Pubblicazioni 2014

1 – Festa C, Renga B, D’Amore C, Sepe V, Finamore C, De Marino S, Carino A, Cipriani S, Monti MC, Zampella A, Fiorucci S. Exploitation of cholane scaffold for the discovery of potent and selective farnesoid X receptor (FXR) and G-protein coupled bile acid receptor 1 (GP-BAR1) ligands. J Med Chem. 2014 Oct 23;57(20):8477-95.

Abstract
Nuclear and G-protein coupled receptors are considered major targets for drug discovery. FXR and GP-BAR1, two bile acid-activated receptors, have gained increasing consideration as druggable receptors. Because endogenous bile acids often target both receptor families, the development of selective ligands has been proven difficult, exposing patients to side effects linked to an unwanted activation of one of the two receptors. In the present study, we describe a novel library of semisynthetic bile acid derivatives obtained by modifications on the cholane scaffold. The pharmacological characterization of this library led to the discovery of 7α-hydroxy-5β-cholan-24-sulfate (7), 6β-ethyl-3α,7β-dihydroxy-5β-cholan-24-ol (EUDCOH, 26), and 6α-ethyl-3α, 7α-dihydroxy-24-nor-5β-cholan-23-ol (NorECDCOH, 30) as novel ligands for FXR and GP-BAR1 that might hold utility in the treatment of FXR and GP-BAR1 mediated disorders

2 – Sepe V, Renga B, Festa C, D’Amore C, Masullo D, Cipriani S, Di Leva FS, Monti MC, Novellino E, Limongelli V, Zampella A, Fiorucci S. Modification on ursodeoxycholic acid (UDCA) scaffold. discovery of bile acid derivatives as selective agonists of cell-surface G-protein coupled bile acid receptor 1 (GP-BAR1). J Med Chem. 2014 Sep 25;57(18):7687-701.

Abstract
Bile acids are signaling molecules interacting with the nuclear receptor FXR and the G-protein coupled receptor 1 (GP-BAR1/TGR5). GP-BAR1 is a promising pharmacological target for the treatment of steatohepatitis, type 2 diabetes, and obesity. Endogenous bile acids and currently available semisynthetic bile acids are poorly selective toward GP-BAR1 and FXR. Thus, in the present study we have investigated around the structure of UDCA, a clinically used bile acid devoid of FXR agonist activity, to develop a large family of side chain modified 3α,7β-dihydroxyl cholanoids that selectively activate GP-BAR1. In vivo and in vitro pharmacological evaluation demonstrated that administration of compound 16 selectively increases the expression of pro-glucagon 1, a GP-BAR1 target, in the small intestine, while it had no effect on FXR target genes in the liver. Further, compound 16 results in a significant reshaping of bile acid pool in a rodent model of cholestasis. These data demonstrate that UDCA is a useful scaffold to generate novel and selective steroidal ligands for GP-BAR1.

3 – Distrutti E, O’Reilly JA, McDonald C, Cipriani S, Renga B, Lynch MA, Fiorucci S. Modulation of intestinal microbiota by the probiotic VSL#3 resets brain gene expression and ameliorates the age-related deficit in LTP. PLoS One. 2014 Sep 9;9(9):e106503.

Abstract
The intestinal microbiota is increasingly recognized as a complex signaling network that impacts on many systems beyond the enteric system modulating, among others, cognitive functions including learning, memory and decision-making processes. This has led to the concept of a microbiota-driven gut-brain axis, reflecting a bidirectional interaction between the central nervous system and the intestine. A deficit in synaptic plasticity is one of the many changes that occurs with age. Specifically, the archetypal model of plasticity, long-term potentiation (LTP), is reduced in hippocampus of middle-aged and aged rats. Because the intestinal microbiota might change with age, we have investigated whether the age-related deficit in LTP might be attenuated by changing the composition of intestinal microbiota with VSL#3, a probiotic mixture comprising 8 Gram-positive bacterial strains. Here, we report that treatment of aged rats with VSL#3 induced a robust change in the composition of intestinal microbiota with an increase in the abundance of Actinobacteria and Bacterioidetes, which was reduced in control-treated aged rats. VSL#3 administration modulated the expression of a large group of genes in brain tissue as assessed by whole gene expression, with evidence of a change in genes that impact on inflammatory and neuronal plasticity processes. The age-related deficit in LTP was attenuated in VSL#3-treated aged rats and this was accompanied by a modest decrease in markers of microglial activation and an increase in expression of BDNF and synapsin. The data support the notion that intestinal microbiota can be manipulated to positively impact on neuronal function.

