SET domain bifurcated 1 (SETDB1) is the only methyltransferase able to trimethylate lysine 9 of the histone 3 tail (H3K9) in euchromatic regions, resulting in gene repression. Its aberrant activity and overexpression have been associated with carcinogenesis and the development of neurodegenerative diseases. However, aside from its methyltransferase activity, SETDB1 is also a methyl lysine reader able to recognize the H3K9Me2K14Ac and H3K9Me3K14Ac marks through its Triple Tudor domain (3TD), whose role is not well understood, and would benefit from a selective chemical probe for this domain.
After screening an in-house library of KMe reader ligands, UNC6535 was identified as a reversible ligand for SETDB1 3TD with an K_d of 3.4 µM by TR-FRET for the displacement of H3K9Me2K14Ac. Interestingly, the crystal structure of this compound with the 3TD of SETDB1 showed an accessible cysteine 5 Å away from the molecule. Synthesis and evaluation of a set of cysteine-targeting analogs of UNC6535 led to the identification of UNC10013 as a lead covalent ligand, targeting C385, with a potent k_inact/K_I of 1.0 x 10⁶ M^-1s^-1. UNC10013 also showed selectivity within the methyl lysine reader and methyltransferase families as well as the whole proteome after cysteine-targeted activity-based protein profiling. In cells, allosteric inhibition of SETDB1-mediated Akt methylation was observed at 30 µM and above. However, cellular target engagement using CETSA, with and without digitonin, confirmed low cell permeability which is currently being addressed using medicinal chemistry. Therefore, we successfully designed the first potent and selective 3TD covalent ligand with cellular activity at concentrations of 30 µM and above. This covalent ligand will help improve understanding of the role of this methyl lysine reader domain independently of the methyltransferase domain of SETDB1.

Devan Shell

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