Targeting the c-Myb-CBP/p300 KIX protein-protein interaction with quinone-methide triterpenes: In silico mechanistic...

The interaction between the transcription factor c-Myb and the CBP/p300 KIX domain is a key regulatory event in transcriptional programs associated with hematologic malignancies, including acute myeloid leukemia, and therefore represents an attractive target for protein-protein interaction (PPI) disruption. In this study, we applied an integrated computational and experimental workflow to identify new quinone-methide triterpenes capable of perturbing the c-Myb-CBP/p300 KIX interface. Using the c-Myb-bound KIX conformation derived from PDB:2AGH as the structural template, a library of 457 quinone-methide-triterpenes was subjected to funnel-based virtual screening. This workflow prioritized isoiguesterin, pristimerin, and tingenone for detailed evaluation. Subsequent 500ns MD simulations revealed distinct ligand-dependent effects on interfacial stability and conformational dynamics. ΔΔG_PPI analysis showed that tingenone produced the strongest predicted weakening of the c-Myb-KIX interaction (+2.31 kcal/mol), whereas pristimerin had only a marginal disruptive effect (+0.28 kcal/mol) and isoiguesterin instead favored stabilization of the complex (-5.18 kcal/mol). Consistently, free-energy-landscape (FEL) analysis showed that tingenone induced the most heterogeneous conformational ensemble with multiple low-energy basins, while isoiguesterin and Naphthol-AS-E-phosphate favored more restricted low-energy states. Although isoiguesterin showed the most favorable direct binding energetics, it tended to stabilize or compact the interface. Pristimerin displayed an intermediate profile. In contrast, tingenone produced the clearest signatures of interfacial weakening and the most favorable disruption metrics. Experimental validation by microscale thermophoresis (MST) confirmed the direct Myb-KIX interaction (K_d = 28.49 ± 2.32 μM) and showed that tingenone was the most potent inhibitor among the tested triterpenes (IC₅₀ = 23.6 ± 3.0 μM), outperforming the reference disruptor Naphthol-AS-E-phosphate (IC₅₀ = 29.0 ± 1.6 μM). These findings identify tingenone as a promising scaffold for the development of new c-Myb-CBP/p300 KIX PPI inhibitors.