Structure‐Guided Discovery of a Potent and Selective Cell‐Active Inhibitor of SETDB1 Tudor Domain

SET domain bifurcated protein 1 (SETDB1) is a histone lysine methyltransferase that promotes the silencing of some tumour suppressor genes and is overexpressed in many cancers. SETDB1 contains a unique tandem tudor domain (TTD) that recognizes histone H3 sequences containing both methylated and acetylated lysines. Beginning with the identification of a hit compound (Cpd1), we discovered the first potent and selective small molecule SETDB1-TTD inhibitor (R,R)-59 through stepwise structure-guided optimization. (R,R)-59 showed a K_D value of 0.088±0.045 μM in the ITC assay. The high potency of (R,R)-59 was well explained by the cocrystal structure of the (R,R)-59-TTD complex. (R,R)-59 is an endogenous binder competitive inhibitor. Evidence has also demonstrated its cellular target engagement. Interestingly, the enantiomer (S,S)-59 did not show activity in all the assays, highlighting the potential of (R,R)-59 as a tool compound in exploring the biological functions of SETDB1-TTD.