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.