| Summary: | <p>Epigenetic signalling is regulated by ‘writer’, ‘reader’, and ‘eraser’ proteins, which add, identify and remove histone modifications, respectively. Simultaneous recognition of multiple epigenetic marks by proteins containing tandem reader domains increases the complexity of the information transmitted, making these proteins interesting targets for probing epigenetic regulation. Tripartite motif-containing (TRIM) protein 24 and 33 are transcriptional regulators, each containing a C-terminal tandem PHD-bromodomain motif which recognises methylation states and acetylation of modified histone lysine residues. TRIM33 functions within the PARP-dependent DNA damage response pathway, where it is responsible for the timely removal of chromatin-remodelling enzyme ALC1 from sites of DNA damage through its bromodomain. Loss of TRIM33 causes prolonged association of ALC1, and therefore extended chromatin relaxation, which leaves DNA exposed and increases the susceptibility of cells to further DNA damage. </p>
<p>The aim of this study was to develop tool compounds to probe the functions of TRIM33 in epigenetic regulation, and to validate this protein as a therapeutic target in situations where DNA damage is increased. This work describes the two approaches taken; targeting the bromodomain with small molecule ligands, and incorporating these ligands into the design of proteolysis-targeting chimeras (PROTACs) to induce degradation of TRIM33 in cells. Initial work identified a compound as binding to the bromodomains of TRIM33 and TRIM24. Subsequent removal of the sulfonamide moiety and extension of the amine chain gave selective binding of the TRIM33 bromodomain over the KAc binding pocket of TRIM24. The ionic interaction between E981 and
the protonated benzylic amine proved essential for both TRIM33 affinity and selectivity. Dose-response AlphaScreen™, WaterLOGSY NMR experiments, and ITC were used to evaluate ligand binding. Two additional acetyl-lysine mimicking head groups were assessed, but the original benzimidazolone core appeared to produce the optimal orientation of the head group relative to N1039, the residue responsible for recognition of acetylated lysine residues. Probing the hydrophobic region above the KAc binding pocket through lipophilic extension of the head group,
sulfonamide projection adjacent to the benzylic amine, and projection of small groups from an alternative vector, all reduced affinity for TRIM33β. Focusing on the amine chain, aiming to bias the ‘pincer’ interaction of the two amines around E981, produced several low micromolar ligands with selectivity over TRIM24. These are the first confirmed ligands for the TRIM33 bromodomain. </p>
<p>These ligands, and knowledge gained from the SAR studies, were subsequently used in the design
of a series of TRIM33-targeting PROTACs. The initial PROTAC series incorporating the lead ligand had low cell permeability, with only three of the 17 PROTACs synthesised appearing in the cell at low concentrations. Consequently, no degradation of TRIM33 was observed in RKO cells. A second series was designed to increase cLogD values by focusing on alkyl linkers and masking hydrogen bond donors. This aimed to increase the concentration of PROTAC within the cell, thus
improving the likelihood of ternary complex formation and degradation. Of the six PROTACs synthesised, three were shown to have high cell permeability. Assessment of these three PROTACs in RKO cells revealed that one PROTAC showed time-dependent reduction in TRIM33 levels, with the lowest levels showing around 70% reduction at 16 h after treatment. Therefore, this work describes the discovery of a range of chemical tools that can be used as a basis for further optimisation. This will ultimately enable us to establish the impact of either degrading or inhibiting the bromodomain of TRIM33 in cancer cells, in situations where DNA damage is increased such as radiotherapy. </p>
<p>Additionally, work to determine if TRIM24 can be recruited as an E3 ligase in PROTAC-mediated degradation of BRD4 was also explored, which has the potential to allow context-specific degradation, as TRIM24 is overexpressed in many cancers.</p>
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