Combinatorial targeting of G-protein-coupled bile acid receptor 1 and cysteinyl leukotriene receptor 1 reveals a mechanistic role for bile acids and leukotrienes in drug-induced liver injury.

BACKGROUND AND AIM: Drug-induced liver injury (DILI) is a common disorder that involves both direct liver cell toxicity and immune activation. The bile acid receptor, G-protein-coupled bile acid receptor 1 (GPBAR1; Takeda G-protein-coupled receptor 5 [TGR5]), and cysteinyl leukotriene receptor (CYSLTR) 1 are G-protein-coupled receptors activated by bile acids and leukotrienes, exerting opposite effects on cell-to-cell adhesion, inflammation, and immune cell activation. To investigate whether GPBAR1 and CYSLTR1 mutually interact in the development of DILI, we developed an orally active small molecule, CHIN117, that functions as a GPBAR1 agonist and CYSLTR1 antagonist. APPROACH AND RESULTS: RNA-sequencing analysis of liver explants showed that acetaminophen (APAP) intoxication positively modulates the leukotriene pathway, CYSLTR1, 5-lipoxygenase, and 5-lipoxygenase activating protein, whereas GPBAR1 gene expression was unchanged. In mice, acute liver injury induced by orally dosing APAP (500 mg/kg) was severely exacerbated by Gpbar1 gene ablation and attenuated by anti-Cysltr1 small interfering RNA pretreatment. Therapeutic dosing of wild-type mice with CHIN117 reversed the liver damage caused by APAP and modulated up to 1300 genes, including 38 chemokines and receptors, that were not shared by dosing mice with a selective GPBAR1 agonist or CYSLTR1 antagonist. Coexpression of the two receptors was detected in liver sinusoidal endothelial cells (LSECs), monocytes, and Kupffer cells, whereas combinatorial modulation of CYSLTR1 and GPBAR1 potently reversed LSEC/monocyte interactions. CHIN117 reversed liver damage and liver fibrosis in mice administered CCl 4 . CONCLUSIONS: By genetic and pharmacological approaches, we demonstrated that GPBAR1 and CYSLTR1 mutually interact in the development of DILI. A combinatorial approach designed to activate GPBAR1 while inhibiting CYSLTR1 reverses liver injury in models of DILI.

Analysis of Hsp90 allosteric modulators interactome reveals a potential dual action mode involving mitochondrial MDH2.

Hsp90 (i.e., Heat shock protein 90) is a well-established therapeutic target for several diseases, ranging from misfolding-related disfunctions to cancer. In this framework, we have developed in recent years a family of benzofuran compounds that act as Hsp90 allosteric modulators. Such molecules can interfere with the stability of some relevant Hsp90 client oncoproteins, showing a low µM cytotoxic activity in vitro in cancer cell lines. Here we identify the target profile of these chemical probes by means of chemical proteomics, which established MDH2 (mitochondrial malate dehydrogenase) as an additional relevant cellular target that might help elucidate the molecular mechanism of their citotoxicity. Western blotting, DARTS (i.e., Drug Affinity Responsive Target Stability) and enzymatic assays data confirmed a dose-dependent interaction of MDH2 with several members of the benzofuran Hsp90 modulators family and a computational model allowed to interpret the observed interactions.

Molecular Network and Culture Media Variation Reveal a Complex Metabolic Profile in Pantoea cf. eucrina D2 Associated with an Acidified Marine Sponge.

The Gram-negative Pantoea eucrina D2 was isolated from the marine sponge Chondrosia reniformis. Sponges were collected in a shallow volcanic vents system in Ischia island (South Italy), influenced by CO2 emissions and lowered pH. The chemical diversity of the secondary metabolites produced by this strain, under different culture conditions, was explored by a combined approach including molecular networking, pure compound isolation and NMR spectroscopy. The metabolome of Pantoea cf. eucrina D2 yielded a very complex molecular network, allowing the annotation of several metabolites, among them two biosurfactant clusters: lipoamino acids and surfactins. The production of each class of metabolites was highly dependent on the culture conditions, in particular, the production of unusual surfactins derivatives was reported for the first time from this genus; interestingly the production of these metabolites only arises by utilizing inorganic nitrogen as a sole nitrogen source. Major components of the extract obtained under standard medium culture conditions were isolated and identified as N-lipoamino acids by a combination of 1D and 2D NMR spectroscopy and HRESI-MS analysis. Assessment of the antimicrobial activity of the pure compounds towards some human pathogens, indicated a moderate activity of leucine containing N-lipoamino acids towards Staphylococcus aureus, Staphylococcus epidermidis and a clinical isolate of the emerging food pathogen Listeria monocytogenes.

