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1.
Vaccines (Basel) ; 9(5)2021 May 17.
Article in English | MEDLINE | ID: mdl-34067514

ABSTRACT

Viruses have developed incredibly creative ways of making a virtue out of necessity, including taking full advantage of their small genomes. Indeed, viruses often encode multiple proteins within the same genomic region by using two or more reading frames in both orientations through a process called overprinting. Complex retroviruses provide compelling examples of that. The human immunodeficiency virus type 1 (HIV-1) genome expresses sixteen proteins from nine genes that are encoded in the three positive-sense reading frames. In addition, the genome of some HIV-1 strains contains a tenth gene in one of the negative-sense reading frames. The so-called Antisense Protein (ASP) gene overlaps the HIV-1 Rev Response Element (RRE) and the envelope glycoprotein gene, and encodes a highly hydrophobic protein of ~190 amino acids. Despite being identified over thirty years ago, relatively few studies have investigated the role that ASP may play in the virus lifecycle, and its expression in vivo is still questioned. Here we review the current knowledge about ASP, and we discuss some of the many unanswered questions.

2.
Biochim Biophys Acta Gen Subj ; 1864(10): 129656, 2020 10.
Article in English | MEDLINE | ID: mdl-32512169

ABSTRACT

BACKGROUND: Intracellular protein trafficking is crucial for survival of cell and proper functioning of the organelles; however, these pathways are not well studied in the malaria parasite. Its unique cellular architecture and organellar composition raise an interesting question to investigate. METHODS: The interaction of Plasmodium falciparum Rab7 (PfRab7) with vacuolar protein sorting-associated protein 26 (PfVPS26) of retromer complex was shown by coimmunoprecipitation (co-IP). Confocal microscopy was used to show the localization of the complex in the parasite with respect to different organelles. Further chemical tools were employed to explore the role of digestive vacuole (DV) in retromer trafficking in parasite and GTPase activity of PfRab7 was examined. RESULTS: PfRab7 was found to be interacting with retromer complex that assembled mostly near DV and the Golgi in trophozoites. Chemical disruption of DV by chloroquine (CQ) led to its disassembly that was further validated by using compound 5f, a heme polymerization inhibitor in the DV. PfRab7 exhibited Mg2+ dependent weak GTPase activity that was inhibited by a specific Rab7 GTPase inhibitor, CID 1067700, which prevented the assembly of retromer complex in P. falciparum and inhibited its growth suggesting the role of GTPase activity of PfRab7 in retromer assembly. CONCLUSION: Retromer complex was found to be interacting with PfRab7 and the functional integrity of the DV was found to be important for retromer assembly in P. falciparum. GENERAL SIGNIFICANCE: This study explores the retromer trafficking in P. falciparum and describes amechanism to validate DV targeting antiplasmodial molecules.


Subject(s)
Plasmodium falciparum/metabolism , Vacuoles/metabolism , rab GTP-Binding Proteins/metabolism , Antimalarials/pharmacology , Chloroquine/pharmacology , Humans , Magnesium/metabolism , Malaria, Falciparum/drug therapy , Malaria, Falciparum/metabolism , Malaria, Falciparum/parasitology , Models, Molecular , Plasmodium falciparum/drug effects , Protein Interaction Maps/drug effects , Vacuoles/drug effects , rab7 GTP-Binding Proteins
5.
Biochim Biophys Acta Proteins Proteom ; 1866(5-6): 722-730, 2018.
Article in English | MEDLINE | ID: mdl-29654975

ABSTRACT

Retromer complex plays a crucial role in intracellular protein trafficking and is conserved throughout the eukaryotes including malaria parasite, Plasmodium falciparum, where it is partially conserved. The assembly of retromer complex in RBC stages of malarial parasite is extremely difficult to explore because of its complicated physiology, small size, and intra-erythrocytic location. Nonetheless, understanding of retromer assembly may pave new ways for the development of novel antimalarials targeting parasite-specific protein trafficking pathways. Here, we investigated the assembly of retromer complex in P. falciparum, by an immunosensing method through highly sensitive Surface Plasmon Resonance (SPR) technique. After taking leads from the bioinformatics search and literature, different interacting proteins were identified and specific antibodies were raised against them. The sensor chip was prepared by covalently linking antibody specific to one component and the whole cell lysate was passed through it in order to trap the interacting complex. Antibodies raised against other interacting components were used to detect them in the trapped complex on the SPR chip. We were able to detect three different components in the retromer complex trapped by the immobilized antibody specific against a different component on a sensor chip. The assay was reproduced and validated in a different two-component CD74-MIF system in mammalian cells. We, thus, illustrate the assembly of retromer complex in P. falciparum through a bio-sensing approach that combines SPR with immunosensing requiring a very small amount of sample from the native source.


