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.