Novel bisubstrate inhibitors for protein N-terminal acetyltransferase D

Protein N-terminal acetyltransferase D (NatD, NAA40, Nat4) that specifically acetylates the N-terminus of histone H4 and H2A has been implicated in various diseases, but no inhibitor has been reported for this important enzyme. Based on the acetyl transfer mechanism of NatD, we designed and prepared a series of highly potent NatD bisubstrate inhibitors by covalently linking coenzyme A to different peptide substrates via an acetyl or propionyl spacer. The most potent bisubstrate inhibitor displayed a Ki of 170 {+/-} 16 pM. We also demonstrated that these inhibitors are highly specific towards NatD, displaying 10,000-fold selectivity over other closely-related acetyltransferases. High resolution crystal structures of NatD bound to two of these inhibitors revealed the molecular basis for their selectivity and inhibition mechanisms, providing a rational path for future inhibitor development.