Subject(s)
Biosensing Techniques , Multiprotein Complexes/metabolism , Plasmodium falciparum/metabolism , Protozoan Proteins/metabolism , Surface Plasmon Resonance , Vesicular Transport Proteins/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Blotting, Western , Computational Biology , Hep G2 Cells , Humans , Immunoprecipitation , Kinetics , Mice , Multiprotein Complexes/genetics , Multiprotein Complexes/immunology , NIH 3T3 Cells , Plasmodium falciparum/genetics , Plasmodium falciparum/immunology , Protein Binding , Protein Transport , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/immunology
6.
Biochem Pharmacol ; 121: 33-51, 2016 Dec 01.
Article in English | MEDLINE | ID: mdl-27693316

ABSTRACT

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat multiple inflammatory diseases and pain but severe gastric mucosal damage is the worst outcome of NSAID-therapy. Here we report that mitoTEMPO, a mitochondrially targeted superoxide (O2-) scavenger protected as well as healed gastric injury induced by diclofenac (DCF), the most commonly used NSAID. Common existing therapy against gastric injury involves suppression of gastric acid secretion by proton pump inhibitors and histamine H2 receptor antagonists; however, dyspepsia, vitamin B12 deficiency and gastric microfloral dysbalance are the major drawbacks of acid suppression. Interestingly, mitoTEMPO did not inhibit gastric acid secretion but offered gastroprotection by preventing DCF-induced generation of O2- due to mitochondrial respiratory chain failure and by preventing mitochondrial oxidative stress (MOS)-mediated mitopathology. MitoTEMPO even restored DCF-stimulated reduced fatty acid oxidation, mitochondrial depolarization and bioenergetic crisis in gastric mucosa. MitoTEMPO also prevented the activation of mitochondrial pathway of apoptosis and MOS-mediated proinflammatory signaling through NF-κB by DCF. Furthermore, mitoTEMPO when administered in rats with preformed gastric lesions expedited the healing of gastric injury and the healed stomach exhibited its normal physiology as evident from gastric acid and pepsin secretions under basal or stimulated conditions. Thus, in contrast to the existing antiulcer drugs, mitochondrially targeted O2- scavengers like mitoTEMPO may represent a novel class of gastroprotective molecules that does not affect gastric acid secretion and may be used in combination with DCF, keeping its anti-inflammatory action intact, while reducing its gastrodamaging effects.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Diclofenac/adverse effects , Gastric Mucosa/drug effects , Gastritis/prevention & control , Mitochondria/metabolism , Organophosphorus Compounds/therapeutic use , Piperidines/therapeutic use , Superoxides/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Line , Chemotaxis, Leukocyte/drug effects , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Gastric Acid/metabolism , Gastric Mucosa/injuries , Gastric Mucosa/metabolism , Gastritis/metabolism , Gastritis/pathology , Humans , Microscopy, Fluorescence , Neutrophil Infiltration/drug effects , Organophosphorus Compounds/administration & dosage , Oxidative Stress/drug effects , Piperidines/administration & dosage , Rats, Sprague-Dawley
7.
Protein Expr Purif ; 120: 7-15, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26690372

