Exploiting the potential of gallium for the treatment of Pseudomonas aeruginosa pulmonary infection

Project Summary (English) - Background Morbidity and mortality in cystic fibrosis (CF) patients is ultimately attributable to persistent pulmonary infection mainly caused by bacteria belonging to different species, the most worrisome one being Pseudomonas aeruginosa (Pa). The emergence of antibiotic resistance in CF pathogens has become a serious problem that must be addressed by developing new antimicrobials and identifying new drug targets. Gallium (Ga3+) inhibits bacterial growth, acting as an iron mimetic, and is a drug already used in clinical practice (brand name Ganite®) for the treatment of non-infectious diseases. The pharmacological properties of Ga3+ rely on the chemical resemblance between the Ga3+ and the ferric (Fe3+) ions. - Hypothesis and objectives Bacterial cells are unable to discriminate between these two ions, and erroneously incorporates Ga3+ instead of Fe3+ within essential molecules, resulting in suppression of bacterial growth. The main aims of this project is to: i) generate new gallium complexes with pyochelin-based deriovatives; ii) perform a comparative evaluation of the antibacterial activity of Ga(III) complexes/formulations against CF pathogens, and to investigate the toxicology and pharmacokinetics of the Ga(III) complexes/formulations. - Material, patients, methods We shall capitalize upon expertise in organic synthesis, pharmaceutical chemistry, microbiology and pharmacology to: i) synthesize gallium complexes ex novo; ii) evaluate the effect of Ga(III) complexes/formulations on CF pathogens; iii) determine their toxicity and pharmacological properties. In this project, the antibacterial properties of Ga3+ will be potentiated by conjugation with ligands that facilitate the stability/solubility, in order to lower the therapeutic dosage. The pharmacological activities of the new compounds will be assessed by: i) inhibition of CF-pathogens growth in vitro; ii) lack of toxicity in cellular and animal systems; iii) evaluation of biodistribution in animal models. - Expected results and spin-offs We expect to identify new Ga3+ formulations endowed with more potent antibacterial properties, high bioavailability, low toxicity and suited for aerosol administration, thus ready for clinical testing. In the worrying scenario of increasing antibiotic resistance CF pathogens, the identification of new antimicrobials such as gallium-based compounds, is highly desirable. These compounds hold great promise for the progression into drugs with potential clinical applicability in the short-medium perspective

Uterine Dysfunction in Diabetic Mice: The Role of Hydrogen Sulfide

It is well-known that the physiological uterine peristalsis, related to several phases of reproductive functions, plays a pivotal role in fertility and female reproductive health. Here, we have addressed the role of hydrogen sulfide (H2S) signaling in changes of uterine contractions driven by diabetes in non-obese diabetic (NOD) mice, a murine model of type-1 diabetes mellitus. The isolated uterus of NOD mice showed a significant reduction in spontaneous motility coupled to a generalized hypo-contractility to uterotonic agents. The levels of cyclic nucleotides, cAMP and cGMP, notoriously involved in the regulation of uterus homeostasis, were significantly elevated in NOD mouse uteri. This increase was well-correlated with the higher levels of H2S, a non-specific endogenous inhibitor of phosphodiesterases. The exposure of isolated uterus to L-cysteine (L-Cys), but not to sodium hydrogen sulfide, the exogenous source of H2S, showed a weak tocolytic effect in the uterus of NOD mice. Western blot analysis revealed a reorganization of the enzymatic expression with an upregulation of 3-mercaptopyruvate-sulfurtransferase (3-MST) coupled to a reduction in both cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) expression. In conclusion, the increased levels of cyclic nucleotides dysregulate the uterus peristalsis and contractility in diabetic mice through an increase in basal H2S synthesis suggesting a role of 3-MST

Involvement of Hydrogen Sulfide (H2S) in Dexamethasone (DEX) Induced Hypertension in Rat

