1. Identification and characterization of a new potent inhibitor targeting CtBP1/BARS in melanoma cells.
- Author
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Filograna A, De Tito S, Monte ML, Oliva R, Bruzzese F, Roca MS, Zannetti A, Greco A, Spano D, Ayala I, Liberti A, Petraccone L, Dathan N, Catara G, Schembri L, Colanzi A, Budillon A, Beccari AR, Del Vecchio P, Luini A, Corda D, and Valente C
- Subjects
- Humans, Animals, Mice, Cell Line, Tumor, Cell Proliferation drug effects, Antineoplastic Agents pharmacology, Epithelial-Mesenchymal Transition drug effects, Xenograft Model Antitumor Assays, Alcohol Oxidoreductases antagonists & inhibitors, Alcohol Oxidoreductases metabolism, Alcohol Oxidoreductases genetics, Melanoma drug therapy, Melanoma pathology, Melanoma metabolism, Melanoma genetics, DNA-Binding Proteins metabolism, DNA-Binding Proteins antagonists & inhibitors, DNA-Binding Proteins genetics
- Abstract
Background: The C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with "cancer hallmarks" and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT). Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions. Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities., Methods: Structured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model., Results: We identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models. CONCLUSIONS: This study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments., (© 2024. The Author(s).)
- Published
- 2024
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