ABSTRACT

Translocation of various proteins to the subcellular organelles is an essential mechanism to regulate the metabolic pathways and often vacuolar protein sorting (VPS) proteins are involved in this transportation. Plasmodium falciparum VPS29 (PfVPS29) is predicted to be a functional component in the assembly of the retromer complex; however, so far detailed characterization of PfVPS29 in its native form is not yet done. We report the successful expression and purification of tag-free recombinant PfVPS29 with a yield of 5.6 mg from 1 L of Escherichia coli culture. PfVPS29 was purified by combined anion-exchange and size exclusion chromatography. The protein showed a single band in SDS-PAGE and it exhibited molecular mass of 21.7 kDa as measured by MALDI-TOF mass spectrometry. Secondary structure was elucidated by circular dichroism spectroscopy. It was found to be a monomeric protein in solution as evident from dynamic light scattering studies, chemical cross-linking experiments and size exclusion chromatography. Subsequently, polyclonal anti-PfVPS29 antibody was generated and used for evaluating protein expression by western blot and following subcellular localization in P. falciparum by confocal immunofluoroscence microscopy. PfVPS29 was found to be located in cytoplasm and expressed from early trophozoite to schizont stages with maximum expression in trophozoite stage. This study provides purification, biophysical characterization and subcellular localization of PfVPS29 in different asexual stages of P. falciparum.


Subject(s)
Plasmodium falciparum/metabolism , Protozoan Proteins/metabolism , Vesicular Transport Proteins/metabolism , Amino Acid Sequence , Blotting, Western , Circular Dichroism , Cloning, Molecular , Cytoplasm/metabolism , Electrophoresis, Polyacrylamide Gel , Gene Expression , Life Cycle Stages , Mass Spectrometry , Molecular Sequence Data , Molecular Weight , Plasmodium falciparum/physiology , Protein Structure, Secondary , Protozoan Proteins/genetics , Protozoan Proteins/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/isolation & purification
9.
Free Radic Biol Med ; 65: 456-467, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23892052

ABSTRACT

Nonsteroidal anti-inflammatory drug (NSAID)-induced mitochondrial oxidative stress (MOS) is an important prostaglandin (PG)-independent pathway of the induction of gastric mucosal injury. However, the molecular mechanism behind MOS-mediated gastric pathology is still obscure. In various pathological conditions of tissue injury oxidative stress is often linked with inflammation. Here we report that MOS induced by indomethacin (an NSAID) induces gastric mucosal inflammation leading to proinflammatory damage. Indomethacin, time dependently stimulated the expression of proinflammatory molecules such as intercellular adhesion molecule 1(ICAM-1), vascular cell adhesion molecule 1(VCAM-1), interleukin1ß (IL-1ß), and monocyte chemotactic protein-1 (MCP-1) in gastric mucosa in parallel with the increase of neutrophil infiltration and injury of gastric mucosa in rat. Western immunoblotting and confocal microscopic studies revealed that indomethacin induced nuclear translocation of nuclear factor kappa-B (NF-κB) in gastric mucosal cells, which resulted in proinflammatory signaling. The prevention of MOS by antioxidant tryptamine-gallic acid hybrid (SEGA) inhibited indomethacin-induced expression of ICAM-1, VCAM-1, IL-1ß, and MCP-1. SEGA also prevented indomethacin-induced NF-κB activation and neutrophil infiltration as documented by chromatin immunoprecipitation studies and neutrophil migration assay, respectively. Heme oxygenase-1 (HO-1), a cytoprotective enzyme associated with tissue repair mechanisms is stimulated in response to oxidative stress. We have investigated the role of HO-1 against MOS and MOS-mediated inflammation in recovering from gastropathy. Indomethacin stimulated the expression of HO-1 and indomethacin-stimulated HO-1 expression was reduced by SEGA, an antioxidant, which could prevent MOS. Thus, the data suggested that the induction of HO-1 was a protective response against MOS developed by indomethacin. Moreover, the induction of HO-1 by cobalt protoporphyrin inhibited inflammation and chemical silencing of HO-1 by zinc protoporphyrin aggravated the inflammation by indomethacin. Thus, NSAID by promoting MOS-induced proinflammatory response damaged gastric mucosa and HO-1 protected NSAID-induced gastric mucosal damage by preventing NF-κB activation and proinflammatory activity.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/toxicity , Gastric Mucosa/drug effects , Gastric Mucosa/metabolism , Heme Oxygenase-1/metabolism , NF-kappa B/metabolism , Animals , Blotting, Western , Chromatin Immunoprecipitation , Gastric Mucosa/injuries , Indomethacin/adverse effects , Inflammation/chemically induced , Microscopy, Confocal , Mitochondria/drug effects , Mitochondria/metabolism , Neutrophil Infiltration/drug effects , Oxidative Stress , Polymerase Chain Reaction , Rats , Rats, Sprague-Dawley
10.
J Biol Chem ; 287(32): 26630-46, 2012 Aug 03.
Article in English | MEDLINE | ID: mdl-22696214