Introduction: Glucocorticoid (GC) excess is related to hypertension. The deletion of endothelial GC-receptors abrogates the blood pressure increase, suggesting GC-induced hypertension is endothelium-dependent. In response to shear stress endothelium releases nitric oxide, endothelial derived hyperpolarizing factor (EDHF) and prostacyclin. Recently H2S has been proposed as a candidate for EDHF. H2S is mainly produced by the enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) from L-cysteine. The aim of this study was to investigate the EDHF/H2S signaling in GC-hypertension. Methods: Male Wistar rats were treated with DEX (1.5 mg/kg/sc) or vehicle (VEH) for 8 days. Systolic blood pressure (SBP) was monitored every 2 days. EDHF was evaluated in mesenteric plexus and carotid artery performing a concentration-effect curve of acetylcholine in presence of indomethacin (INDO) and nitro-L-arginine methyl ester (L-NAME). Apamin (APA) plus charibdotoxin (CTX), SKCa and BKCa inhibitors, or propargylglycine (PAG), CSE inhibitor, were used. CBS and CSE levels were analyzed by immunoblot. H2S levels were measured by a colorimetric assay. Results: DEX treatment significantly increased SBP compared to VEH (*p<0.05, **p<0.01, ***p<0.001 at days 2-4, 6, 8 respectively). EDHF-mediated relaxation of mesenteric bed or carotid artery was markedly reduced in DEX group compared to VEH (***p<0.001). APA and CTX as well as PAG abolished EDHF-mediated relaxation in DEX or VEH group (***,°°°p<0.001 respectively). CBS and CSE levels were significantly reduced in mesenteric plexus and carotid artery in DEX group (*p<0.05). The H2S production was markedly reduced in mesenteric plexus and carotid artery (*p<0.05, **p<0.01 respectively) as well as plasmatic H2S levels (*p<0.05) in DEX rats compared to VEH. Conclusions: Our data demonstrate that GC-excess induces an impairment of H2S/EDHF signaling indicating an additional cause of GC-mediated hypertension

Erucin, an H₂S-Releasing Isothiocyanate, Exerts Anticancer Effects in Human Triple-Negative Breast Cancer Cells Triggering Autophagy-Dependent Apoptotic Cell Death

Breast cancer is the most frequent form of cancer occurring in women of any age. Among the different types, the triple-negative breast cancer (TNBC) subtype is recognized as the most severe form, being associated with the highest mortality rate. Currently, there are no effective treatments for TNBC. For this reason, the research of novel therapeutics is urgently needed. Natural products and their analogs have historically made a major contribution to pharmacotherapy and the treatment of various human diseases, including cancer. In this study, we explored the potential anti-cancer effects of erucin, the most abundant H2S-releasing isothiocyanate present in arugula (Eruca sativa) in MDA-MB-231 cells, a validated in vitro model of TNBC. We found that erucin, in a concentration-dependent manner, significantly inhibited MDA-MB-231 cell proliferation by inducing apoptosis and autophagy. Additionally, erucin prevented intracellular ROS generation promoting the expression of key antioxidant genes and halted MDA-MB-231 cell migration, invasion, and colony formation. In conclusion, using a cellular and molecular biology approach, we show that the consumption of erucin could represent a novel and promising strategy for intervention against TNBC

Some remarks on Hattori's theory for mapping cones of coexact squares

SIGLETIB: RO 1945 (109) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman

Effects of Subtoxic Concentrations of Atrazine, Cypermethrin, and Vinclozolin on microRNA-Mediated PI3K/Akt/mTOR Signaling in SH-SY5Y Cells

Endocrine-disrupting chemicals (EDCs) are different natural and synthetic chemicals that may interfere with several mechanisms of the endocrine system producing adverse developmental, metabolic, reproductive, and neurological effects in both human beings and wildlife. Among pesticides, numerous chemicals have been identified as EDCs. MicroRNAs (miRNAs) can regulate gene expression, making fine adjustments in mRNA abundance and regulating proteostasis. We hypothesized that exposure to low doses of atrazine, cypermethrin, and vinclozolin may lead to effects on miRNA expression in SH-SY5Y cells. In particular, the exposure of SH-SY5Y cells to subtoxic concentrations of vinclozolin is able to downregulate miR-29b-3p expression leading to the increase in the related gene expression of ADAM12 and CDK6, which may promote a pro-oncogenic response through the activation of the PI3K/Akt/mTOR pathway and counteracting p53 activity. A better understanding of the molecular mechanisms of EDCs could provide important insight into their role in human disease

Cystathionine γ-lyase, a H<inf>2</inf>S-generating enzyme, is a GPBAR1-regulated gene and contributes to vasodilation caused by secondary bile acids

