Discovery and Characterization of Halogenated Xanthene Inhibitors of DUSP5 as Potential Photodynamic Therapeutics.
ABSTRACT: Dual specific phosphatases (DUSPs) are an important class of mitogen-activated protein kinase (MAPK) regulators, and are drug targets for treating vascular diseases. Previously we had shown that DUSP5 plays a role in embryonic vertebrate vascular patterning. Herein, we screened a library of FDA-approved drugs and related compounds, using a para-nitrophenylphosphate substrate (pNPP)-based assay. This assay identified merbromin (also known as mercurochrome) as targeting DUSP5; and, we subsequently identified xanthene-ring based merbromin analogs eosin Y, erythrosin B, and rose bengal, all of which inhibit DUSP5 in vitro. Inhibition was time-dependent for merbromin, eosin Y, 2',7'-dibromofluorescein, and 2',7'-dichlorofluorescein, with enzyme inhibition increasing over time. Reaction progress curve data fit best to a slow-binding model of irreversible enzyme inactivation. Potency of the time-dependent compounds, except for 2',7'-dichlorofluorescein, was diminished when dithiothreitol (DTT) was present, suggesting thiol reactivity. Two additional merbromin analogs, erythrosin B and rose bengal also inhibit DUSP5, but have the therapeutic advantage of being less sensitive to DTT and exhibiting little time dependence for inhibition. Inhibition potency is correlated with the xanthene dye's LUMO energy, which affects ability to form light-activated radical anions, a likely active inhibitor form. Consistent with this hypothesis, rose bengal inhibition is light-dependent and demonstrates the expected red shifted spectrum upon binding to DUSP5, with a Kd of 690 nM. These studies provide a mechanistic foundation for further development of xanthene dyes for treating vascular diseases that respond to DUSP5 inhibition, with the following relative potencies: rose bengal > merbromin > erythrosin B > eosin Y.
Project description:Enoate reductases from the family of old yellow enzymes (OYEs) can catalyze stereoselective trans-hydrogenation of activated C=C bonds. Their application is limited by the necessity for a continuous supply of redox equivalents such as nicotinamide cofactors [NAD(P)H]. Visible light-driven activation of OYEs through NAD(P)H-free, direct transfer of photoexcited electrons from xanthene dyes to the prosthetic flavin moiety is reported. Spectroscopic and electrochemical analyses verified spontaneous association of rose bengal and its derivatives with OYEs. Illumination of a white light-emitting-diode triggered photoreduction of OYEs by xanthene dyes, which facilitated the enantioselective reduction of C=C bonds in the absence of NADH. The photoenzymatic conversion of 2-methylcyclohexenone resulted in enantiopure (ee>99?%) (R)-2-methylcyclohexanone with conversion yields as high as 80-90?%. The turnover frequency was significantly affected by the substitution of halogen atoms in xanthene dyes.
Project description:We used a pulsed dye laser working at 540 nm to excite triplet-state formation of eosin and erythrosin, either bound or unbound to bovine serum albumin, in aqueous solution anaerobically at pH 8 and 20-22 degrees C. Delayed emission from radiative transitions of the triplet state was readily detectable, both as delayed fluorescence and as red phosphorescence. Detection of the triplet state by measurement of phosphorescence at 645 nm upwards was at least 100-fold more sensitive than by absorbance measurements of ground-state depletion at 500 nm. When immobilized in poly(methyl methacrylate), the phosphorescence of eosin and erythrosin was polarized with an anisotropy parameter [Jablonski (1961) Z. Naturforsch. A16, 1-4] of about 0.25. The phosphorescence of erythrosin is sufficiently intense to be distinguishable from the long-wavelength end of fluorescence under conditions of continuous rather than pulsed excitation. Our observations suggest that phosphorescence depolarization of eosin or erythrosin probes could be used as a highly sensitive method of measuring rotational relaxation times in region from 10(-5) to 10(-3) s, such as those of the uniaxial rotation of membrane proteins.
