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 (ΔΔGPPI = +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 (Kd = 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.
H. Alfassam, Emadeldin M. Kamel, Sarah I. Othman et al.· Archiv der Pharmazie· 0 citations
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.
Emadeldin M. Kamel, H. Rudayni, A. A. Allam et al.· Journal of Molecular Graphic...· 0 citations
The protein-protein interaction (PPI) between metadherin (MTDH) and staphylococcal nuclease and tudor domain-containing 1 (SND1) drives oncogenic signaling and tumor progression in multiple cancer types, yet remains an underexploited therapeutic target. Here, we report an integrated computational-experimental strategy to identify small-molecule disruptors of the MTDH-SND1 interface. A focused PubChem library of 4,149 2-arylbenzothiazoles was screened using a tiered workflow combining drug-likeness/PAINS filtering, hierarchical docking into the MTDH-binding groove on SND1 (PDB 4QMG), and MM/PBSA-based refinement. Top-ranked candidates were further evaluated by 1-μs molecular dynamics (MD) simulations and prioritized based on pose stability and interfacial engagement metrics. Four representative hits-L1 (2-(4-aminophenyl)benzothiazole), L2 (2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole), L3 (YL-109), and L4 (2-(3,4,5-trimethoxyphenyl)benzothiazole)-were advanced for experimental validation. MD analyses indicated ligand-dependent modulation of PPI stability and conformational landscapes, while MM/PBSA decomposition suggested binding was driven predominantly by short-range hydrophobic packing within the interfacial groove. A quantitative split-luciferase complementation assay confirmed dose-dependent disruption of MTDH-SND1 in a cell-free format with low-micromolar potency (IC50: L1 10.72 ± 1.12 μM, L2 5.72 ± 0.92 μM, L3 11.77 ± 1.42 μM, L4 7.41 ± 1.01 μM) and in a cell-based reporter (IC50: L1 35.71 ± 2.80 μM, L2 16.44 ± 0.24 μM, L3 32.96 ± 2.58 μM, L4 21.93 ± 0.9 μM). Importantly, a linked-luciferase counterscreen (IC50 > 1,000 μM) supported minimal luciferase interference. MST analysis further confirmed direct binding of L1-L4 to purified SND1, yielding low-micromolar KD values. Together, these results establish 2-arylbenzothiazoles as a promising chemotype for MTDH-SND1 PPI inhibition and provide a validated computational-to-experimental framework for discovering and optimizing MTDH-directed therapeutics.
Emadeldin M. Kamel, S. Khadrawy, A. A. Allam et al.· Archives of Biochemistry and...· 0 citations
The S-phase kinase-associated protein 2 (Skp2)-cyclin-dependent kinase subunit 1 (Cks1) protein-protein interaction (PPI) plays a central role in recognition of phosphorylated p27 and therefore represents an attractive target for anticancer drug discovery. Herein, we sought to identify new small-molecule disruptors of the Skp2-Cks1 interface from a focused fumiquinazoline-scaffold library by combining virtual screening, molecular simulation, and experimental validation. Docking showed that all selected compounds occupied the Skp2-Cks1 interfacial hotspot. However, longer 1000-ns MD simulations, comparative MM/PBSA calculations, and interface-related metrics revealed distinct structural, energetic, and dynamic binding profiles among the selected compounds. Consistent with these analyses, comparative MM/PBSA evaluation of PPI stability showed that Fumiquinazoline D produced a positive Δ Δ G PPI , indicating weakening of the Skp2-Cks1 interface relative to the apo complex, whereas Ardeemin, Fiscalin A, and Fumiquinazoline F generally yielded negative Δ Δ G PPI values consistent with interfacial stabilization. Free energy landscape analysis further supported a more weakly confined and dynamically disruptive binding mode for Fumiquinazoline- D. In vitro homogeneous time-resolved fluorescence assays validated these predictions: Fumiquinazoline D inhibited the Skp2-Cks1 interaction with an IC50 of 6.33 ± 0.44 µM, whereas Ardeemin, Fiscalin A, and Fumiquinazoline F were substantially weaker. Although ADMET profiling identified substantial absorption and toxicity liabilities requiring future optimization, these 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
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