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Cystic fibrosis (CF) is a genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator ( CFTR ), a selective anion channel expressed in the epithelium of various organs. The most frequent mutation is F508del. This mutation leads to a misfolded CFTR protein quickly degraded via ubiquitination in the endoplasmic reticulum. Although preventing ubiquitination stabilizes the protein, functionality is not restored due to impaired plasma membrane transport. However, inhibiting the ubiquitination process can improve the effectiveness of correctors which act as chemical chaperones, facilitating F508del CFTR trafficking to the plasma membrane. Previous studies indicate a crosstalk between SUMOylation and ubiquitination in the regulation of CFTR. In this study, we investigated the potential of inhibiting SUMOylation to increase the effects of correctors and enhance the rescue of the F508del mutant across various cell models. In the widely used CFBE41o-cell line expressing F508del-CFTR, inhibiting SUMOylation substantially boosted F508del expression, thereby increasing the efficacy of correctors. Interestingly, this outcome did not result from enhanced stability of the mutant channel, but rather from augmented cytomegalovirus (CMV) promoter-mediated gene expression of F508del-CFTR. Notably, CFTR regulated by endogenous promoters in multiple cell lines or patient cells was not influenced by SUMOylation inhibitors.

Cystic fibrosis (CF) is caused by mutations in the gene encoding of the cystic fibrosis transmembrane conductance regulator (CFTR), an anion-selective plasma membrane channel that mainly regulates chloride transport in a variety of epithelia. More than 2000 mutations, most of which presumed to be disease-relevant, have been identified in the CFTR gene. The single CFTR mutation F508del (deletion of phenylalanine in position 508) is present in about 90% of global CF patients in at least one allele. F508del is responsible for the defective folding and processing of CFTR, failing to traffic to the plasma membrane and undergoing premature degradation via the ubiquitin-proteasome system. CFTR is subjected to different post-translational modifications (PTMs), and the possibility to modulate these PTMs has been suggested as a potential therapeutic strategy for the functional recovery of the disease-associated mutants. Recently, the PTM mapping of CFTR has identified some lysine residues that may undergo methylation or ubiquitination, suggesting a competition between these two PTMs. Our work hypothesis moves from the idea that favors methylation over ubiquitination, e.g., inhibiting demethylation could be a successful strategy for preventing the premature degradation of unstable CFTR mutants. Here, by using a siRNA library against all the human demethylases, we identified the enzymes whose downregulation increases F508del-CFTR stability and channel function. Our results show that KDM2A and KDM3B downregulation increases the stability of F508del-CFTR and boosts the functional rescue of the channel induced by CFTR correctors.

Protein kinase CK2 (CK2) is a highly conserved and ubiquitous kinase is involved in crucial biological processes, including proliferation, migration, and differentiation. CK2 holoenzyme is a tetramer composed by two catalytically active (α/α') and two regulatory (β) subunits and exerts its function on a broad range of targets. In the brain, it regulates different steps of neurodevelopment, such as neural differentiation, neuritogenesis, and synaptic plasticity. Interestingly, CK2 mutations have been recently linked to neurodevelopmental disorders; however, the functional requirements of the individual CK2 subunits in neurodevelopment have not been yet investigated. Here, we disclose the role of CK2 on the migration and adhesion properties of GN11 cells, an established model of mouse immortalized neurons, by different in vitro experimental approaches. Specifically, the cellular requirement of this kinase has been assessed pharmacologically and genetically by exploiting CK2 inhibitors and by generating subunit-specific CK2 knockout GN11 cells (with a CRISPR/Cas9-based approach). We show that CK2α' subunit has a primary role in increasing cell adhesion and reducing migration properties of GN11 cells by activating the Akt-GSK3β axis, whereas CK2α subunit is dispensable. Further, the knockout of the CK2β regulatory subunits counteracts cell migration, inducing dramatic alterations in the cytoskeleton not observed in CK2α' knockout cells. Collectively taken, our data support the view that the individual subunits of CK2 play different roles in cell migration and adhesion properties of GN11 cells, supporting independent roles of the different subunits in these processes.

