Identification of small molecules as potential inhibitors of NAT10 for the treatment of colon cancer

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality globally, with limited therapeutic options for advanced or metastatic disease, highlighting the urgent need for novel targeted therapies. N-acetyltransferase 10 (NAT10), an RNA acetyltransferase mediating N4-acetylcytidine (ac4C) modification, has emerged as a critical oncogenic driver in CRC, contributing to tumor progression, metastasis, immune evasion, and chemoresistance. The recent resolution of the NAT10 crystal structure (PDB: 9J3C) provides a pivotal foundation for structure-based drug discovery. In this study, we employed an integrated computational-experimental approach to identify potent NAT10 inhibitors. A library of ~ 300,000 compounds was subjected to multi-tiered virtual screening (HTVS, SP, XP docking), followed by ADMET property prediction, 100-ns molecular dynamics (MD) simulations, and MM/PBSA binding free energy calculations. Two lead compounds, G856-6814 and S396-0093, exhibited stable binding to the NAT10 active site, interacting with key residues (Val632, Ser643, Phe723) primarily via van der Waals and hydrophobic forces, with binding free energies of -26.33 ± 3.37 kcal/mol and − 22.31 ± 4.19 kcal/mol, respectively. In vitro validation in HCT116 CRC cells demonstrated that G856-6814 exerted superior cytotoxicity (IC ₅₀ =211.6 ± 14.8 nM) compared to S396-0093 (IC ₅₀ =477.7 ± 31.7 nM) and the positive control 5-fluorouracil (IC ₅₀ =4.46 ± 0.28 µM). Critically, NAT10 knockdown via shRNA significantly attenuated G856-6814’s cytotoxicity, confirming target-dependent activity. Mechanistic studies (WB and flow cytometry) further revealed that G856-6814 downregulated NAT10 and downstream cell cycle regulators (c-Myc, Cyclin A2, Cyclin B1), inducing G2/M phase arrest and mild apoptosis—effects reversed by NAT10 depletion. This study validates NAT10 as a tractable therapeutic target and identifies G856-6814 as a promising lead compound with a well-characterized mechanism of action. The integrated strategy provides a robust framework for future NAT10 inhibitor optimization, offering new hope for developing targeted therapies against CRC and other NAT10-dependent malignancies.