A-family anti-inflammatory cyclopentenone prostaglandins: A novel class of non-statin inhibitors of HMG-CoA reductase.

Disruption of the intracellular lipid balance leading to cholesterol accumulation is one of the features of cells that participate in the development of atherosclerotic lesions. Evidence form our laboratory indicates that anti-inflammatory cyclopentenone prostaglandins (cyPGs) of A- and J-family deviate lipid metabolism from the synthesis of cholesterol and cholesteryl esters to the synthesis of phospholipids in foam-cell macrophages. cyPGs possessing an α,ß-unsaturated cyclopentane ring are highly electrophilic substances able to promptly react with reactive cysteines of intracellular molecules through Michael addition. On the other hand, HMG-CoA reductase (HMGCR), the enzyme responsible for the rate-limiting step in cholesterol biosynthesis, presents critically reactive cysteines at the entry of catalytic domain, particularly Cys561, that could be target of cyPG inhibition. In the present study, we showed that cyPGs (but not other non-α,ß-unsaturated PGs) physically interact with HMGCR, in a dithiothreitol- and ß-mercaptoethanol-sensitive way, and block the activity of the catalytic subunit of the enzyme (IC50 for PGA2 = 0.17 µM). PGA2 inhibits HMGCR activity in cultured rat and human macrophages/macrophage-foam cells and leads to enhanced expression of HMGCR protein, as observed with statins. In cell culture models, PGA2 effectively inhibits the reductase at non-toxic doses (e.g., 1 µM) that block cell proliferation thus suggesting that part of the well-known antiproliferative effect of PGA2 may be due to its ability of blocking HMGCR activity, as cells cannot proliferate without a robust cholesterogenesis. Therefore, besides the powerfully anti-inflammatory and antiproliferative effects, the anticholesterogenic effects of PGA2 should be exploited in atherosclerosis therapeutics.

Development of Rifapentine-Loaded PLGA-Based Nanoparticles: In vitro Characterisation and in vivo Study in Mice.

BACKGROUND: Tuberculosis (TB) is a leading cause of death amongst infectious diseases. The poor response to antitubercular agents necessitates the long-term use of high drug doses, resulting in low patient compliance, which is the main reason for chemotherapy failure and contributes to the development of multidrug-resistant TB. Patient non-compliance has been a major obstacle in the successful management of TB. The aim of this work was to develop and characterise rifapentine (RPT)-loaded PLGA-based nanoparticles (NPs) for reducing dosing frequency. METHODS: RPT-loaded PLGA and PLGA-PEG NPs were prepared using premix membrane homogenisation combined with solvent evaporation method. The resulting NPs were characterised in terms of physicochemical characteristics, toxicity, cellular uptake and antitubercular activity. NPs were further evaluated for pharmacokinetic and biodistribution studies in mice. RESULTS: The resulting NPs showed suitable and safe physicochemical characteristics and could be taken up by macrophages. RPT-loaded NPs were more effective against Mycobacterium tuberculosis than free RPT. In vivo studies revealed that NPs could improve pharmacokinetic parameters, particularly for RPT/PLGA-PEG NPs. Moreover, both formulations had no toxicity to the organs of mice and could reduce hepatotoxicity. CONCLUSION: The application of PLGA-based NPs as sustained-release delivery vehicles for RPT could prolong drug release, modify pharmacokinetics, increase antitubercular activity and diminish toxicity, thereby allowing low dosage and frequency.

Twin-arginine signal peptide of Bacillus licheniformis GlmU efficiently mediated secretory expression of protein glutaminase

Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA.

Molecular, chemical, and structural characterization of prostaglandin A2 as a novel agonist for Nur77.

Nur77 is a transcription factor belonging to the NR4A subfamily of nuclear hormone receptors. Upon induction, Nur77 modulates the expression of its target genes and controls a variety of biological and pathophysiological processes. Prior research that revealed a structurally atypical ligand-binding domain (LBD) and failed to locate an endogenous ligand had led to a classification of Nur77 as an orphan receptor. However, several more recent studies indicate that small synthetic molecules and unsaturated fatty acids can bind to Nur77. Discovery of additional endogenous ligands will facilitate our understanding of the receptor's functions and regulatory mechanisms. Our data have identified prostaglandin A2 (PGA2), a cyclopentenone prostaglandin (PG), as such a ligand. Cyclopentenone PGs exert their biological effects primarily by forming protein adducts via the characteristic electrophilic ß-carbon(s) located in their cyclopentenone rings. Our data show that PGA2 induces Nur77 transcriptional activity by forming a covalent adduct between its endocyclic ß-carbon, C9, and Cys566 in the receptor's LBD. The importance of this endocyclic ß-carbon was substantiated by the failure of PGs without such electrophilic properties to react with Nur77. Calculated chemical properties and data from reactive molecular dynamic simulations, intrinsic reaction co-ordinate modeling, and covalent molecular docking also corroborate the selectivity of PGA2's C9 ß-carbon towards Nur77's Cys. In summary, our molecular, chemical, and structural characterization of the PGA2-Nur77 interaction provides the first evidence that PGA2 is an endogenous Nur77 agonist.