ABSTRACT

We have investigated the impact of persistent intravascular hemolysis on liver dysfunction using the mouse malaria model. Intravascular hemolysis showed a positive correlation with liver damage along with the increased accumulation of free heme and reactive oxidants in liver. Hepatocytes overinduced heme oxygenase-1 (HO-1) to catabolize free heme in building up defense against this pro-oxidant milieu. However, in a condition of persistent free heme overload in malaria, the overactivity of HO-1 resulted in continuous transient generation of free iron to favor production of reactive oxidants as evident from 2',7'-dichlorofluorescein fluorescence studies. Electrophoretic mobility shift assay documented the activation of NF-κB, which in turn up-regulated intercellular adhesion molecule 1 as evident from chromatin immunoprecipitation studies. NF-κB activation also induced vascular cell adhesion molecule 1, keratinocyte chemoattractant, and macrophage inflammatory protein 2, which favored neutrophil extravasation and adhesion in liver. The infiltration of neutrophils correlated positively with the severity of hemolysis, and neutrophil depletion significantly prevented liver damage. The data further documented the elevation of serum TNFα in infected mice, and the treatment of anti-TNFα antibodies also significantly prevented neutrophil infiltration and liver injury. Deferoxamine, which chelates iron, interacts with free heme and bears antioxidant properties that prevented oxidative stress, NF-κB activation, neutrophil infiltration, hepatocyte apoptosis, and liver damage. Furthermore, the administration of N-acetylcysteine also prevented NF-κB activation, neutrophil infiltration, hepatocyte apoptosis, and liver damage. Thus, hepatic free heme accumulation, TNFα release, oxidative stress, and NF-κB activation established a link to favor neutrophil infiltration in inducing liver damage during hemolytic conditions in malaria.


Subject(s)
Heme/metabolism , Hemolysis , Liver/physiopathology , Malaria/physiopathology , NF-kappa B/metabolism , Neutrophil Infiltration , Animals , Base Sequence , Blotting, Western , DNA Primers , Disease Models, Animal , Electrophoretic Mobility Shift Assay , Heme Oxygenase (Decyclizing)/metabolism , In Situ Nick-End Labeling , Liver/metabolism , Malaria/parasitology , Male , Mice , Mice, Inbred BALB C , Oxidative Stress , Plasmodium yoelii/isolation & purification , Tumor Necrosis Factor-alpha/blood , Tumor Necrosis Factor-alpha/metabolism
11.
J Biol Chem ; 287(29): 24844-61, 2012 Jul 13.
Article in English | MEDLINE | ID: mdl-22645149

ABSTRACT

Macrophage migration inhibitory factor (MIF) is responsible for proinflammatory reactions in various infectious and non-infectious diseases. We have investigated the mechanism of anti-inflammatory activity of epoxyazadiradione, a limonoid purified from neem (Azadirachta indica) fruits, against MIF. Epoxyazadiradione inhibited the tautomerase activity of MIF of both human (huMIF) and malaria parasites (Plasmodium falciparum (PfMIF) and Plasmodium yoelii (PyMIF)) non-competitively in a reversible fashion (K(i), 2.11-5.23 µm). Epoxyazadiradione also significantly inhibited MIF (huMIF, PyMIF, and PfMIF)-mediated proinflammatory activities in RAW 264.7 cells. It prevented MIF-induced macrophage chemotactic migration, NF-κB translocation to the nucleus, up-regulation of inducible nitric-oxide synthase, and nitric oxide production in RAW 264.7 cells. Epoxyazadiradione not only exhibited anti-inflammatory activity in vitro but also in vivo. We tested the anti-inflammatory activity of epoxyazadiradione in vivo after co-administering LPS and MIF in mice to mimic the disease state of sepsis or bacterial infection. Epoxyazadiradione prevented the release of proinflammatory cytokines such as IL-1α, IL-1ß, IL-6, and TNF-α when LPS and PyMIF were co-administered to BALB/c mice. The molecular basis of interaction of epoxyazadiradione with MIFs was explored with the help of computational chemistry tools and a biological knowledgebase. Docking simulation indicated that the binding was highly specific and allosteric in nature. The well known MIF inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) inhibited huMIF but not MIF of parasitic origin. In contrast, epoxyazadiradione inhibited both huMIF and plasmodial MIF, thus bearing an immense therapeutic potential against proinflammatory reactions induced by MIF of both malaria parasites and human.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Limonins/pharmacology , Macrophage Migration-Inhibitory Factors/pharmacology , Macrophages/drug effects , Animals , Azadirachta/chemistry , Cell Line , Electrophoretic Mobility Shift Assay , Humans , Male , Mice , Mice, Inbred BALB C , NF-kappa B/metabolism , Nitric Oxide/metabolism , Plasmodium falciparum/metabolism , Plasmodium yoelii/metabolism
12.
Free Radic Biol Med ; 53(1): 129-42, 2012 Jul 01.
Article in English | MEDLINE | ID: mdl-22588006