GPBAR1 is a bile acid-activated receptor (BAR) for secondary bile acids, lithocholic (LCA) and deoxycholic acid (DCA), expressed in the enterohepatic tissues and in the vasculature by endothelial and smooth muscle cells. Despite that bile acids cause vasodilation, it is unclear why these effects involve GPBAR1, and the vascular phenotype of GPBAR1 deficient mice remains poorly defined. Previous studies have suggested a role for nitric oxide (NO) in regulatory activity exerted by GPBAR1 in liver endothelial cells. Hydrogen sulfide (H2S) is a vasodilatory agent generated in endothelial cells by cystathionines-γ-lyase (CSE). Here we demonstrate that GPBAR1 null mice had increased levels of primary and secondary bile acids and impaired vasoconstriction to phenylephrine. In aortic ring preparations, vasodilation caused by chenodeoxycholic acid (CDCA), a weak GPBAR1 ligand and farnesoid-x-receptor agonist (FXR), was iberiotoxin-de-pendent and GPBAR1-independent. In contrast, vasodilation caused by LCA was GPBAR1 dependent and abrogated by propargyl-gly-cine, a CSE inhibitor, and by 5β-cholanic acid, a GPBAR1 antagonist, but not by N5-(1-iminoethyl)-L-ornithine (l-NIO), an endothelial NO synthase inhibitor, or iberiotoxin, a large-conductance calcium-activated potassium (BKCa) channels antagonist. In venular and aortic endothelial (HUVEC and HAEC) cells GPBAR1 activation increases CSE expression/activity and H2S production. Two cAMP response element binding protein (CREB) sites (CREs) were identified in the CSE promoter. In addition, TLCA stimulates CSE phosphorylation on serine residues. In conclusion we demonstrate that GPBAR1 mediates the vasodilatory activity of LCA and regulates the expression/activity of CSE. Vasodilation caused by CDCA involves BKCa channels. The GPBAR1/CSE pathway might contribute to endothelial dysfunction and hyperdynamic circulation in liver cirrhosis

Hydrogen sulfide donor AP123 restores endothelial nitric oxide-dependent vascular function in hyperglycemia via a CREB-dependent pathway

Diabetes is associated with severe vascular complications involving the impairment of endothelial nitric oxide synthase (eNOS) as well as cystathionine γ-lyase (CSE) activity. eNOS function is suppressed in hyperglycaemic conditions, resulting in reduced NO bioavailability, which is paralleled by reduced levels of hydrogen sulfide (H2S). Here we have addressed the molecular basis of the interplay between the eNOS and CSE pathways. We tested the impact of H2S replacement by using the mitochondrial-targeted H2S donor AP123 in isolated vessels and cultured endothelial cells in high glucose (HG) environment, at concentrations not causing any vasoactive effect per se. Aorta exposed to HG displayed a marked reduction of acetylcholine (Ach)-induced vasorelaxation that was restored by the addition of AP123 (10 nM). In HG condition, bovine aortic endothelial cells (BAEC) showed reduced NO levels, downregulation of eNOS expression, and suppression of CREB activation (p-CREB). Similar results were obtained by treating BAEC with propargylglycine (PAG), an inhibitor of CSE. AP123 treatment rescued eNOS expression, as well as NO levels, and restored p-CREB expression in both the HG environment and the presence of PAG. This effect was mediated by a PI3K-dependent activity since wortmannin (PI3K inhibitor) blunted the rescuing effects operated by the H2S donor. Experiments performed in the aorta of CSE−/− mice confirmed that reduced levels of H2S not only negatively affect the CREB pathway but also impair Ach-induced vasodilation, significantly ameliorated by AP123. We have demonstrated that the endothelial dysfunction due to HG involves H2S/PI3K/CREB/eNOS route, thus highlighting a novel aspect of the H2S/NO interplay in the vasoactive response

New Insight into the Binding Mode of Peptide Ligands at Urotensin-II Receptor: Structure−Activity Relationships Study on P5U and Urantide

Urotensin II (U-II) is a disulfide bridged peptide hormone identified as the ligand of a G protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of hU-II termed P5U (H-Asp-c [Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and the compound termed urantide (H-Asp-c[Pen-Phe-DTrp-Orn-Tyr-Cys]-Val-OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized several analogues of P5U and urantide in which the Asp(4) residue in N-terminus position was replaced with coded and noncoded amino acids. The replacement of the Asp(4) residue by Tic led to an analogue, compound 14, more potent as antagonist (pK(B) = 8.94) compared to urantide. Furthermore, a different SAR was observed for the P5U compared to the urantide analogues. NMR and docking studies revealed a different binding mode for the agonist and antagonist ligands which could explain the observed SAR