4.– Hodnik Ž, Peterlin Mašič L, Tomašić T, Smodiš D, D’Amore C, Fiorucci S, Kikelj D. Bazedoxifene-scaffold-based mimetics of solomonsterols A and B as novel pregnane X receptor antagonists. J Med Chem. 2014 Jun 12;57(11):4819-33.

Abstract
Pregnane X receptor (PXR), a member of the NR1I nuclear receptor family, acts as a xenobiotic sensor and a paramount transcriptional regulator of drug-metabolizing enzymes and transporters. The overexpression of PXR in various cancer cells indicates the importance of PXR as a drug target for countering multidrug resistance in anticancer treatments. We describe the discovery of novel bazedoxifene-scaffold-based PXR antagonists inspired by the marine sulfated steroids solomonsterol A and B as natural leads. A luciferase reporter assay on a PXR-transfected HepG2 cell line identified compounds 19-24 as promising PXR antagonists. Further structure-activity relationship studies of the most active PXR antagonist from the series (compound 20, IC50 = 11 μM) revealed the importance of hydroxyl groups as hydrogen-bond donors for PXR antagonistic activity. PXR antagonists 20 and 24 (IC50 = 14 μM), in addition to the downregulation of PXR expression, exhibited inhibition of PXR-induced CYP3A4 expression, which illustrates their potential to suppress PXR-regulated phase-I drug metabolism.

5 – Renga B, Francisci D, Schiaroli E, Carino A, Cipriani S, D’Amore C, Sidoni A, Sordo RD, Ferri I, Lucattelli M, Lunghi B, Baldelli F, Fiorucci S. The HIV matrix protein p17 promotes the activation of human hepatic stellate cells through interactions with CXCR2 and Syndecan-2. PLoS One. 2014 Apr 15;9(4):e94798.

Abstract
BACKGROUND:
The human immunodeficiency virus type 1 (HIV-1) p17 is a matrix protein involved in virus life’s cycle. CXCR2 and Syndecan-2, the two major coreceptors for the p17 protein, are expressed in hepatic stellate cells (HSCs), a key cell type involved in matrix deposition in liver fibrotic disorders.
AIM:
In this report we have investigated the in vitro impact of p17 on HSCs transdifferentiation and function and underlying signaling pathways involved in these processes.
METHODS:
LX-2 cells, a human HSC line, and primary HSC were challenged with p17 and expressions of fibrogenic markers and of p17 receptors were assessed by qRT-PCR and Western blot. Downstream intracellular signaling pathways were evaluated with qRT-PCR and Western blot as well as after pre-treatment with specific pathway inhibitors.
RESULTS:
Exposure of LX2 cells to p17 increases their contractile force, reshapes the cytoskeleton fibers and upregulates the expression of transdifferentiation markers including αSMA, COL1α1 and endothelin-1 through the activation of Jak/STAT and Rho signaling pathways. These effects are lost in HSCs pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide. Confocal laser microscopy studies demonstrates that CXCR2 and syndecan-2 co-associate at the plasma membrane after exposure to p17. Immunostaining of HIV/HCV liver biopsies from co-infected patients reveals that the progression of liver fibrosis correlates with a reduced expression of CXCR2.

CONCLUSIONS:

The HIV matrix protein p17 is pro-fibrogenic through its interactions both with CXCR2 and syndecan-2 on activated HSCs.

6 – D’Amore C, Di Leva FS, Sepe V, Renga B, Del Gaudio C, D’Auria MV, Zampella A, Fiorucci S, Limongelli V. Design, synthesis, and biological evaluation of potent dual agonists of nuclear and membrane bile acid receptors. J Med Chem. 2014 Feb 13;57(3):937-54.

Abstract
Bile acids exert genomic and nongenomic effects by interacting with membrane G-protein-coupled receptors, including the bile acid receptor GP-BAR1, and nuclear receptors, such as the farnesoid X receptor (FXR). These receptors regulate overlapping metabolic functions; thus, GP-BAR1/FXR dual agonists, by enhancing the biological response, represent an innovative strategy for the treatment of enteroendocrine disorders. Here, we report the design, total synthesis, and in vitro/in vivo pharmacological evaluation of a new generation of dual bile acid receptor agonists, with the most potent compound, 19, showing promising pharmacological profiles. We show that compound 19 activates GP-BAR1, FXR, and FXR regulated genes in the liver, increases the intracellular concentration of cAMP, and stimulates the release of the potent insulinotropic hormone GLP-1, resulting in a promising drug candidate for the treatment of metabolic disorders. We also elucidate the binding mode of the most potent dual agonists in the two receptors through a series of computations providing the molecular basis for dual GP-BAR1/FXR agonism.