ABSTRACT

We have synthesized a new series of aryl aryl methyl thio arenes (AAMTAs) and evaluated antimalarial activity in vitro and in vivo against drug-resistant malaria. These compounds interact with free heme, inhibit hemozoin formation, and prevent Plasmodium falciparum growth in vitro in a concentration-dependent manner. These compounds concentration dependently promote oxidative stress in Plasmodium falciparum as evident from the generation of intraparasitic oxidants, protein carbonyls, and lipid peroxidation products. Furthermore, AAMTAs deplete intraparasite GSH levels, which is essential for antioxidant defense and survival during intraerythrocytic stages. These compounds displayed potent antimalarial activity not only in vitro but also in vivo against multidrug-resistant Plasmodium yoelii dose dependently in a mouse model. The mixtures of enantiomers of AAMTAs containing 3-pyridyl rings were found to be more efficient in providing antimalarial activity. Efforts have been made to synthesize achiral AAMTAs 17-23 and among them, compound 18 showed significant antimalarial activity in vivo.


Subject(s)
Antimalarials/therapeutic use , Drug Resistance, Multiple/drug effects , Hydrocarbons, Aromatic/chemistry , Malaria/prevention & control , Oxidative Stress/drug effects , Plasmodium falciparum/drug effects , Animals , Glutathione/metabolism , Heme/metabolism , Malaria/parasitology , Male , Mice , Mice, Inbred BALB C
13.
J Biol Chem ; 287(5): 3495-509, 2012 Jan 27.
Article in English | MEDLINE | ID: mdl-22157011

ABSTRACT

We have investigated the gastroprotective effect of SEGA (3a), a newly synthesized tryptamine-gallic acid hybrid molecule against non-steroidal anti-inflammatory drug (NSAID)-induced gastropathy with mechanistic details. SEGA (3a) prevents indomethacin (NSAID)-induced mitochondrial oxidative stress (MOS) and dysfunctions in gastric mucosal cells, which play a pathogenic role in inducing gastropathy. SEGA (3a) offers this mitoprotective effect by scavenging of mitochondrial superoxide anion (O(2)(·-)) and intramitochondrial free iron released as a result of MOS. SEGA (3a) in vivo blocks indomethacin-mediated MOS, as is evident from the inhibition of indomethacin-induced mitochondrial protein carbonyl formation, lipid peroxidation, and thiol depletion. SEGA (3a) corrects indomethacin-mediated mitochondrial dysfunction in vivo by restoring defective electron transport chain function, collapse of transmembrane potential, and loss of dehydrogenase activity. SEGA (3a) not only corrects mitochondrial dysfunction but also inhibits the activation of the mitochondrial pathway of apoptosis by indomethacin. SEGA (3a) inhibits indomethacin-induced down-regulation of bcl-2 and up-regulation of bax genes in gastric mucosa. SEGA (3a) also inhibits indometacin-induced activation of caspase-9 and caspase-3 in gastric mucosa. Besides the gastroprotective effect against NSAID, SEGA (3a) also expedites the healing of already damaged gastric mucosa. Radiolabeled ((99m)Tc-labeled SEGA (3a)) tracer studies confirm that SEGA (3a) enters into mitochondria of gastric mucosal cell in vivo, and it is quite stable in serum. Thus, SEGA (3a) bears an immense potential to be a novel gastroprotective agent against NSAID-induced gastropathy.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Apoptosis/drug effects , Gallic Acid/pharmacology , Indomethacin/adverse effects , Mitochondria/metabolism , Stomach Diseases/chemically induced , Stomach Diseases/drug therapy , Tryptamines/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Electron Transport Chain Complex Proteins/metabolism , Gastric Mucosa/metabolism , Gene Expression Regulation/drug effects , Humans , Indomethacin/pharmacology , Lipid Peroxidation/drug effects , Membrane Potential, Mitochondrial/drug effects , Oxidative Stress/drug effects , Rats , Rats, Sprague-Dawley , Superoxides/metabolism , bcl-2-Associated X Protein/biosynthesis
14.
Nucleic Acids Res ; 40(3): 1174-90, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22006844

