Breaking the Myb-KIX Alliance: Structure-Guided Discovery of Biaryl Hydroxy-Naphthamides as Disruptors of the...

The interaction between the transcription factor c-Myb and the CBP/p300 KIX domain is a critical regulatory node in hematopoietic gene expression and an attractive target in MYB-dependent leukemia. Herein, we aimed to identify new small-molecule disruptors of the c-Myb-CBP/p300 KIX protein-protein interaction using integrated in silico and experimental workflow. A focused library of 1143 biaryl hydroxy-naphthamides (naphthanilides) was subjected virtual screening filters yielding five prioritized candidates (C1-C5). Docking showed that all selected compounds occupied the c-Myb-facing groove of the KIX domain and reproduced key interfacial contacts. Subsequent 1000 ns molecular dynamics simulations revealed distinct stability and disruption profiles, with C1 and C4 showing the most favorable combination of stable groove occupancy, protein-protein interface perturbation, and ligand-binding energetics. Free-energy landscape analysis further supported these compounds as the most conformationally stable interfacial binders. Differential MM/PBSA analysis of the protein-protein interface showed that C1 produced the largest predicted weakening of the c-Myb-KIX interaction (ΔΔG_PPI = +38.81 kcal/mol), followed by C4 (+29.90), C5 (+27.78), and C3 (+25.81 kcal/mol), whereas C2 was predicted to stabilize the complex (-5.41 kcal/mol). In silico ADMET profiling indicated that the series was drug-like by Lipinski criteria but carried liabilities related to solubility, metabolism, and predicted toxicity. Experimental validation by microscale thermophoresis confirmed direct binding of the assay proteins (K_d = 26.72 ± 0.82 µM) and demonstrated that the naphthanilides disrupt the interaction in vitro. C1 was the most potent disruptor (IC₅₀ = 9.50 ± 0.22 µM), outperforming Naphthol AS-E phosphate (IC₅₀ = 32.84 ± 9.46 µM). These findings establish 3-hydroxy-2-naphthamides as promising scaffolds for targeting the c-Myb-CBP/p300 KIX interface.