| Summary: | <p>Tripartite motif-containing protein 24 (TRIM24) is an epigenetic transcriptional regulator involved in reading histone 3 (H3) methylated (KMe<sub><em>x</em></sub>) and acetylated (KAc) lysine post-translational modifications (PTMs) through an adjacent plant homeodomain-bromodomain (PHD-BRD) cassette, respectively. The PHD recognises trimethylated K9 (K9Me<sub>3</sub>) through a non-canonical cation-π interaction with a solvent-exposed tryptophan (W828); while the BRD reads K18/23Ac <em>via</em> a hydrogen bonding interaction with an asparagine (N980). TRIM24 has been implicated in breast cancer development, and genetic knockdown of the protein was shown to have an antiproliferative effect in breast cancer cell lines, making it an attractive therapeutic target. Currently, only two high-affinity TRIM24 BRD ligands exist in the literature, and no small-molecule TRIM24 PHD inhibitors have been reported. The two BRD ligands are also non-selective over BRPF1 (a homologous BRD-containing protein), and importantly, have no significant cytotoxic or antiproliferative effects on breast cancer cell lines.</p>
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<p>While TRIM24 BRD inhibition alone has proven ineffective against breast cancer cells, simultaneous PHD and BRD inhibition remains unexplored. The aims of this project were to synthesise and evaluate a series of bivalent TRIM24 chemical tools in order to investigate the effects of inhibiting <em>both</em> the PHD and BRD on breast cancer cells.</p>
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<p>Given the close proximity of the PHD and BRD, and lack of TRIM24 PHD ligands, it was decided that a TRIM24 BRD ligand would be linked to a H3-mimicking peptide containing a KMe<sub>3</sub> isostere, to generate bivalent peptide-drug conjugates (PDCs) (Figure 0.1a). KMe<sub>3</sub> mimics, which could interact with W828 in the TRIM24 PHD, were screened by incorporation into a K9C mutant H3 peptide using cysteine-selective alkylation reactions (Figure 0.1b). An AlphaScreen<sup>®</sup> competition assay identified isoindoline as one of the highest affinity and most synthetically tractable KMe<sub>3</sub> mimics, which was subsequently taken forward for incorporation into an unnatural amino acid in the PDCs (Figure 0.1c).</p>
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<p>The PDCs showed low nanomolar<sup>®</sup> IC<sub>50</sub> values for displacement of a TRIM24 PHD- and BRD-binding peptide (H3K9Me<sub>3</sub>K18Ac), and exhibited avidity for TRIM24, which arises from simultaneous in <em>cis</em> binding to the PHD and BRD. Surface plasmon resonance (SPR) assays revealed the PDCs have picomolar affinity for TRIM24, resulting from a slow dissociation rate, consistent with a bivalent binding mode. Furthermore, by BROMO<em>scan</em> the PDCs demonstrated 13-fold selectivity for TRIM24 (PHD-BRD) over BRPF1 (BRD). PDC5 demonstrated some cell permeability, despite its peptidomimetic structure, and so was evaluated in proliferation assays. Although, no significant antiproliferative effects were observed in the ER<sup>+</sup> MCF-7 breast cancer cell line, the triple negative CAL-51 cell line appeared sensitive to simultaneous TRIM24 PHD and BRD inhibition. Future work with these high-affinity bivalent PDCs will enable investigation of TRIM24 function in more detail.</p>
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