Novel Bisubstrate Inhibitors for Protein N‑Terminal Acetyltransferase D

Protein
N-terminal acetyltransferase D (NatD, NAA40) that specifically
acetylates the alpha-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 an apparent Ki value of 1.0
nM. Biochemical studies indicated that bisubstrate inhibitors are
competitive to the peptide substrate and noncompetitive to the cofactor,
suggesting that NatD undergoes an ordered Bi–Bi mechanism.
We also demonstrated that these inhibitors are highly specific toward
NatD, displaying about 1000-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 mechanism, providing a rational path for
future inhibitor development.