Astrocytes synthesize primary and cyclopentenone prostaglandins that are negative regulators of their proliferation.

Recently, the modulation of cellular inflammatory responses via endogenous regulators became a major focus of medically relevant investigations. Prostaglandins (PGs) are attractive regulatory molecules, but their synthesis and mechanisms of action in brain cells are still unclear. Astrocytes are involved in manifestation of neuropathology and their proliferation is an important part of astrogliosis, a cellular neuroinflammatory response. The aims of our study were to measure synthesis of PGs by astrocytes, and evaluate their influence on proliferation in combination with addition of inflammatory pathway inhibitors. With UPLC-MS/MS analysis we detected primary PGs (1410 ±â€¯36 pg/mg PGE2, 344 ±â€¯24 PGD2) and cyclopentenone PGs (cyPGs) (87 ±â€¯17 15d-PGJ2, 308 ±â€¯23 PGA2) in the extracellular medium after 24-h lipopolysaccharide (LPS) stimulation of astrocytes. PGs reduced astrocytic proliferation with the following order of potencies (measured as inhibition at 20 µM): most potent 15d-PGJ2 (90%) and PGA2 (80%), > PGD2 (40%) > 15d-PGA2 (20%) > PGE2 (5%), the least potent. However, PGF2α and 2-cyclopenten-1-one, and ciglitazone and rosiglitazone (synthetic agonists of PPARγ) had no effect. Combinations of cyPGs with SC-560 or NS-398 (specific anti-inflammatory inhibitors of cyclooxygenase-1 and -2, respectively) were not effective; while GW9662 (PPARγ antagonist) or MK-741 (inhibitor of multidrug resistance protein-1, MRP1, and CysLT1 receptors) amplified the inhibitory effect of PGA2 and 15d-PGJ2. Although concentrations of individual PGs and cyPGs are low, all of them, as well as primary PGs suppress proliferation. Thus, the effects are potentially additive, and activated PGs synthesis suppresses proliferation in astrocytes.

Pentablock copolymer dexamethasone nanoformulations elevate MYOC: in vitro liberation, activity and safety in human trabecular meshwork cells.

AIM: The aim of this study is to examine the elevation of MYOC in long-term treatment of human trabecular meshwork (HTM) cells using dexamethasone (DEX) encapsulated pentablock (PB) copolymer-based nanoparticles (NPs) (DEX-PB-NPs). MATERIALS & METHODS: PB copolymers and DEX-PB-NPs were synthesized and characterized using nuclear magnetic resonance, gel permeation chromatography, and X-ray diffraction analyses. MYOC levels secreted from HTM cells were measured by western blot (WB) analysis. RESULTS: DEX-PB-NPs were formulated in the size range of 109 ± 3.77 nm (n = 3). A long term DEX release from the NPs was observed over three months. Cell viability and cytotoxicity were not affected up to 12 weeks of treatment with PB-copolymer or DEX-PB-NPs. WB data from five HTM cell strains showed that MYOC levels increased by 5.2 ± 1.3, 7.4 ± 4.3, and 2.8 ± 1.1-fold in the presence of DEX-PB-NPs compared with 9.2 ± 3.8, 2.2 ± 0.5, and 1.5 ± 0.3-fold at 4, 8 and 12 weeks in control-DEX treatment group, respectively (n = 5). Based on the decline in MYOC levels after withdrawal of DEX from control wells, DEX-PB-NPs released the DEX for at least 10 weeks. CONCLUSION: The treatment of HTM cells using DEX-PB-NPs were analyzed in this study. The in vitro cell-based system developed here is a valuable tool for determining the safety and effects of steroids released from polymeric NPs.

Synthesis of Chiral Cyclopentenones.