Determining the Effect of Pterostilbene on Insulin Secretion Using Chemoproteomics.

Pterostilbene, the 3,5-dimethoxy derivative of resveratrol, is a well-known polyphenolic compound, mainly found in blueberries, grapevines, and Pterocarpus marsupium heartwood, which has recently attracted a great deal of attention due to its wide bio-pharmacological profile. Moreover, pterostilbene is more lipophilic than resveratrol, with a consequently better bioavailability and a more interesting therapeutic potential. In this work, a chemoproteomic approach, based on affinity chromatography, was applied on pterostilbene in the attempt to identify the biological targets responsible for its bioactivity. On this basis, syntaxins, a group of proteins involved in the formation of SNARE complexes mediating vesicles exocytosis, were selected among the most interesting pterostilbene interactors. In vitro and in cell assays gave evidence of the pterostilbene ability to reduce insulin secretion on glucose-stimulated pancreatic beta cells, opening the way to potential applications of pterostilbene as a supplement in the care of insulin-dependent metabolic disorders.

[Validation of surgical masks during COVID19 emergency: activities at the University of Napoli Federico II]. / Validazione di maschere chirurgiche nella fase di emergenza COVID19: l'esperienza dell'Università degli Studi di Napoli Federico II.

SUMMARY: During COVID-19 pandemic crisis, Italian Government has approved Law Decree no. 18 of 17 march 2020, in which art. 15 allows enterprises to produce, import and commercialize surgical masks notwithstanding the current rules of product certification. It is just required that the interested enterprises send to the Italian National Institute of Health a selfcertification in which they declare the technical characteristics of the masks and that masks are produced according to the safety requirements. In this context, a technical-scientific unit was established at the University of Napoli Federico II to provide interested enterprises with state-of-the-art consultancy, testing and measurement services, adhering to rigorous scientific protocols. Characterization tests were carried out on 163 surgical masks and/or materials for their construction and they have enabled the identification of pre-screening criteria to simplify the procedure for evaluating surgical masks using methods for assessing the filtration efficiency of particles and aerosols. Based on experimental results, it has been observed that a filtration efficiency for particles with sizes larger that 650 nm (PFE>650) exceeding 35% might guarantees a bacterial filtration efficiency (BFE) higher than 95% while BFE values higher than 98% are obtained when the PFE>650 is larger than 40%. PFE measurement is extremely simpler with respect to BFE, the latter being time-consuming and requiring specific equipment and methods for its realization. Many tested materials have shown the capability to assure high filtration efficiencies but Spundonded-Meltblown-Spunbonded (SMS), that are layers of non-woven fabric with different weights of Meltblown, can simultaneously guarantee high particle filtration efficiencies with pressure drop values (breathability) in the limits to classify the surgical masks as Type II/IIR. In fact, the fabric products analyzed so far have not been able to simultaneously guarantee adequate BFE and breathability values. On the contrary, Spunbonds of adequate weights can virtually verify both requirements and accredit themselves as possible materials for the production of surgical masks, at least of Type I. Further studies are needed to verify the possibility of producing low-cost, reusable surgical masks that could meet the criteria of circular economy.

Scalarane sesterterpenes from Thorectidae sponges as inhibitors of TDP-43 nuclear factor.

The analysis of two Thorectidae sponge samples, Hyrtios sp. and Petrosaspongia sp., collected at Fiji Islands, led to the isolation of five new scalarane derivatives along with fifteen known compounds. Their structures were elucidated on the basis of NMR and MS spectroscopic data. The small library of natural scalarane derivatives was investigated for their ability to modulate the activity of trans-activation response DNA-binding protein of 43 kDa (TDP-43), a key factor in several neurodegenerative conditions and the study resulted in the identification of potent inhibitors of TDP-43 protein.

Monitoring of seven pesticide residues by LC-MS/MS in extra virgin olive oil samples and risk assessment for consumers.

