Mechanistic Insights into the c-MYC G-Quadruplex and Berberine Binding inside an Aqueous Two-Phase System Mimicking Biomolecular Condensates.

We investigated the binding between the c-MYC G-quadruplex (GQ) and berberine chloride (BCl) in an aqueous two-phase system (ATPS) with 12.3 wt % polyethylene glycol and 5.6 wt % dextran, mimicking the highly crowded intracellular biomolecular condensates formed via liquid-liquid phase separation. We found that in the ATPS, complex formation is significantly altered, leading to an increase in affinity and a change in the stoichiometry of the complex with respect to neat buffer conditions. Thermodynamic studies reveal that binding becomes more thermodynamically favorable in the ATPS due to entropic effects, as the strong excluded volume effect inside ATPS droplets reduces the entropic penalty associated with binding. Finally, the binding affinity of BCl for the c-MYC GQ is higher than those for other DNA structures, indicating potential specific interactions. Overall, these findings will be helpful in the design of potential drugs targeting the c-MYC GQ structures in cancer-related biocondensates.

Identification and characterization of a new potent inhibitor targeting CtBP1/BARS in melanoma cells.

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.

Morphological Transformations of SARS-CoV-2 Nucleocapsid Protein Biocondensates Mediated by Antimicrobial Peptides.

Recently, the discovery of antimicrobial peptides (AMPs) as excellent candidates for overcoming antibiotic resistance has attracted significant attention. AMPs are short peptides active against bacteria, cancer cells, and viruses. It has been shown that the SARS-CoV-2 nucleocapsid protein (N-P) undergoes liquid-liquid phase separation in the presence of RNA, resulting in biocondensate formation. These biocondensates are crucial for viral replication as they concentrate the viral RNA with the host cell's protein machinery required for viral protein expression. Thus, N-P biocondensates are promising targets to block or slow down viral RNA transcription and consequently virion assembly. We investigated the ability of three AMPs to interfere with N-P/RNA condensates. Using microscopy techniques, supported by biophysical characterization, we found that the AMP LL-III partitions into the condensate, leading to clustering. Instead, the AMP CrACP1 partitions into the droplets without affecting their morphology but reducing their dynamics. Conversely, GKY20 leads to the formation of fibrillar structures after partitioning. It can be expected that such morphological transformation severely impairs the normal functionality of the N-P droplets and thus virion assembly. These results could pave the way for the development of a new class of AMP-based antiviral agents targeting biocondensates.

Structural characterization of the antimicrobial peptides myxinidin and WMR in bacterial membrane mimetic micelles and bicelles.

Antimicrobial peptides are a promising class of potential antibiotics that interact selectively with negatively charged lipid bilayers. This paper presents the structural characterization of the antimicrobial peptides myxinidin and WMR associated with bacterial membrane mimetic micelles and bicelles by NMR, CD spectroscopy, and molecular dynamics simulations. Both peptides adopt a different conformation in the lipidic environment than in aqueous solution. The location of the peptides in micelles and bicelles has been studied by paramagnetic relaxation enhancement experiments with paramagnetic tagged 5- and 16-doxyl stearic acid (5-/16-SASL). Molecular dynamics simulations of multiple copies of the peptides were used to obtain an atomic level of detail on membrane-peptide and peptide-peptide interactions. Our results highlight an essential role of the negatively charged membrane mimetic in the structural stability of both myxinidin and WMR. The peptides localize predominantly in the membrane's headgroup region and have a noticeable membrane thinning effect on the overall bilayer structure. Myxinidin and WMR show a different tendency to self-aggregate, which is also influenced by the membrane composition (DOPE/DOPG versus DOPE/DOPG/CL) and can be related to the previously observed difference in the ability of the peptides to disrupt different types of model membranes.