Claudio D’Amore

Efficacy of the CCR5 Antagonist Maraviroc in Reducing Early, Ritonavir-Induced Atherogenesis and Advanced Plaque Progression in Mice

Background— CCR5 plays an important role in atherosclerosis and ischemic cardiovascular diseases, as well as in HIV replication and diffusion. HIV infection is characterized by a high burden of cardiovascular diseases, particularly in subjects exposed to ritonavir-boosted protease inhibitors. Maraviroc, a CCR5 antagonist antiretroviral drug, might provide benefit for patients with M-tropic HIV infections at high risk for cardiovascular diseases. Methods and Results— Exposure to maraviroc limits the evolution and associated systemic inflammation of ritonavir-induced atherosclerotic in ApoE−/− mice and inhibits plaques development in a late model of atherosclerosis in which dyslipidemia plays the main pathogenic role. In ritonavir-treated mice, maraviroc reduced plaque areas and macrophage infiltration; downregulated the local expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and interleukin-17A; and reduced tumor necrosis factor-&agr; and RANTES (regulated on activation, normal T cell expressed, and secreted). Moreover, maraviroc counterregulated ritonavir-induced lipoatrophy and interlelukin-6 gene expression in epididymal fat, along with the splenic proinflammatory profile and expression of CD36 on blood monocytes. In the late model, maraviroc inhibited atherosclerotic progression by reducing macrophage infiltration and lowering the expression of adhesion molecules and RANTES inside the plaques. However, limited systemic inflammation was observed. Conclusions— In a mouse model of genetic dyslipidemia, maraviroc reduced the atherosclerotic progression by interfering with inflammatory cell recruitment into plaques. Moreover, in mice characterized by a general ritonavir-induced inflammation, maraviroc reversed the proinflammatory profile. Therefore, maraviroc could benefit HIV-positive patients with residual chronic inflammation who are at a high risk of acute coronary disease despite a suppressive antiretroviral therapy. To determine these benefits, large clinical studies are needed.

Theonellasterols and Conicasterols from Theonella swinhoei. Novel Marine Natural Ligands for Human Nuclear Receptors

Silica gel column chromatography, followed by HPLC purification on the apolar fraction of the methanol extract of marine sponge Theonella swinhoei, resulted in the isolation of a library of 10 polyhydroxylated steroids which we named theonellasterols B-H (1-7) and conicasterols B-D (8-10). The structures were determined on the basis of extensive spectroscopic data (MS, (1)H and (13)C NMR, COSY, HSQC, HMBC, and ROESY) analysis, and the putative binding mode to nuclear receptors (NRs) has been obtained through docking calculations. Pharmacological and structure-activity relationship analysis demonstrate that these natural polyhydroxylated steroids are potent ligands of human nuclear pregnane receptor (PXR) and modulator of farnesoid-X-receptor (FXR). In addition, the molecular characterization of theonellasterol G allowed the identification of the first FXR modulator and PXR ligand so far identified. Exposure of liver cells to this agent resulted in potent induction of PXR-regulated genes and modulation of FXR-regulated genes, highlighting its pharmacological potential in the treatment of liver disorders.

Binding Mechanism of the Farnesoid X Receptor Marine Antagonist Suvanine Reveals a Strategy To Forestall Drug Modulation on Nuclear Receptors. Design, Synthesis, and Biological Evaluation of Novel Ligands

Here, we report suvanine, a marine sponge sesterterpene, as an antagonist of the mammalian bile acid sensor farnesoid-X-receptor (FXR). Using suvanine as a template, we shed light on the molecular bases of FXR antagonism, identifying the essential conformational changes responsible for the transition from the agonist to the antagonist form. Molecular characterization of the nuclear corepressor NCoR and coactivator Src-1 revealed that receptor conformational changes are associated with a specific dynamic of recruitment of these cofactors to the promoter of OSTα, a FXR regulated gene. Using suvanine as a novel hit, a library of semisynthetic derivatives has been designed and prepared, leading to pharmacological profiles ranging from agonism to antagonism toward FXR. Deep pharmacological evaluation demonstrated that derivative 19 represents a new chemotype of FXR modulator, whereas alcohol 6, with a simplified molecular scaffold, exhibits excellent antagonistic activity.

Dissociation of Intestinal and Hepatic Activities of FXR and LXRα Supports Metabolic Effects of Terminal Ileum Interposition in Rodents

The farnesoid X receptor (FXR) and the liver x receptors (LXRs) are bile acid–activated receptors that are highly expressed in the enterohepatic tissues. The mechanisms that support the beneficial effects of bariatric surgery are only partially defined. We have investigated the effects of ileal interposition (IT), a surgical relocation of the distal ileum into the proximal jejunum, on FXR and LXRs in rats. Seven months after surgery, blood concentrations of total bile acids, taurocholic acid, an FXR ligand, and taurohyocholic acid, an LXRα ligand, were significantly increased by IT (P < 0.05). In contrast, liver and intestinal concentrations of conjugated and nonconjugated bile acids were decreased (P < 0.05). These changes were associated with a robust induction of FXR and FXR-regulated genes in the intestine, including Fgf15, a negative regulator of bile acid synthesis. IT repressed the liver expression of glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase (Pepck), two gluconeogenetic genes, along with the expression of LXRα and its target genes sterol regulatory element-binding protein (Srebp) 1c and fatty acid synthase (Fas) in the liver. Treating IT rats with chenodeoxycholic acid ameliorated insulin signaling in the liver. Whether confirmed in human settings, these results support the association of pharmacological therapies with bariatric surgeries to exploit the selective activation of intestinal nuclear receptors.