Residue levels of seven pesticides were analyzed in thirty-five samples of Extra Virgin Olive Oil to assess the health risk of consuming Italian oils correlated with the presence of these pesticides. An in-house analytical procedure was developed and validated, consisting of a specific dispersive solid-phase extraction using the QuEChERS technique and a qualitative-quantitative analysis using liquid chromatography coupled with tandem mass spectrometry. Thirty-four percent of the samples were contaminated with pesticide residues; in the concentration range of 0.53-0.56 ng/mL for imazalil, 1.11-1.56 ng/mL for acetamiprid-N-desmethyl, 1.28-1.46 ng/mL for clothianidin, 0.94-1.49 ng/mL for thiacloprid, 1.08-4.64 ng/mL for dinotefuran, 0.42-1.47 ng/mL for thiamethoxam, 0.42-6.14 ng/mL for imidacloprid). Risk assessment was evaluated using the hazard quotient, hazard index, and Pesticide Residue Intake Model by EFSA. All hazard indices confirmed that the concentrations of pesticides detected in the oil samples did not represent a short or long-term risk for consumers' health.

BAR502/fibrate conjugates: synthesis, biological evaluation and metabolic profile.

BAR502, a bile acid analogue, is active as dual FXR/GPBAR1 agonist and represents a promising lead for the treatment of cholestasis and NASH. In this paper we report the synthesis and the biological evaluation of a library of hybrid compounds prepared by combining, through high-yield condensation reaction, some fibrates with BAR502.The activity of the new conjugates was evaluated towards FXR, GPBAR1 and PPARα receptors, employing transactivation or cofactor recruitment assays. Compound 1 resulted as the most promising of the series and was subjected to further pharmacological investigation, together with stability evaluation and cell permeation assessment. We have proved by LCMS analysis that compound 1 is hydrolyzed in mice releasing clofibric acid and BAR505, the oxidized metabolite of BAR502, endowed with retained dual FXR/GPBAR1 activity.

Discovery of a Novel Class of Dual GPBAR1 Agonists-RORγt Inverse Agonists for the Treatment of IL-17-Mediated Disorders.

Retinoic acid receptor-related orphan receptor γ-t (RORγt) and GPBAR1, a transmembrane G-protein-coupled receptor for bile acids, are attractive drug targets to develop clinically relevant small modulators as potent therapeutics for autoimmune diseases. Herein, we designed, synthesized, and evaluated several new bile acid-derived ligands with potent dual activity. Furthermore, we performed molecular docking and MD calculations of the best dual modulators in the two targets to identify the binding modes as well as to better understand the molecular basis of the inverse agonism of RORγt by bile acid derivatives. Among these compounds, 7 was identified as a GPBAR1 agonist (EC50 5.9 µM) and RORγt inverse agonist (IC50 0.107 µM), with excellent pharmacokinetic properties. Finally, the most promising ligand displayed robust anti-inflammatory activity in vitro and in vivo in a mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis.

Correction to "Discovery of a Novel Class of Dual GPBAR1 Agonists-RORγt Inverse Agonists for the Treatment of IL-17-Mediated Disorders".

[This corrects the article DOI: 10.1021/acsomega.2c07907.].

Chemical proteomics reveals heat shock protein 60 to be the main cellular target of the marine bioactive sesterterpene suvanine.

Marine bioactive compounds are potential drug leads because of their diverse pharmacological effects against human diseases. The identification of their cellular targets is crucial for a rational approach to their application in medicinal chemistry. Thus, we have analyzed the cell interactome of suvanine, a sulfated tricyclic terpenoid of marine origin endowed with an interesting anti-inflammatory activity, by application of a chemical proteomic approach. Heat Shock Protein 60, a chaperone involved in the inflammatory response, is the main cellular target of suvanine, which is also able to interfere with protein chaperone activity, giving evidence for its anti-inflammatory properties.

Quantitative Determination of Bisphenol A and Its Congeners in Plant-Based Beverages by Liquid Chromatography Coupled to Tandem Mass Spectrometry.

The consumption of plant-based beverages as an alternative to cow's milk has recently gained vast attention worldwide. The aim of this work is to monitor the intake of Bisphenol A (BPA), Bisphenol B (BPB) and Bisphenol S (BPS) in the Italian population through the consumption of these foodstuffs. Specifically, the development and validation of an analytical procedure for the quantitative determination of the analytes by liquid chromatography coupled to tandem mass spectrometry was reported. Thirty-four samples of plant-based beverages (soya, coconut, almond, oats and rice) of popular brands marketed in Italy were analyzed. BPA was found in 32% of the samples, while BPB was found in 3% of the samples. The risk assessment using the Rapid Assessment of Contaminant Exposure (RACE) tool demonstrated that there was no risk for all population groups, when using the current Tolerable Daily Intake (TDI) of 4 ng/kg body weight (bw)/day as a toxicological reference point. In contrast, using the new temporary TDI of 0.04 ng/kg bw/day, the existing risk was found to be real for all population groups. If this value were to become final, even more attention would have to be paid to the possible presence of BPA in food to protect consumer health.

