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
| Identifer | oai:union.ndltd.org:pacific.edu/oai:scholarlycommons.pacific.edu:uop_etds-4994 |
| Date | 01 January 2023 |
| Creators | Gu, Li |
| Publisher | Scholarly Commons |
| Source Sets | University of the Pacific |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | University of the Pacific Theses and Dissertations |
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