A Cell-Potent Bisubstrate Inhibitor to Probe NatD Acetyltransferase Activity

N-terminal acetyltransferase D (NatD) is a highly selective
enzyme
that acetylates the α-N-terminal amine of histones H4 and H2A,
which share the SGRGK motif. Elevated NatD expression has been observed
in lung, colorectal, breast, and bone cancer tissues, and is correlated
with poor patient survival in these cancer types. In non-small cell
lung cancer, NatD depletion reduces progression by repressing the
epithelial-to-mesenchymal transition (EMT). Hence, NatD is a potential
epigenetic target for lung cancer. To unravel the functions of NatD,
a cell-potent and selective NatD inhibitor is needed to investigate
the acetyltransferase activity of NatD in cancer progression. We previously
reported potent and selective NatD bisubstrate inhibitors, designed
by covalently linking coenzyme A to peptide substrates via an acetyl
and propionyl linker. However, these inhibitors are not cell-active,
limiting their application for cellular studies. Here, we designed
cell-permeable bisubstrate analogs by attaching cell-penetrating peptides
(CPP) to the bisubstrate inhibitor. The inhibitor displayed a Ki value of 23 nM and effectively reduced cellular
Nα-acetylation on histone H4, leading to reduced migration in
lung cancer cells by modulating the expression of E-cadherin, N-cadherin, and vimentin. Our findings
demonstrate that the CPP-conjugated NatD inhibitor serves as a valuable
chemical probe for elucidating the biological roles of NatD in lung
cancer, laying the groundwork for future therapeutic strategies targeting
NatD.