们将使用小的DNA寡核苷酸作为靶向配体 与E3连接酶招募分子结合，作为降解EBNA1的化学工具化合物。在项目中 2，我们将开发选择性PARP1降解剂，并将这些降解剂与已知的PARP抑制剂进行比较 PARP1蛋白降解与抑制PARP1酶活性的比较。我们已经证明了 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。 PARP1选择性PROTACs对SNU719细胞杀伤力与奥拉帕利相当或更强的观察 这表明生物活性可能不需要诱捕。我们已经开发出使用PARP1抑制剂的原型 临床相关的PARP抑制剂奥拉帕利与E3连接酶募集配体结合募集Cereblon(CRBN) 或MDM2。我们发现我们的原型化合物选择性地降解PARP1，并观察到 利用MDM2招募配体的降解剂对SNU719 EBV胃癌细胞具有有效的杀灭作用。我们利用 一种新的基于平板的细胞内Western检测方法以优化PROTACs的活性以提高检测效率 吞吐量和准确性，并确定化合物的优先级以进行蛋白质印迹确认。在项目3中，我们将 开发uhrf1的新型抑制剂和降解剂，uhrf1是一种在维持DNA方面起关键作用的重要蛋白质 哺乳动物细胞中的甲基化。核心B已经开发了一种HTRF检测方法，用于测量uhrf1与特定的 三甲基赖氨酸配体，以进一步促进uhrf1降解物和抑制物的开发，从而阻断其 CIMP中的机制。初步筛选结果已经确定了有效的亚微摩尔UHRF1配体， 包括被评价为新型uhrf1降解剂的双功能分子。 核心B还将为所有项目提供化验开发和筛选。例如，协同效应 利用已知泛癌NCI文库筛选EBV阳性SNU719胃癌细胞系 药物和已知临床药物的表观基因库。核心B的具体目标是(1)提供最新的 ART合成化学/药物化学设计和合成化学探针的能力 项目，以及(2)开发适当的生化和基于细胞的分析，以支持探针优化工作和 提供协同筛选，以增强我们目前的探测器的活性。

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.