The cyclopentenone unit is a very powerful synthon for the synthesis of a variety of bioactive target molecules. This is due to the broad diversity of chemical modifications available for the enone structural motif. In particular, chiral cyclopentenones are important precursors in the asymmetric synthesis of target chiral molecules. This Review provides an overview of reported methods for enantioselective and asymmetric syntheses of cyclopentenones, including chemical and enzymatic resolution, asymmetric synthesis via Pauson-Khand reaction, Nazarov cyclization and organocatalyzed reactions, asymmetric functionalization of the existing cyclopentenone unit, and functionalization of chiral building blocks.

[An example of oscillatory (cyclic) reaction for prostaglandin chemistry].

It is shown an ability of prostaglandin A1 to the oscillatory reaction which has significance both the theoretical and practical if prostaglandins are used for therapy.

A-class prostaglandins: early findings and new perspectives for overcoming tumor chemoresistance.

The antiproliferative properties of cyclopentenone prostaglandins of the A-class have long been known. Considerable research has led to the elucidation of some of the mechanisms of action of these pleiotropic compounds. A-class prostaglandins or derived molecules (A-PG) may block the cell cycle, inhibit anti-apoptotic transcription factors, activate apoptotic cascades, induce a stress response and inhibit protein synthesis in a cell type-dependent manner. In addition, recent reports indicate that A-class PG may interact with various cellular detoxification systems and drug metabolizing enzymes used by cancer cells as mechanisms of chemoresistance. Some of these findings may open new perspectives for the development of strategies aimed at overcoming cancer resistance to widely used antitumor drugs. Here we outline the mechanisms of action for the antitumoral effects of PGA and related compounds, emphasizing those with impact on cellular defence systems which may contribute to cancer chemoresistance. The ability of A-PG to form covalent adducts with thiol groups in proteins and in glutathione is essential for their biological actions. Therefore, identification of the protein targets and elucidation of the interactions of A-PG with the glutathione biotransformation system will be critical for understanding the antitumoral effects of these compounds per se or through their ability to sensitize cancer cells towards other drugs.

A biotinylated analog of the anti-proliferative prostaglandin A1 allows assessment of PPAR-independent effects and identification of novel cellular targets for covalent modification.

The cyclopentenone prostaglandin (cyPG) PGA(1) displays potent anti-proliferative and anti-inflammatory effects. Therefore, PGA(1) derivatives are being studied as therapeutic agents. One major mechanism for cyPG action is the modification of protein cysteine residues, the nature of the modified proteins being highly dependent on the structure of the cyPG. Biotinylated cyPGs may aid in the proteomic identification of cyPG targets of therapeutic interest. However, for the identified targets to be relevant it is critical to assess whether biotinylated cyPGs retain the desired biological activity. Here we have explored the anti-inflammatory, anti-proliferative and cell stress-inducing effects of a biotinylated analog of PGA(1) (PGA(1)-biotinamide, PGA(1)-B), to establish its validity to identify cyPG-protein interactions of potential therapeutic interest. PGA(1) and PGA(1)-B displayed similar effects on cell viability, Hsp70 and heme oxygenase-1 induction and pro-inflammatory gene inhibition. Remarkably, PGA(1)-B did not activate PPAR. Therefore, this biotinylated analog can be useful to identify PPAR-independent effects of cyPGs. Protein modification and subcellular distribution of PGA(1)-B targets were cell-type-dependent. Through proteomic and biochemical approaches we have identified a novel set of PGA(1)-B targets including proteins involved in stress response, protein synthesis, cytoskeletal regulation and carbohydrate metabolism. Moreover, the modification of several of the targets identified could be reproduced in vitro. These results unveil novel interactions of PGA(1) that will contribute to delineate the mechanisms for the anti-proliferative and metabolic actions of this cyPG.

Effects of 15-deoxy-delta 12, 14-prostaglandin J2 on the expression of p53 in MCF-7 cells.

Cyclopentenone prostaglandins (cyPGs) exert diverse cellular functions, such as anti-inflammatory and cytoprotective effects, via multiple mechanisms. CyPGs, especially those of the A and J series, are characterized by the presence of a chemically reactive alpha,beta-unsaturated carbonyl group. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a representative cyPG of the J series, has been reported to directly inhibit the activity of redox-sensitive transcription factors, such as activator protein-1 and nuclear factor-kappaB. In the present study, we examined the effects of 15d-PGJ(2) on activation of p53 tumor suppressor in human breast cancer (MCF-7) cells. MCF-7 cells treated with 15d-PGJ(2) exhibited elevated p53 protein expression in time- and concentration-related manners, whereas prostaglandin A(2) (PGA(2)) and the nonprostaglandin derivative 2-cyclopenten-1-one exerted an effect to a lesser extent than did 15d-PGJ(2). In addition, MCF-7 cells exposed to 15d-PGJ(2) significantly accumulated p53 in both cytosolic and nuclear fractions. Despite the elevated levels of p53, its DNA-binding activity was reduced in 15d-PGJ(2)-treated MCF-7 cells. Moreover, isolated MCF-7 nuclear extracts directly treated with 15d-PGJ(2) exhibite diminished DNA-binding ability of p53, while the same concentration of PGA(2) or 2-cyclopenten-1-one was much less inhibitory. Thus, the electrophilic carbon center located in the alpha,beta-unsaturated carbonyl moiety of the cyclopentenone ring might be critical for the control of DNA-binding activity as well as cellular levels of p53 by 15d-PGJ(2).

