Development of Inhibitors for N-Terminal Acetyltransferase D

N-terminal acetyltransferase D (NatD) is a highly selective enzyme that acetylates the SGRGK motif on histones H4 and H2A. Elevated NatD expression has been observed in lung, colorectal, breast, and bone cancer tissues with poor survival outcomes. Knockdown of NatD suppresses lung cancer progression by inhibiting the epithelial-to-mesenchymal transition (EMT) through the suppression of transcription factor Slug. In colorectal cancer, NatD knockdown induces p53-independent apoptosis, downregulation of one-carbon metabolism to decrease chemoresistance, highlighting its potential as a new therapeutic target. Therefore, potent and selective NatD inhibitors are needed to elucidate its acetyltransferase activity and role in cancer progression.

To characterize NatD activity, we developed two biochemical assays: (1) a continuous fluorescence assay that monitors CoA production and (2) a fluorescence polarization (FP) assay for ligand binding. These assays enable kinetic characterization of NatD and inhibitor screening. Using fluorescence assay, we examined how the local chemical environment on the N-terminal SGRGK sequence regulates NatD-catalyzed Nα-acetylation on histone H4/H2A, including oncohistone mutations and post-translational modifications (PTMs). To develop NatD inhibitors, we employed three strategies: (1) peptidomimetic, (2) bisubstrate inhibitors, and (3) high-throughput screening (HTS). We explored the N-terminal modifications of the SGRGK sequence and substitutions at Ser1, all of which exhibited inhibitory activity over 100 μM. To improve cell uptake, NatD bisubstrate inhibitors were conjugated with cell-penetrating peptides, leading to a reduction in histone H4 Nα-acetylation and a concurrent increase in histone H4 serine 1 phosphorylation (H4S1ph). This inhibitor also demonstrated similar phenotypes as NatD knockdown results in lung cancer cells by suppressing migration without affecting cell viability and altering EMT markers.

Through high-throughput screening of 39000 compounds, we identified 10 hits with IC50 < 30 μM. After validation, we structurally optimized one hit to yield YH501, a small-molecule allosteric inhibitor with micromolar activity in biochemical assay. In cellular assays, YH501 reduced histone H4 Nα-acetylation while increasing H4S1ph, demonstrating an inhibitory effect on cell migration and EMT markers similar to that of bisubstrate inhibitors.

Overall, our studies establish a foundation for the development of NatD inhibitors, providing valuable tools for elucidating NatD’s physiological and pharmacological roles in cancer progression and other biological processes.