ABSTRACT

We have investigated the DNA-binding nature as well as the function of a putative Alba (Acetylation lowers binding affinity) family protein (PfAlba3) from Plasmodium falciparum. PfAlba3 possesses DNA-binding property like Alba family proteins. PfAlba3 binds to DNA sequence non-specifically at the minor groove and acetylation lowers its DNA-binding affinity. The protein is ubiquitously expressed in all the erythrocytic stages of P. falciparum and it exists predominantly in the acetylated form. PfAlba3 inhibits transcription in vitro by binding to DNA. Plasmodium falciparum Sir2 (PfSir2A), a nuclear localized deacetylase interacts with PfAlba3 and deacetylates the lysine residue of N-terminal peptide of PfAlba3 specific for DNA binding. PfAlba3 is localized with PfSir2A in the periphery of the nucleus. Fluorescence in situ hybridization studies revealed the presence of PfAlba3 in the telomeric and subtelomeric regions. ChIP and ChIP ReChIP analyses further confirmed that PfAlba3 binds to the telomeric and subtelomeric regions as well as to var gene promoter.


Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Plasmodium falciparum , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Amino Acid Sequence , Chromatin Immunoprecipitation , DNA/chemistry , DNA/metabolism , DNA-Binding Proteins/analysis , Lysine/metabolism , Models, Molecular , Molecular Sequence Data , Peptides/metabolism , Protein Binding , Protozoan Proteins/analysis , RNA-Binding Proteins/chemistry , Sirtuin 2/metabolism , Transcription, Genetic
15.
Bioorg Med Chem ; 19(24): 7365-73, 2011 Dec 15.
Article in English | MEDLINE | ID: mdl-22088307

ABSTRACT

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is involved in the development of an array of inflammatory disorders including rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis and sepsis. The synthesis of MIF-inhibitor is a rationale approach to develop novel anti-inflammatory agent to treat multitude of inflammatory diseases. In this work, we have synthesized and evaluated MIF-inhibitory activity of a series of small molecules containing isoxazoline skeleton. Mode of binding of this inhibitor to human MIF (huMIF) was determined by docking studies. The synthesized molecules inhibit tautomerase activity of huMIF. The anti-inflammatory activity of the most active inhibitor, 4-((3-(4-hydroxy-3-methoxyphenyl)-4, 5-dihydroisoxazol-5-yl) methoxy) benzaldehyde (4b) was evaluated against huMIF-induced inflammation in a cellular model (RAW 264.7 cell). Compound 4b significantly inhibits huMIF-mediated NF-κB translocation to the nucleus, up-regulation of inducible nitric oxide synthase and nitric oxide production in RAW 264.7 cell which are the markers for inflammation. The compound 4b is not cytotoxic as evident from cell viability assay. Hence, the compound 4b has potential to be a novel anti-inflammatory agent.


Subject(s)
Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Isoxazoles/chemistry , Isoxazoles/pharmacology , Macrophage Migration-Inhibitory Factors/antagonists & inhibitors , Active Transport, Cell Nucleus/drug effects , Animals , Anti-Inflammatory Agents/chemical synthesis , Binding Sites , Cell Line , Cell Survival/drug effects , Humans , Isoxazoles/chemical synthesis , Macrophage Migration-Inhibitory Factors/chemistry , Macrophage Migration-Inhibitory Factors/immunology , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Mice , Models, Molecular , NF-kappa B/metabolism , Nitric Oxide/immunology , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/immunology , Up-Regulation
16.
J Biol Chem ; 286(45): 39387-402, 2011 Nov 11.
Article in English | MEDLINE | ID: mdl-21908612