Neurotoxic lipid peroxidation species formed by ischemic stroke increase injury.

Stroke is the third leading cause of death in the United States, yet no neuroprotective agents for treatment are clinically available. There is a pressing need to understand the signaling molecules that mediate ischemic cell death and identify novel neuroprotective targets. Cyclopentenone isoprostanes (IsoPs), formed after free radical-mediated peroxidation of arachidonic acid, are used as markers of stress, but their bioactivity is poorly understood. We have recently shown that 15-A(2t)-IsoP is a potent neurotoxin in vitro and increases the free radical burden in neurons. In this work, we demonstrate that 15-A(2t)-IsoP is abundantly produced in stroke-infarcted human cortical tissue. Using primary neuronal cultures we found that minimally toxic exposure to 15-A(2t)-IsoP does not alter ATP content, but in combination with oxygen glucose deprivation resulted in a significant hyperpolarization of the mitochondrial membrane and dramatically increased neuronal cell death. In the presence of Ca(2+), 15-A(2t)-IsoP led to a rapid induction of the permeability transition pore and release of cytochrome c. Taken with our previous work, these data support a model in which ischemia causes generation of reactive oxygen species, calcium influx, lipid peroxidation, and 15-A(2t)-IsoP formation. These factors combine to enhance opening of the permeability transition pore leading to cell death subsequent to mitochondrial cytochrome c release. These data are the first documentation of significant 15-A(2t)-IsoP formation after acute ischemic stroke and suggest that the addition of 15-A(2t)-IsoP to in vitro models of ischemia may help to more fully recapitulate stroke injury.

Study of protein targets for covalent modification by the antitumoral and anti-inflammatory prostaglandin PGA1: focus on vimentin.

Prostaglandins with cyclopentenone structure (cyPG) display potent antiproliferative actions that have elicited their study as potential anticancer agents. Several natural and synthetic analogs of the cyPG prostaglandin A(1) (PGA(1)) have proven antitumoral efficacy in cancer cell lines and animal models. In addition, PGA(1) has been used as an inhibitor of transcription factor NF-kappaB-mediated processes, including inflammatory gene expression and viral replication. An important determinant for these effects is the ability of cyPG to form Michael adducts with free thiol groups. The chemical nature of this interaction implies that PGA(1) could covalently modify cysteine residues in a large number of cellular proteins potentially involved in its beneficial effects. However, only a few targets of PGA(1) have been identified. In previous work, we have observed that a biotinylated analog of PGA(1) that retains the cyclopentenone moiety (PGA(1)-B) binds to multiple targets in fibroblasts. Here, we have addressed the identification of these targets through a proteomic approach. Cell fractionation followed by avidin affinity chromatography yielded a fraction enriched in proteins modified by PGA(1)-B. Analysis of this fraction by SDS-PAGE and LC-MS/MS allowed the identification of the chaperone Hsp90, elongation and initiation factors for protein synthesis and cytoskeletal proteins including actin, tubulin and vimentin. Furthermore, we have characterized the modification of vimentin both in vitro and in intact cells. Our observations indicate that cysteine 328 is the main site for PGA(1) addition. These results may contribute to a better understanding of the mechanism of action of PGA(1) and the potential of cyPG-based therapeutic strategies.

Addition of electrophilic lipids to actin alters filament structure.

Pathophysiological processes associated with oxidative stress lead to the generation of reactive lipid species. Among them, lipids bearing unsaturated aldehyde or ketone moieties can form covalent adducts with cysteine residues and modulate protein function. Through proteomic techniques we have identified actin as a target for the addition of biotinylated analogs of the cyclopentenone prostaglandins 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and PGA(1) in NIH-3T3 fibroblasts. This modification could take place in vitro and mapped to the protein C-terminal end. Other electrophilic lipids, like the isoprostane 8-iso-PGA(1) and 4-hydroxy-2-nonenal, also bound to actin. The C-terminal region of actin is important for monomer-monomer interactions and polymerization. Electron microscopy showed that actin treated with 15d-PGJ(2) or 4-hydroxy-2-nonenal formed filaments which were less abundant and displayed shorter length and altered structure. Streptavidin-gold staining allowed mapping of biotinylated 15d-PGJ(2) at sites of filament disruption. These results shed light on the structural implications of actin modification by lipid electrophiles.

