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 <i>K</i>_i 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 <i>E</i>-cadherin, <i>N</i>-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.