ABSTRACT

The mechanism of action of heme oxygenase-1 (HO-1) in mitochondrial oxidative stress (MOS)-mediated apoptotic tissue injury was investigated. MOS-mediated gastric mucosal apoptosis and injury were introduced in rat by indomethacin, a non-steroidal anti-inflammatory drug. Here, we report that HO-1 was not only induced but also translocated to mitochondria during gastric mucosal injury to favor repair mechanisms. Furthermore, mitochondrial translocation of HO-1 resulted in the prevention of MOS and mitochondrial pathology as evident from the restoration of the complex I-driven mitochondrial respiratory control ratio and transmembrane potential. Mitochondrial translocation of HO-1 also resulted in time-dependent inhibition of apoptosis. We searched for the plausible mechanisms responsible for HO-1 induction and mitochondrial localization. Free heme, the substrate for HO-1, was increased inside mitochondria during gastric injury, and mitochondrial entry of HO-1 decreased intramitochondrial free heme content, suggesting that a purpose of mitochondrial translocation of HO-1 is to detoxify accumulated heme. Heme may activate nuclear translocation of NF-E2-related factor 2 to induce HO-1 through reactive oxygen species generation. Electrophoretic mobility shift assay and chromatin immunoprecipitation studies indicated nuclear translocation of NF-E2-related factor 2 and its binding to HO-1 promoter to induce HO-1 expression during gastric injury. Inhibition of HO-1 by zinc protoporphyrin aggravated the mucosal injury and delayed healing. Zinc protoporphyrin further reduced the respiratory control ratio and transmembrane potential and enhanced MOS and apoptosis. In contrast, induction of HO-1 by cobalt protoporphyrin reduced MOS, corrected mitochondrial dysfunctions, and prevented apoptosis and gastric injury. Thus, induction and mitochondrial localization of HO-1 are a novel cytoprotective mechanism against MOS-mediated apoptotic tissue injury.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Apoptosis/drug effects , Gastric Mucosa/enzymology , Gastric Mucosa/injuries , Heme Oxygenase (Decyclizing)/metabolism , Indomethacin/adverse effects , Mitochondria/enzymology , Oxidative Stress/drug effects , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Indomethacin/pharmacology , Membrane Potential, Mitochondrial/drug effects , NF-E2 Transcription Factor/metabolism , Protein Transport/drug effects , Rats , Rats, Sprague-Dawley
17.
Free Radic Biol Med ; 50(11): 1659-68, 2011 Jun 01.
Article in English | MEDLINE | ID: mdl-21406224

ABSTRACT

Plasmodium falciparum macrophage migration inhibitory factor (PfMIF) exhibits thioredoxin (Trx)-like oxidoreductase activity but the active site for this activity and its function have not been evaluated. A bioinformatics search revealed that the conserved CXXC motif, which is responsible for Trx-like oxidoreductase activity, is absent from PfMIF. In contrast, the adjacent N-terminal Cys-3 and Cys-4 are conserved in MIF across species of malarial parasites. Mutation of either vicinal Cys-3 or Cys-4 of PfMIF abolished the Trx-like activity, whereas the mutation of the remaining Cys-59 or Cys-103 did not affect it. PfMIF has an antioxidant function. It prevents reactive oxygen species-mediated lipid peroxidation and oxidative damage of DNA as evident from DNA nicking assay. Interestingly, chemical modification of the vicinal cysteines by phenylarsine oxide (PAO), a specific vicinal thiol modifier, significantly prevented this antioxidant activity. Modification of Cys-3 and Cys-4 was confirmed by MALDI-TOF mass spectroscopy of peptide fragments obtained after cyanogen bromide digestion of PAO-modified PfMIF. Furthermore, mutation of either Cys-3 or Cys-4 of PfMIF resulted in the loss of both Trx-like oxidoreductase and antioxidant activities of PfMIF. Altogether, our results suggest that the vicinal Cys-3 and Cys-4 play a critical role in the Trx-like oxidoreductase activity and antioxidant property of PfMIF.