Sweat urea bioassay based on degradation of Prussian Blue as the sensing architecture.

Urea provides pathophysiological information on renal and hepatic disorders. Among the secretion pathways via several bio-fluids, sweat urea represent an important and non-invasive strategy to evaluate kidney and liver pathologies. In this work, a rapid and reliable colorimetric urea bioassay assisted by chemometrics design of experiments has been obtained. The sensing method is based on the hydrolysis of urea to ammonia catalyzed by urease. If urea is present in the sample, the enzymatic reaction leads to an alkaline environment that is capable to ruin the structure of Prussian Blue. For the first time Prussian Blue is adopted as a precision pH sensing element. Alkaline solution destroys the structure of Prussian Blue, leading to a dramatic colorimetric response from blue to colorless. Design of experiments has been adopted to optimize experimental setup by evaluating correlation among variables. The colorimetric method was shown to be able to detect urea down to 0.05 mM, with a linearity up to 2 mM and a relative standard deviation (RSD) lower than 10%. Sweat samples have been satisfactorily analyzed, and the high accuracy of the portable device has been demonstrated with liquid chromatography with tandem mass spectrometry analysis of the same sweat samples.

Effects of Hst3p inhibition in Candida albicans: a genome-wide H3K56 acetylation analysis.

Candida spp. represent the third most frequent worldwide cause of infection in Intensive Care Units with a mortality rate of almost 40%. The classes of antifungals currently available include azoles, polyenes, echinocandins, pyrimidine derivatives, and allylamines. However, the therapeutical options for the treatment of candidiasis are drastically reduced by the increasing antifungal resistance. The growing need for a more targeted antifungal therapy is limited by the concern of finding molecules that specifically recognize the microbial cell without damaging the host. Epigenetic writers and erasers have emerged as promising targets in different contexts, including the treatment of fungal infections. In C. albicans, Hst3p, a sirtuin that deacetylates H3K56ac, represents an attractive antifungal target as it is essential for the fungus viability and virulence. Although the relevance of such epigenetic regulator is documented for the development of new antifungal therapies, the molecular mechanism behind Hst3p-mediated epigenetic regulation remains unrevealed. Here, we provide the first genome-wide profiling of H3K56ac in C. albicans resulting in H3K56ac enriched regions associated with Candida sp. pathogenicity. Upon Hst3p inhibition, 447 regions gain H3K56ac. Importantly, these genomic areas contain genes encoding for adhesin proteins, degradative enzymes, and white-opaque switching. Moreover, our RNA-seq analysis revealed 1330 upregulated and 1081 downregulated transcripts upon Hst3p inhibition, and among them, we identified 87 genes whose transcriptional increase well correlates with the enrichment of H3K56 acetylation on their promoters, including some well-known regulators of phenotypic switching and virulence. Based on our evidence, Hst3p is an appealing target for the development of new potential antifungal drugs.

Discovery of a Potent and Orally Active Dual GPBAR1/CysLT1R Modulator for the Treatment of Metabolic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are two highly prevalent human diseases caused by excessive fat deposition in the liver. Although multiple approaches have been suggested, NAFLD/NASH remains an unmet clinical need. Here, we report the discovery of a novel class of hybrid molecules designed to function as cysteinyl leukotriene receptor 1 (CysLT1R) antagonists and G protein bile acid receptor 1 (GPBAR1/TGR5) agonists for the treatment of NAFLD/NASH. The most potent of these compounds generated by harnessing the scaffold of the previously described CystLT1R antagonists showed efficacy in reversing liver histopathology features in a preclinical model of NASH, reshaping the liver transcriptome and the lipid and energy metabolism in the liver and adipose tissues. In summary, the present study described a novel orally active dual CysLT1R antagonist/GPBAR1 agonist that effectively protects against the development of NAFLD/NASH, showing promise for further development.

Structural Basis for Developing Multitarget Compounds Acting on Cysteinyl Leukotriene Receptor 1 and G-Protein-Coupled Bile Acid Receptor 1.

G-protein-coupled receptors (GPCRs) are the molecular target of 40% of marketed drugs and the most investigated structures to develop novel therapeutics. Different members of the GPCRs superfamily can modulate the same cellular process acting on diverse pathways, thus representing an attractive opportunity to achieve multitarget drugs with synergic pharmacological effects. Here, we present a series of compounds with dual activity toward cysteinyl leukotriene receptor 1 (CysLT1R) and G-protein-coupled bile acid receptor 1 (GPBAR1). They are derivatives of REV5901─the first reported dual compound─with therapeutic potential in the treatment of colitis and other inflammatory processes. We report the binding mode of the most active compounds in the two GPCRs, revealing unprecedented structural basis for future drug design studies, including the presence of a polar group opportunely spaced from an aromatic ring in the ligand to interact with Arg792.60 of CysLT1R and achieve dual activity.

