UHRF1 is an E3 ubiquitin ligase and a key epigenetic regulator establishing a crosstalk between DNA methylation and histone modification. Despite the important biochemical role of UHRF1 in cells, its overexpression has been found in almost all primary cancer types including breast cancer, lung cancer and so on. Numerous evidence indicates a strong link between tumorigenesis and UHRF1 overexpression, supporting its potential as a universal biomarker for cancer. However, UHRF1 is “yet-to-be drugged” and no highly potent chemical probes have been developed to target UHRF1 to date. In this study, we proposed two drug design approaches for UHRF1. The first approach is to construct multivalent DNA-peptide nucleosome mimetics that can target UHRF1 directly. For UHRF1 to promote DNA methylation, the interaction with nucleosomes, both through a DNA-binding (SRA) and histone-binding domain (TTD-PHD), and ubiquitylation of histone H3 are necessary to recruit DNA methyltransferase. We utilized the natural binding activity between UHRF1 and nucleosome in cells to develop a DNA-peptide hybrid that mimics UHRF1’s interaction with nucleosomes, thereby inhibiting UHRF1-dependent histone ubiquitylation and impairing its function in controlling DNA methylation. Here, we described the synthesis of the DNA-peptide hybrids using different lengths of PEG linkers including PEG2, 6, 8, 16 and 24. We purified and characterized the molecules with RP-HPLC and ESI-MS. Biophysical assays such as ITC and METRIS were conducted to study about the binding affinities of these DNA-peptide hybrids. In vitro UHRF1 ubiquitylation assays were performed to investigate the inhibition efficacy of these inhibitors, and pull-down assays were conducted to study their selectivity. In addition, mass photometry assays were used to study the stoichiometry of the binding between UHRF1 and the DNA-peptide hybrids. We demonstrated that multivalent DNA-peptide hybrids possess high affinity for UHRF1 and can inhibit histone ubiquitylation. Among them, In16 can form a 1:1 binding complex with UHRF1, substantiating its ability to be used as a molecular tool for structural analysis of UHRF1. In the second approach, we designed and constructed three generations of multi-domain protein inhibitors of E2 enzyme Ube2D, including RING-UBL (RU), UBOX-UBL (UU) and UBOX-UbvD1.1 short/long (UD1 and UD2). Through targeting both the RING- and backside-binding sites on Ube2D, UHRF1 enzymatic function can be indirectly inhibited as Ube2D is the only cognate E2 enzyme that cooperates with UHRF1 for histone H3 ubiquitylation. In this study, ITC was used to measure the binding affinities of these inhibitors, showing an increasing affinity from the first inhibitor RU to the last one UD2, ranging from 10-6 M to

In most eukaryotic organisms, the ubiquitination pathway is one of the most important and versatile signaling systems in use. It is integral to processes such as protein degradation and homeostasis, DNA repair cell cycle regulation, signaling and regulation, epigenetics, and many more. Ubiquitin (Ub) is a short polypeptide of 8.6 kDa, 76 residues that functions as a reversible post-translation modification (PTM). It furthermore contains 7 different lysine residues (K6, K11, K27, K29, K33, K48, K63), all of which can form isopeptide linkages with one another to link individual Ub moieties to form unique polyUb chains onto substrates. The type of polyUb chain a substrate gets labeled with can determine the subsequent activity of that substrate. Substrate ubiquitination is achieved through an enzymatic cascade. First, an E1-activating enzyme activates a free Ub moiety. Then Ub is transferred onto an E2-conjugating enzyme, and finally an E3 ligase interacts with both substrate and E2~Ub complex to facilitate Ub transfer onto a substrate. Within this scheme, the E2-enzyme acts as a master manipulator in that, it controls when, where and how a ubiquitin chain is transferred onto a substrate. Irregular activity of E2-conjugating enzyme has been implicated in a wide variety of diseases such as cancer, neurodegenerative diseases, muscular dystrophy, genetic azoospermia and more. While attempts have been made to inhibit other ubiquitination cascade enzymes such as E3 ligases and E1-activating enzymes, there is a strikingly small number of inhibitors specifically targeting E2 enzymes mainly due to the high degree of structural conservation that exists among members of the E2 enzyme family. In this work, we introduce 3 novel linked-domain protein inhibitors of the E2-conjugating enzyme Ube2D. We covalently attached either UHRF1 RING domain or an affinity optimized U-box domain, with UHRF1 UBL domain or UbvD1.1 (A ubiquitin variant specific for Ube2D), through a glycine-serine linker, producing 3 unique inhibitors: Ring-UBL (RU), U-box-UBL (UU), and U-box-UbvD1.1 (UUD1.1). In this way, we attempt to specifically inhibit Ube2D for two purposes : 1) While Ube2D can interact with the largest number of E3 ligases and facilitate the largest number of polyUb chains, very little is known about cellular phenotypes specifically associated with Ube2D; 2) We want to establish whether targeting the E2 enzyme in general can be utilized as a viable therapeutic treatment for cancer. We show that all three inhibitors are able to inhibit ubiquitin assays using Ube2D and using ITC we measured binding affinities of UUD1.1 (5 nM) > UUWT (300 nM) > RU WT (3 µM). Furthermore, we found that all inhibitors could prevent E1, E3 and backside binding domain interactions simultaneously, which single domain UBL could not. UU and RU showed specificity towards Ube2D when tested against APC/C and Cullin1 E3 ligases and their cognate E2 enzymes. We propose that linking domains in this way, by targeting the backside binding domains of E2 enzymes, could be a strategy that can be standardized and applied to the rest of the E2 enzyme family as well. In vivo testing must now elucidate whether these inhibitors can provide more information about the cellular role of Ube2D and whether it is a viable therapeutic target to treat cancer.