Subject(s)
Cysteine/metabolism , Erythrocytes/metabolism , Macrophage Migration-Inhibitory Factors/metabolism , Malaria/metabolism , Plasmodium falciparum/enzymology , Protozoan Proteins/metabolism , Animals , Arsenicals/pharmacology , Computational Biology , Cysteine/chemistry , Cysteine/genetics , DNA Damage/drug effects , Enzyme Activation/drug effects , Erythrocytes/parasitology , Erythrocytes/pathology , Macrophage Migration-Inhibitory Factors/chemistry , Malaria/genetics , Malaria/parasitology , Mutagenesis, Site-Directed , Mutation/genetics , Oxidative Stress/drug effects , Oxidoreductases/metabolism , Plasmodium falciparum/pathogenicity , Protozoan Proteins/chemistry , Rabbits , Stereoisomerism , Thioredoxins/chemistry , Thioredoxins/metabolism
18.
Free Radic Biol Med ; 49(2): 258-67, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20406680

ABSTRACT

Nonsteroidal anti-inflammatory drug (NSAID)-induced oxidative stress plays a critical role in gastric mucosal cell apoptosis and gastropathy. NSAIDs induce the generation of hydroxyl radical ((*)OH) through the release of free iron, which plays an important role in developing gastropathy. Thus, molecules having both iron-chelating and antiapoptotic properties will be beneficial in preventing NSAID-induced gastropathy. Gallic acid (GA), a polyphenolic natural product, has the capacity to chelate free iron. Here, we report that GA significantly prevents, as well as heals, NSAID-induced gastropathy. In vivo, GA blocks NSAID-mediated mitochondrial oxidative stress by preventing mitochondrial protein carbonyl formation, lipid peroxidation, and thiol depletion. In vitro, GA scavenges free radicals and blocks (*)OH-mediated oxidative damage. GA also attenuates gastric mucosal cell apoptosis in vivo as well as in vitro in cultured gastric mucosal cells as evident from the TUNEL assay. GA prevents NSAID-induced activation of caspase-9, a marker for the mitochondrial pathway of apoptosis, and restores NSAID-mediated collapse of the mitochondrial transmembrane potential and dehydrogenase activity. Thus, the inhibition of mitochondrial oxidative stress by GA is associated with the inhibition of NSAID-induced mitochondrial dysfunction and activation of apoptosis in gastric mucosal cells, which are responsible for gastric injury or gastropathy.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Apoptosis , Gallic Acid/pharmacology , Iron Chelating Agents/pharmacology , Mitochondria/drug effects , Stomach Diseases/drug therapy , Animals , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Apoptosis/drug effects , Caspase 9/genetics , Caspase 9/metabolism , Cells, Cultured , Enzyme Activation/drug effects , Gallic Acid/administration & dosage , Gastric Mucosa/drug effects , Gastric Mucosa/metabolism , Gastric Mucosa/pathology , Iron Chelating Agents/administration & dosage , Membrane Potential, Mitochondrial/drug effects , Mitochondria/metabolism , Protein Carbonylation/drug effects , Rats , Rats, Sprague-Dawley , Stomach Diseases/chemically induced , Stomach Diseases/pathology , Stomach Diseases/physiopathology
19.
Expert Rev Clin Pharmacol ; 2(5): 469-89, 2009 Sep.
Article in English | MEDLINE | ID: mdl-22112223

ABSTRACT

Malaria is a major global threat, that results in more than 2 million deaths each year. The treatment of malaria is becoming extremely difficult due to the emergence of drug-resistant parasites, the absence of an effective vaccine, and the spread of insecticide-resistant vectors. Thus, malarial therapy needs new chemotherapeutic approaches leading to the search for new drug targets. Here, we discuss different approaches to identifying novel antimalarial drug targets. We have also given due attention to the existing validated targets with a view to develop novel, rationally designed lead molecules. Some of the important parasite proteins are claimed to be the targets; however, further in vitro or in vivo structure-function studies of such proteins are crucial to validate these proteins as suitable targets. The interactome analysis among apicoplast, mitochondrion and genomic DNA will also be useful in identifying vital pathways or proteins regulating critical pathways for parasite growth and survival, and could be attractive targets. Molecules responsible for parasite invasion to host erythrocytes and ion channels of infected erythrocytes, essential for intra-erythrocyte survival and stage progression of parasites are also becoming attractive targets. This review will discuss and highlight the current understanding regarding the potential antimalarial drug targets, which could be utilized to develop novel antimalarials.

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