GPBAR1 Activation by C6-Substituted Hyodeoxycholane Analogues Protect against Colitis.

GPBAR1 agonists have been identified as potential leads for the treatment of diseases related to colon inflammation such as Crohn's and ulcerative colitis. In this paper, we report the discovery of a small library of hyodeoxycholane analogues, decorated at C-6 with different substituents, as potent and selective GPBAR1 agonists. In vitro pharmacological assays showed that compound 6 selectively activates GPBAR1 (EC50 = 0.3 µM) and reduces the production of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) in THP1 cells. The binding mode of compound 6 in GPBAR1 was elucidated by docking calculations. Moreover, compound 6 protects against TNBS-induced colitis in Gpbar1+/+ rodent model, representing an intriguing lead for the treatment of these inflammatory disorders.

Chemical Proteomics-Guided Identification of a Novel Biological Target of the Bioactive Neolignan Magnolol.

Understanding the recognition process between bioactive natural products and their specific cellular receptors is of key importance in the drug discovery process. In this outline, some potential targets of Magnolol, a natural bioactive compound, have been identified by proteomic approaches. Among them, Importin-ß1 has been considered as the most relevant one. A direct binding between Magnolol and this nuclear chaperone has been confirmed by DARTS and molecular docking, while its influence on Importin-ß1 translocation has been evaluated by in vitro assays.

Novel Isoxazole Derivatives with Potent FXR Agonistic Activity Prevent Acetaminophen-Induced Liver Injury.

Acetaminophen misuse is a leading cause of acute liver failure and liver transplantation for which therapy is poorly effective. FXR ligands have shown effective in reducing liver injury in several experimental and clinical settings. In this Letter, we have elaborated on the structure of GW4064, the first nonsteroidal agonist for FXR, to identify novel isoxazoles endowed with FXR agonistic activity and improved ADME properties. The pharmacological characterization and molecular docking studies for the structure-activity rationalization allowed the identification of several FXR agonists with nanomolar potency in transactivation and SRC-1 recruitment assays. This characterization resulted in the identification of a potent FXR agonist, compound 20 that was orally active, and rescued mice from acute liver failure caused by acetaminophen overdose in a FXR-dependent manner.

Tumor-associated macrophage-secreted 14-3-3ζ signals via AXL to promote pancreatic cancer chemoresistance.

Pancreatic ductal adenocarcinoma (PDAC) is an inherently chemoresistant tumor. Chemotherapy leads to apoptosis of cancer cells, and in previous studies we have shown that tumor-associated macrophage (TAM) infiltration increases following chemotherapy in PDAC. Since one of the main functions of macrophages is to eliminate apoptotic cells, we hypothesized that TAMs phagocytose chemotherapy-induced apoptotic cells and secrete factors, which favor PDAC chemoresistance. To test this hypothesis, primary human PDAC cultures were treated with conditioned media (CM) from monocyte-derived macrophage cultures incubated with apoptotic PDAC cells (MØApopCM). MØApopCM pretreatment rendered naïve PDAC cells resistant to Gemcitabine- or Abraxane-induced apoptosis. Proteomic analysis of MØApopCM identified YWHAZ/14-3-3 protein zeta/delta (14-3-3ζ), a major regulator of apoptotic cellular pathways, as a potential mediator of chemoresistance, which was subsequently validated in patient transcriptional datasets, serum samples from PDAC patients and using recombinant 14-3-3ζ and inhibitors thereof. Moreover, in mice bearing orthotopic PDAC tumors, the antitumor potential of Gemcitabine was significantly enhanced by elimination of TAMs using clodronate liposomes or by pharmacological inhibition of the Axl receptor tyrosine kinase, a 14-3-3ζ interacting partner. These data highlight a unique regulatory mechanism by which chemotherapy-induced apoptosis acts as a switch to initiate a protumor/antiapoptotic mechanism in PDAC via 14-3-3ζ/Axl signaling, leading to phosphorylation of Akt and activation of cellular prosurvival mechanisms. The data presented therefore challenge the idea that apoptosis of tumor cells is therapeutically beneficial, at least when immune sensor cells, such as macrophages, are present.