7 – Sepe V, D’Amore C, Ummarino R, Renga B, D’Auria MV, Novellino E, Sinisi A, Taglialatela-Scafati O, Nakao Y, Limongelli V, Zampella A, Fiorucci S. Insights on pregnane-X-receptor modulation. Natural and semisynthetic steroids from Theonella marine sponges. Eur J Med Chem. 2014 Feb 12;73:126-34.

Abstract
Pregnane-X-receptor (PXR) is a member of nuclear receptors superfamily that activates gene transcription by binding to responsive elements in the promoter of target genes. PXR is a master gene orchestrating the expression/activity of genes involved in the metabolism of endobiotics including bilirubin, bile acids, glucose and lipid. In addition PXR oversights the metabolism of the large majority of xenobiotics including a large amount of prescribing drugs. Thus, developing PXR ligands represents a great opportunity for a therapeutic intervention on human diseases including diabetes, obesity, dyslipidemias and liver disorders. To this end, natural compounds represent an arsenal of new chemical scaffolds useful for the identification of novel PXR ligands. Here, we report a series of 4-methylenesteroid derivatives isolated from Theonella marine sponges as novel PXR modulators. In addition, combining medicinal chemistry, pharmacological experiments and computational studies, we have investigated the effects of different modifications on ring A and on the side chain of 4-methylenesteroid derivatives toward PXR modulation. This study provides the molecular bases of ligand/PXR interaction useful for designing novel PXR modulators.

8 – Di Micco S, Renga B, Carino A, D’Auria MV, Zampella A, Riccio R, Fiorucci S, Bifulco G. Structural insights into Estrogen Related Receptor-β modulation: 4-methylenesterols from Theonella swinhoei sponge as the first example of marine natural antagonists. Steroids. 2014 Feb;80:51-63.

Abstract
In this paper, we report the first evidence of 4-methylenesterols, isolated from the marine sponge Theonella swinhoei, as antagonists of Estrogen Related Receptors (ERRs). The interactions of 4-methylenesterols with ERRs were investigated through a multi-parametric approach involving biological assays and molecular modelling. Here the first homology model of active and inactive conformations of the Estrogen Related Receptor β (ERRβ) is also reported, benchmarked with the well known agonists gsk4716 and genistein, and the antagonists 4-hydroxytamoxifen and diethylstilbestrol. Our proposed model could contribute to the clarification of small molecule interaction mode in the ERRβ and, notably, to the rational design of new potential and selective modulators of this emerging therapeutic target.

9 – Renga B, Mencarelli A, Cipriani S, D’Amore C, Francisci D, Santucci L, Baldelli F, Distrutti E, Fiorucci S. In vivo administration of ritonavir worsens intestinal damage caused by cyclooxygease inhibitors. Eur J Pharmacol. 2014 Jan 15;723:194-201.

Abstract
The protease inhibitor ritonavir is part of the highly active anti-retroviral therapy (HAART) successfully used in the treatment of human immunodeficiency virus (HIV)-1 infection. There is evidence that ritonavir alters intestinal permeability and induces damage to the small intestine. Because HIV infected patients taking HAART are at high risk for developing cardiovascular complications, there might be a need for the use of low dose of aspirin (ASA) to prevent ischemic events. Similarly, long term survival exposes HIV infected persons to detrimental interactions of ritonavir with non-steroidal anti-inflammatory drugs (NSAIDs). In the present work we tested whether ritonavir worsens intestinal injury caused by NSAIDs and ASA. C57BL6 mice were treated for 25 days with ritonavir and for a further 5 days with the combination of ritonavir plus ASA or ritonavir plus naproxen. In a second set of experiments C57BL6 mice were cotreated with ritonavir plus misoprostol, a PGE1 analog. We found that ritonavir administration caused intestinal damage and its co-administration with naproxen or ASA exacerbated the severity of injury and intestinal inflammation, as assessed by measuring haematocrit, MPO, mucosal levels of PGE2 and mRNA levels of iNOS, MCP-1 and VLA-1. Co-administration of misoprostol protected against intestinal damage induced by naproxen and ritonavir. In conclusion we demonstrated that ritonavir causes intestinal damage and that its association with NSAIDs or ASA worsens the damage caused by COX-inhibitors. Misoprostol rescues from intestinal damage caused by ritonavir. Further studies are need to clarify whether this observation has a clinical readout.