Project description:BACKGROUND:Ficus deltoidea (FD) has been shown to have antidiabetic, anti-inflammatory, antinociceptive and antioxidant properties. However, its effects on key events in the pathogenesis of atherosclerosis are unknown. AIM:To investigate the endothelial activation, inflammation, monocyte-endothelial cell binding and oxidative stress effects of four FD varieties. METHODS:Human coronary artery endothelial cells (HCAEC) were incubated with different concentrations of aqueous ethanolic extracts of FD var. trengganuensis (FDT), var. kunstleri (FDK), var. deltoidea (FDD) and var. intermedia (FDI), together with LPS. Protein and gene expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), endothelial-leukocyte adhesion molecule-1 (E-selectin), interleukin-6 (IL-6), Nuclear factor-?B (NF-?B) p50 and p65 and endothelial nitric oxide synthase (eNOS) were measured using ELISA and QuantiGene plex, respectively. Adhesion of monocyte to HCAEC and formation of reactive oxygen species (ROS) were detected by Rose Bengal staining and 2'-7'-dichlorofluorescein diacetate (DCFH-DA) assay. RESULTS:FDK exhibited the highest inhibition of biomarkers in relation to endothelial activation and inflammation, second in reducing monocyte binding (17.3%) compared to other varieties. FDK (25.6%) was also the most potent at decreasing ROS production. CONCLUSION:FD has anti-atherogenic effects, possibly mediated by NF-?B and eNOS pathways; with FDK being the most potent variety. It is potentially beneficial in mitigating atherogenesis.
Project description:The thermal and chemical-based methods applied for microbial control in the food industry are not always environmentally friendly and may change the nutritional and organoleptic characteristics of the final products. Moreover, the efficacy of sanitizing agents may be reduced when microbial cells are enclosed in biofilms. The objective of this study was to investigate the effect of photodynamic inactivation, using two xanthene dyes (rose bengal and erythrosine) as photosensitizing agents and green LED as a light source, against Staphylococcus aureus, Listeria innocua, Enterococcus hirae and Escherichia coli in both planktonic and biofilm states. Both photosensitizing agents were able to control planktonic cells of all bacteria tested. The treatments altered the physicochemical properties of cells surface and also induced potassium leakage, indicating damage of cell membranes. Although higher concentrations of the photosensitizing agents (ranging from 0.01 to 50.0 ?mol/L) were needed to be applied, the culturability of biofilm cells was reduced to undetectable levels. This finding was confirmed by the live/dead staining, where propidium iodide-labeled bacteria numbers reached up to 100%. The overall results demonstrated that photoinactivation by rose bengal and erythrosine may be a powerful candidate for the control of planktonic cells and biofilms in the food sector.
Project description:Halogenation of organic compounds plays diverse roles in biochemistry, including selective chemical modification of proteins and improved oral absorption/blood-brain barrier permeability of drug candidates. Moreover, halogenation of aromatic molecules greatly affects aromatic interaction-mediated self-assembly processes, including amyloid fibril formation. Perturbation of the aromatic interaction caused by halogenation of peptide building blocks is known to affect the morphology and other physical properties of the fibrillar structure. Consequently, in this article, we investigated the ability of halogenated ligands to modulate the self-assembly of amyloidogenic peptide/protein. As a model system, we chose amyloid-beta peptide (A?), which is implicated in Alzheimer's disease, and a novel modulator of A? aggregation, erythrosine B (ERB). Considering that four halogen atoms are attached to the xanthene benzoate group in ERB, we hypothesized that halogenation of the xanthene benzoate plays a critical role in modulating A? aggregation and cytotoxicity. Therefore, we evaluated the modulating capacities of four ERB analogs containing different types and numbers of halogen atoms as well as fluorescein as a negative control. We found that fluorescein is not an effective modulator of A? aggregation and cytotoxicity. However, halogenation of either the xanthenes or benzoate ring of fluorescein substantially enhanced the inhibitory capacity on A? aggregation. Such A? aggregation inhibition by ERB analogs except rose bengal correlated well to the inhibition of A? cytotoxicity. To our knowledge, this is the first report demonstrating that halogenation of aromatic rings substantially enhance inhibitory capacities of small molecules on A?-associated neurotoxicity via A? aggregation modulation.