A new sulfated sterol, phallusiasterol C (1), has been isolated from the Mediterranean ascidian Phallusia fumigata and its structure has been determined on the basis of extensive spectroscopic (mainly 2D NMR) analysis. The possible role in regulating the pregnane X receptor (PXR) activity of phallusiasterol C has been investigated; although the new sterol resulted inactive, this study adds more items to the knowledge of the structure-PXR regulating activity relationships in the case of sulfated steroids.

In recent years many sterols with unusual structures and promising biological profiles have been identified from marine sources. Here we report the isolation of a series of 24-alkylated-hydroxysteroids from the soft coral Sinularia kavarattiensis, acting as pregnane X receptor (PXR) modulators. Starting from this scaffold a number of derivatives were prepared and evaluated for their ability to activate the PXR by assessing transactivation and quantifying gene expression. Our study reveals that ergost-5-en-3β-ol (4) induces PXR transactivation in HepG2 cells and stimulates the expression of the PXR target gene CYP3A4. To shed light on the molecular basis of the interaction between these ligands and PXR, we investigated, through docking simulations, the binding mechanism of the most potent compound of the series, 4, to the PXR. Our findings provide useful functional and structural information to guide further investigations and drug design.

Purification of the apolar extracts of the marine ascidian Phallusia fumigata, afforded two new sulfated sterols, phallusiasterols A (1) and B (2). The structures of the new compounds have been elucidated using mass spectrometry and NMR experiments. The effects of phallusiasterols A and B as modulators of pregnane-X-receptor (PXR) have been investigated. These studies revealed that phallusiasterol A induces PXR transactivation in HepG2 cells and stimulates the expression of the PXR target genes CYP3A4 and MDR1 in the same cell line. Molecular docking calculations suggested the theoretical binding mode of phallusiasterol A with hPXR and revealed that phallusiasterol A fitted well in the LBD of PXR.

In the present study we provide evidence that solomonsterol A, a selective pregnane X receptor (PXR) agonist isolated from the marine sponge Theonella swinhoei, exerts anti-inflammatory activity and attenuates systemic inflammation and immune dysfunction in a mouse model of rheumatoid arthritis. Solomonsterol A was effective in protecting against the development of arthritis induced by injecting transgenic mice harboring a humanized PXR, with anti-collagen antibodies (CAIA) with beneficial effects on joint histopathology and local inflammatory response reducing the expression of inflammatory markers (TNFα, IFNγ and IL-17 and chemokines MIP1α and RANTES) in draining lymph nodes. Solomonsterol A rescued mice from systemic inflammation were assessed by measuring arthritis score, CRP and cytokines in the blood. In summary, the present study provides a molecular basis for the regulation of systemic local and systemic immunity by PXR agonists.

Further purification of the apolar extracts of the sponge Plakinastrella mamillaris, afforded a new oxygenated polyketide named gracilioether K, together with the previously isolated gracilioethers E-G and gracilioethers I and J. The structure of the new compound has been elucidated by extensive NMR (1H and 13C, COSY, HSQC, HMBC, and ROESY) and ESI-MS analysis. With the exception of gracilioether F, all compounds are endowed with potent pregnane-X-receptor (PXR) agonistic activity and therefore represent a new chemotype of potential anti-inflammatory leads. Docking calculations suggested theoretical binding modes of the identified compounds, compatible with an agonistic activity on hPXR, and clarified the molecular basis of their biological activities.

Using theonellasterol as a novel FXR antagonist hit, we prepared a series of semi-synthetic derivatives in order to gain insight into the structural requirements for exhibiting antagonistic activity. These derivatives are characterized by modification at the exocyclic carbon-carbon double bond at C-4 and at the hydroxyl group at C-3 and were prepared from theonellasterol using simple reactions. Pharmacological investigation showed that the introduction of a hydroxyl group at C-4 as well as the oxidation at C-3 with or without concomitant modification at the exomethylene functionality preserve the ability of theonellasterol to inhibit FXR transactivation caused by CDCA. Docking analysis showed that the placement of these molecules in the FXR-LBD is well stabilized when on ring A functional groups, able to form hydrogen bonds and π interactions, are present.