Induction of Fas clustering and apoptosis by coral prostanoid in human hormone-resistant prostate cancer cells.

Cyclopentenone prostaglandins (PGs) such as PGA1, PGA2 and delta12-PGJ2 have been shown to suppress tumor cell growth and to induce apoptosis in prostate cancer cells. Bromovulone III, which is isolated from the soft coral Clavularia viridis, is a cyclopentenone prostanoid. In this study, the anti-tumor activity as well as action mechanism of bromovulone III was identified in prostate cancer cells. Bromovulone III displayed anti-tumor activity of 30 to 100 times more effective than PGA1, PGA2 and delta12-PGJ2 in PC-3 cells. Several targets of caspases and Bcl-2 family of proteins were detected and the data demonstrated that bromovulone III induced the activation of caspase-8, -9 and -3, and Bid cleavage in which the caspase-8 activation occurred the first. Bromovulone III did not modify the protein levels of death receptors and ligands. Of note, the Fas clustering in PC-3 cells responsive to bromovulone III was observed by confocal immunofluorescence microscopy suggesting the involvement of Fas-mediated pathway. Bromovulone III also induced the cleavage of Mcl-1 in this study. The cleavage fragments (24, 19 and 17 kDa) may partly share the apoptotic insult. Although it has been suggested that Fas-mediated signaling may contribute to the caspase-8 activation induced by DNA-damaging agents; however, bromovulone III did not induce any DNA breakage, suggesting that bromovulone III-induced Fas/caspase-8-dependent signaling is not through the direct target on DNA damage. In summary, the data suggest that bromovulone III causes a rapid redistribution and clustering of Fas in PC-3 cells. Subsequently, the Fas event causes the activation and interaction of caspase-8/Bid/caspase-9 signaling cascades, and the activation of executor caspase-3.

Chemical transformation of prostaglandin-A2: a novel series of C-10 halogenated, C-12 hydroxylated prostaglandin-A2 analogues.

[reaction: see text] Synthesis of a novel class of C-10 halogenated and C-12 oxygenated prostaglandin-A(2) derivatives (6a-6c) has been accomplished. (15S)-Prostaglandin-A(2) (1), from the gorgonian Plexaura homomalla, served as the starting material for the synthesis. The absolute configuration was determined using NMR.

Cyclopentenone isoprostanes are novel bioactive products of lipid oxidation which enhance neurodegeneration.

Oxidative stress and subsequent lipid peroxidation are involved in the pathogenesis of numerous neurodegenerative conditions, including stroke. Cyclopentenone isoprostanes (IsoPs) are novel electrophilic lipid peroxidation products formed under conditions of oxidative stress via the isoprostane pathway. These cyclopentenone IsoPs are isomeric to highly bioactive cyclopentenone prostaglandins, yet it has not been determined if these products are biologically active or are formed in the brain. Here we demonstrate that the major cyclopentenone IsoP isomer 15-A2t-IsoP potently induces apoptosis in neuronal cultures at submicromolar concentrations. We present a model in which 15-A2t-IsoP induced neuronal apoptosis involves initial depletion of glutathione and enhanced production of reactive oxygen species, followed by 12-lipoxygenase activation and phosphorylation of extracellular signal-regulated kinase 1/2 and the redox sensitive adaptor protein p66shc, which results in caspase-3 cleavage. 15-A2t-IsoP application also dramatically potentiates oxidative glutamate toxicity at concentrations as low as 100 nm, demonstrating the functional importance of these molecules in neurodegeneration. Finally, we employ novel mass spectrometric methods to show that cyclopentenone IsoPs are formed abundantly in brain tissue under conditions of oxidative stress. Together these findings suggest that cyclopentenone IsoPs may contribute to neuronal death caused by oxidative insults, and that their activity should perhaps be addressed when designing neuroprotective therapies.

Biochemistry of prostaglandins A.

This review considers modern concepts on the structural-functional properties and antiproliferative, antitumor, and antiviral effects of cyclopentenone prostaglandins A and mechanisms underlying their actions. Possible directions of pharmacological application of these compounds and their analogs are discussed.