Project description:We have been developing the use of plasma-membrane-bound fluorescent probes to measure the pH values at the surfaces of living chondrocytes. For this purpose, three lipophilic pH indicators were made by covalently binding the xanthene dyes fluorescein, eosin or dichlorofluorescein to the amino group of phosphatidylethanolamine. The probes were incorporated into phospholipid vesicles and the effect of pH on the fluorescence was characterized. Fluorescence was measured at a single emission wavelength during excitation at two wavelengths, and the ratio of the intensities was calculated. The experimentally observed pKobs. values were determined by fitting the fluorescence ratios to the Henderson-Hasselbalch equation. All three probes acted as pH indicators, and the eosinyl-, dichlorofluoresceinyl- and fluoresceinylphosphatidylethanolamines had pKobs. values of 3.5, 6.3 and 7.5 respectively. At physiological salt concentrations, changes in the composition of the vesicle membrane had little effect on these values. We concluded that these probes were promising candidates for the measurement of pH values at cell surfaces.
Project description:<h4>Purpose</h4>This study aimed to determine if rebamipide eyedrops can improve ocular surface damage caused by the use of glaucoma eyedrops.<h4>Methods</h4>Female Kbl:Dutch rabbits were used to evaluate glaucoma eyedrop-induced ocular surface damage; one eye of each rabbit was untreated and the other was administered glaucoma eyedrops for 30 days. To evaluate the effects of rebamipide on ocular surface damage, one eye of each rabbit was administered vehicle-treated glaucoma eyedrops and the other was administered rebamipide-treated glaucoma eyedrops for 30 days. Corneal and conjunctival epithelial damage was evaluated using fluorescein and rose bengal staining, respectively. Conjunctival inflammation was observed by light microscopy with hematoxylin-eosin staining. Dark cells (in which the corneal microvilli were damaged) were analyzed by scanning electron microscopy.<h4>Results</h4>There were no significant differences in fluorescein staining between the untreated and glaucoma eyedrop-treated groups; however, rose bengal staining and the number of inflammatory cells in the conjunctiva significantly increased after glaucoma eyedrop treatment. There was a four-fold increase in the number of dark cells in the glaucoma eyedrop-treated group compared to untreated. In contrast, in the conjunctiva of the rebamipide-treated glaucoma eyedrop group, rose bengal staining scores, the number of inflammatory cells, and the number of dark cells were decreased compared to the vehicle-treated glaucoma eyedrop group.<h4>Conclusions</h4>Results from our in vivo rabbit study demonstrated that short-term use of glaucoma eyedrops induces corneal epithelium disorders at the cellular level, but that simultaneous use of rebamipide has the potential to protect and repair the ocular surface.
Project description:Age-associated cellular senescence is thought to promote vascular dysfunction. p16(INK4a) is a cell cycle inhibitor that promotes senescence and is upregulated during normal aging. In this study, we examine the contribution of p16(INK4a) overexpression to venous thrombosis.Mice overexpressing p16(INK4a) were studied with 4 different vascular injury models: (1) ferric chloride (FeCl(3)) and (2) Rose Bengal to induce saphenous vein thrombus formation; (3) FeCl(3) and vascular ligation to examine thrombus resolution; and (4) lipopolysaccharide administration to initiate inflammation-induced vascular dysfunction. p16(INK4a) transgenic mice had accelerated occlusion times (13.1 ± 0.4 minutes) compared with normal controls (19.7 ± 1.1 minutes) in the FeCl(3) model and 12.7 ± 2.0 and 18.6 ± 1.9 minutes, respectively in the Rose Bengal model. Moreover, overexpression of p16(INK4a) delayed thrombus resolution compared with normal controls. In response to lipopolysaccharide treatment, the p16(INK4a) transgenic mice showed enhanced thrombin generation in plasma-based calibrated automated thrombography assays. Finally, bone marrow transplantation studies suggested increased p16(INK4a) expression in hematopoietic cells contributes to thrombosis, demonstrating a role for p16(INK4a) expression in venous thrombosis.Venous thrombosis is augmented by overexpression of the cellular senescence protein p16(INK4a).