Structure-guided identification and experimental validation of NAT10-targeting small molecules in colorectal cancer...

Colorectal cancer (CRC) remains a major cause of cancer-related mortality worldwide, and patients with advanced or metastatic disease still require more effective targeted therapeutic options. N-acetyltransferase 10 (NAT10), an RNA acetyltransferase responsible for N4-acetylcytidine (ac4C) modification, has been implicated in CRC progression, metastasis, immune evasion, and therapy resistance. The recently resolved human NAT10 structure (PDB ID: 9J3C) provides a structural basis for rational discovery of NAT10-directed chemical scaffolds. We combined large-scale virtual screening, molecular simulation, and cellular testing to identify NAT10-targeting compounds. Approximately 300,000 molecules were screened by hierarchical docking, followed by ADMET prediction, 100-ns molecular dynamics simulations, and MM/PBSA analysis. G856-6814 showed a predicted binding free energy of - 26.33 ± 3.37 kcal/mol and inhibited HCT116 cell viability with an IC₅₀ of 211.6 ± 14.8 nM. In a recombinant enzyme assay, G856-6814 inhibited NAT10 acetyltransferase activity with an IC₅₀ of 2.3 ± 0.4 µM. G856-6814 treatment increased the apparent NAT10 melting temperature from 50.4 ± 0.5 to 56.0 ± 0.8 °C. Treatment also reduced global RNA ac4C and ac4C enrichment on c-MYC, CCNA2, and CCNB1 transcripts. G856-6814 was 19.6-fold less potent against TIP60 and more than 43-fold less potent against p300 and GCN5. Together with the attenuation of cellular effects after NAT10 knockdown, these findings support G856-6814 as a NAT10-targeting inhibitory scaffold that requires broader selectivity and in vivo evaluation.