4-Methylenesterols from Theonella swinhoei sponge are natural pregnane-X-receptor agonists and farnesoid-X-receptor antagonists that modulate innate immunity.

We report the isolation and the structural elucidation of a family of polyhydroxylated steroids from the marine sponge Theonella swinhoei. Decodification of interactions of these family with nuclear receptors shows that these steroids are potent agonists of human pregnane-X-receptor (PXR) and antagonists of human farnesoid-X-receptor (FXR) with the putative binding mode to nuclear receptors (NRs) obtained through docking experiments. By using monocytes isolated from transgenic mice harboring hPXR, we demonstrated that swinhosterol B counter-regulates induction of pro-inflammatory cytokines in a PXR-dependent manner. Exposure of CD4(+) T cells to swinhosterol B upregulates the expression of IL-10 causing a shift toward a T cells regulatory phenotype in a PXR dependent manner. These results pave the way to development of a dual PXR agonist/FXR antagonist with a robust immunomodulatory activity and endowed with the ability to modulate the expression of bile acid-regulated genes in the liver.

Plakilactones from the marine sponge Plakinastrella mamillaris. Discovery of a new class of marine ligands of peroxisome proliferator-activated receptor γ.

In this paper we report the isolation and the molecular characterization of a new class of PPARγ ligands from the marine environment. Biochemical characterization of a library of 13 oxygenated polyketides isolated from the marine sponge Plakinastrella mamillaris allowed the discovery of gracilioether B and plakilactone C as selective PPARγ ligands in transactivation assays. Both agents covalently bind to the PPARγ ligand binding domain through a Michael addition reaction involving a protein cysteine residue and the α,β-unsaturated ketone in their side chains. Additionally, gracilioether C is a noncovalent agonist for PPARγ, and methyl esters 1 and 2 are noncovalent antagonists. Structural requirements for the interaction of these agents within the PPARγ ligand binding domain were obtained by docking analysis. Gracilioether B and plakilactone C regulate the expression of PPARγ-dependent genes in the liver and inhibit the generation of inflammatory mediators by macrophages.

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

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.

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)

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.

SHP-dependent and -independent induction of peroxisome proliferator-activated receptor-γ by the bile acid sensor farnesoid X receptor counter-regulates the pro-inflammatory phenotype of liver myofibroblasts

ObjectiveThe regulation of hepatic stellate cells (HSCs) by bacterial lipopolysaccharide (LPS) represents a recently-discovered and novel mechanism for hepatic injury and fibrosis. Stimulation of HSCs with LPS results in a rapid and marked induction of interleukin (IL)1β, IL6 and tumor necrosis factor α. These events lead to the development of the activated phenotype in the HSCs associated with fibrosis and inflammation in the injured liver. We have previously demonstrated that farnesoid X receptor (FXR) activation increases transcription of rat peroxisome proliferator-activated receptor-γ (PPARγ) gene in HSCs. We aimed at evaluating the molecular mechanism of the transcriptional regulation of the PPARγ gene by FXR.MethodsReal-time PCR, ELISA, transactivations, EMSA and ChIP experiments were performed in HSC-T6 cells, in primary HSCs, in HEK293T cells and in CCl4-treated rats.ResultsIn vivo and in vitro activation of FXR downregulates cytokines and collagen(α)1 while inducing PPARγ and small heterodimer partner (SHP). NUBIScan analysis of rat PPARγ promoter revealed the presence of a putative FXR response element. Cotransfection with FXR/retinoic acid receptor significantly enhanced chenodeoxycholic acid-induced luciferase activity. EMSA experiments demonstrated that FXR was able to bind to an inverted repeat-1 sequence and ChIP experiments confirmed that FXR is recruited on the PPARγ promoter.ConclusionThe present study provides a molecular basis for the physiological cross-talk between FXR and PPARγ pathways in HSCs.

Polyhydroxylated sterols from the Indonesian soft coral Sinularia sp. and their effect on farnesoid X-activated receptor

Chemical investigation of the Indonesian soft coral Sinularia sp. resulted in the isolation of three known (1, 2 and 7) and five new (3-6 and 8) sterols, characterized by either 24-methylcholestane or gorgostane skeletons. The stereostructures of the new compounds have been elucidated by application of HR-MS and 2D NMR techniques. The isolated steroids have been evaluated for their interaction with the farnesoid X-activated receptor (FXR) and some of them, including the new compound 3 and gorgosterol (7), showed a consistent antagonistic activity, potentially useful for the treatment of cholestasis. The FXR antagonistic activity of gorgosterol (7) was also supported by gene expression experiments. Our results represent the first evaluation of soft coral steroids for interaction with nuclear receptors and qualify gorgosterol (7) as a new chemotype of FXR antagonist.