Targeting the c-Myb-CBP/p300 KIX protein-protein interaction with quinone-methide triterpenes: In silico mechanistic insights and MST biochemical validation.
Aug 2026· Journal of Molecular Graphics and Modelling· Vol 148, pp.
109553
· 0 citations· 31 references
Medicine
TL;DR
Tingenone is identified as a promising scaffold for the development of new c-Myb-CBP/p300 KIX PPI inhibitors, having produced the clearest signatures of interfacial weakening and the most favorable disruption metrics.
Abstract
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. ΔΔGPPI 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 (Kd = 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.
3‐hydroxy‐2‐naphthamides are established as promising scaffolds for targeting the c‐Myb–CBP/p300 KIX interface and are supported as the most conformationally stable interfacial binders.
H. Alfassam, Emadeldin M. Kamel, Sarah I. Othman et al.· Archiv der Pharmazie· 0 citations
The interaction between c-Myb and the CBP/p300 KIX domain is a critical transcriptional regulatory event and an attractive target for the development of candidate disruptors of the recombinant c-Myb-KIX interaction. In this study, we used an integrated computational and experimental strategy to identify new small molecules capable of disrupting this protein-protein interaction. A focused Umbelliferyl phosphate scaffold library was subjected to stepwise virtual screening via drug-likeness assessment and docking to the c-Myb-binding region of the KIX domain and short molecular dynamics refinement. Selected compounds were then evaluated by 500 ns molecular dynamics simulations, MM/PBSA analysis, free energy landscape (FEL) mapping, and finally by microscale thermophoresis (MST) assay. Computational analyses showed that stable ligand binding did not necessarily translate into disruption of the c-Myb-KIX interface, allowing separation of compounds that stabilized the complex from those predicted to weaken it. Consistent with this distinction, ΔΔGPPIanalysis identified only MUP and Naphthol AS-BI phosphate as protein-protein interaction-weakening ligands, with Naphthol AS-BI phosphate showing the strongest predicted disruptive effect (ΔΔGPPI=+3.25 kcal/mol), whereas DiFMUP and Naphthol AS-D phosphate were predicted to stabilize the complex. Among the tested molecules, Naphthol AS-BI phosphate showed the clearest disruption-like behavior in silico and was the most potent inhibitor in vitro, with an IC₅₀ of 18.9 ± 0.6 μM. Importantly, MUP emerged as the most promising umbelliferyl phosphate-derived hit, displaying measurable inhibitory activity (IC₅₀ = 33.5 ± 0.3 μM) comparable to the reference Naphthol AS-E phosphate (IC₅₀ = 31.2 ± 1.3 μM) and a more favorable predicted ADMET profile. Overall, this work identifies new chemical starting points for targeting the c-Myb-CBP/p300 KIX interaction and supports MUP as an attractive scaffold for further optimization.
Emadeldin M. Kamel, H. Rudayni, A. A. Allam et al.· Biophysical Chemistry· 0 citations
Findings validate the diphenylpyrazine scaffold as a promising chemotype for Skp2–Cks1 inhibition and identify C3 as a strong lead for further optimization.
Emadeldin M. Kamel, A. A. Allam, H. Rudayni et al.· Journal of Computer-Aided Mo...· 0 citations
Findings identify Fumiquinazoline D as an early‐stage biochemical hit for disruption of the Skp2–Cks1 interaction and as a potential scaffold for further mechanistic validation and medicinal‐chemistry investigation.
Emadeldin M. Kamel, A. A. Allam, H. Rudayni et al.· ChemMedChem· 0 citations
Background: Hypoxia-inducible factor-1 alpha (HIF-1α) drives tumor adaptation to hypoxia by promoting angiogenesis, metabolic reprogramming, and survival signaling in clear cell renal cell carcinoma (ccRCC). Transcriptional activity of HIF-1α depends on its interaction with the p300/CBP (CREB-binding protein) coactivator via the C-terminal transactivation domain (C-TAD). Direct targeting of this protein–protein interface remains limited, highlighting the need for novel inhibitors.Methods: A structure-based virtual screening of ~250,000 compounds from the ZINC database was conducted to identify candidates targeting a predicted pocket within the HIF-1α C-TAD. Top hits were evaluated using molecular docking and 100 ns molecular dynamics simulations. The lead compound, HFS764, was further characterized using in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling, homogeneous time-resolved fluorescence (HTRF) assays, and differential scanning fluorimetry (DSF). Functional effects were assessed in Caki-1 cells under hypoxia using hypoxia response element (HRE)-luciferase assays, cell viability and vascular endothelial growth factor (VEGF) secretion.Results: HFS764 demonstrated stable binding within the HIF-1α pocket, with ligand root-mean-square deviation (RMSD) <0.20 nm and a predicted binding free energy of –22.38 kcal/mol. HTRF assays confirmed inhibition of the HIF-1α–p300 interaction (IC50 = 4.89 μM), while DSF showed ligand-induced stabilization (ΔTm = +2.0 °C). In Caki-1 cells, HFS764 suppressed HRE-driven transcription (IC50 = 4.27 μM), and reduced VEGF secretion under hypoxia. Selective cytotoxicity was observed (GI50: 14.70 μM in Caki-1 vs. 37.98 μM in HK-2; therapeutic index = 2.58).Conclusion: HFS764 disrupts the HIF-1α–p300 interface and suppresses hypoxia-driven signaling in ccRCC, supporting the use of transcriptional complex inhibition as a promising therapeutic strategy.
Mesfer Al Shahrani· Discover medicine· 0 citations
The phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (p110α) is a key lipid kinase encoded by PIK3CA gene that regulates multiple intracellular signaling pathways. The nsSNPs within PIK3CA can alter protein structure and function, thereby influencing cellular processes and increasing susceptibility to various types of cancers. This study aimed to systematically evaluate the consequences of nsSNPs in PIK3CA, and their potential influence on protein behavior and drug interaction. A comprehensive set of in silico tools-including functional impact predictors, structural stability analyzers, conservation-based algorithms, and protein property assessment tools-was employed to investigate the consequences of nsSNPs. Structural modeling was performed to generate native and mutant p110α structures, followed by molecular docking to evaluate how these variants influence binding affinity with Alpelisib. Ten nsSNPs-G363V, R398C, R555K, C769G, F801C, R808W, R808Q, E849K, E849G, and R992Q-were consistently predicted to be deleterious across all functional prediction platforms and were found to affect evolutionarily conserved residues or domains. Among these, six variants (R398C, C769G, F801C, R808W, R808Q, and R992Q) were further predicted to significantly alter physicochemical protein properties. Docking analyses revealed variant-specific shifts in binding affinity. R555K displaying the highest affinity even higher than native and R398C showing lowest affinity toward Alpelisib. These suggest a potentially altered or dysregulated drug-protein interaction. However, the biological benefit or detriment of this shift warrants further experimental evaluation. The findings of this study may enhances our understanding of how PIK3CA variants contribute to predict disease risk and may support future precision-medicine approaches targeting the PI3K pathway.
Rajtilak Detroja, Joydeep Chakraborty, Mandar Kulkarni et al.· Journal of Biomolecular Stru...· 0 citations
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