10 – Fiorucci S, Distrutti E, Ricci P, Giuliano V, Donini A, Baldelli F. Targeting FXR in cholestasis: hype or hope. Expert Opin Ther Targets. 2014 Dec;18(12):1449-59.

Abstract
INTRODUCTION:
Bile acids, the end product of cholesterol metabolism, are signaling molecules. The farnesoid X receptor (FXR) is a bile acid sensor and is part of a network of nuclear receptors that regulate bile acid homeostasis. In addition to FXR, bile acids activate other nuclear receptors (CAR, PXR and VDR), cell surface receptors including the G protein-coupled bile acid receptor 1 (GP-BAR1/TGR5), muscarinic receptor and calcium-gated potassium channels.
AREAS COVERED:
The semisynthetic bile acid derivative 6-ethyl chenodeoxycholic acid (6-ECDCA, INT-747 later christened obeticholic acid) is a dual FXR/GP-BAR1 ligand that attenuates bile flow impairment in cholestasis induced by 17β-estradiol; a model of pregnancy-induced cholestasis. Phase II trials with this agent in early stage primary biliary cirrhosis have shown beneficial effects on surrogate markers of damage progression, specifically alkaline phosphatase, with a dose-dependent itching being the most severe and common side effect (up to 70% of patients) leading to therapy discontinuation in 38% of patients. GP-BAR1 activation in the skin triggers itching, thus providing a molecular explanation for this side effect.
EXPERT OPINION:
While the role of FXR activation in treating severe cholestasis needs confirmation, the activation of GP-BAR1 is likely involved in pruritus development that associates with clinical use of dual FXR/GP-BAR1 ligands. FXR antagonist could be an interesting opportunity for treatment of severe/obstructive cholestasis.

11 – Sepe V, Di Leva FS, D’Amore C, Festa C, De Marino S, Renga B, D’Auria MV, Novellino E, Limongelli V, D’Souza L, Majik M, Zampella A, Fiorucci S. Marine and semi-synthetic hydroxysteroids as new scaffolds for pregnane X receptor modulation. Mar Drugs. 2014 May 27;12(6):3091-115.

Abstract
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.

12 – Chianese G, Sepe V, Limongelli V, Renga B, D’Amore C, Zampella A, Taglialatela-Scafati O, Fiorucci S. Incisterols, highly degraded marine sterols, are a new chemotype of PXR agonists. Steroids. 2014 May;83:80-5.

Abstract
During the chromatographic purification of organic extracts obtained from Plakortis cfr. lita we obtained three highly degraded steroid derivatives, the known incisterol A2 (1) and the new incisterols A5 (2) and A6 (3). The new compounds were characterized basing on NMR and MS evidences along with comparison with model compounds. Incisterol A5 proved to bear a 17S-ethyl-15E,18-diene (incisterol numbering system) side chain, found for the first time in a marine organism. The new incisterols A5 and A6 proved to be potent inducers of transactivation of the pregnane X receptor (PXR) and they also stimulate the expression of CYP7A4 and MDR1 with a potency comparable to that of Rifaximin. These observations prompt to consider incisterols A5 and A6 as new potent agonists of PXR. On the other hand, the 17R-methyl analogue incisterol A2 shows only a poor PXR agonist activity. Molecular docking simulations elucidated the binding mechanism of the active incisterols in the ligand binding domain of PXR.

13 – Mencarelli A, D’Amore C, Renga B, Cipriani S, Carino A, Sepe V, Perissutti E, D’Auria MV, Zampella A, Distrutti E, Fiorucci S. Solomonsterol A, a marine pregnane-X-receptor agonist, attenuates inflammation and immune dysfunction in a mouse model of arthritis. Mar Drugs. 2013 Dec 24;12(1):36-53.

Abstract
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.

14 – Imperatore C, D’Aniello F, Aiello A, Fiorucci S, D’Amore C, Sepe V, Menna M. Phallusiasterols A and B: two new sulfated sterols from the Mediterranean tunicate Phallusia fumigata and their effects as modulators of the PXR receptor. Mar Drugs. 2014 Apr 3;12(4):2066-78.