scholarly journals Ascorbate Peroxidase, a Key Molecule Regulating Amphotericin B Resistance in Clinical Isolates of Leishmania donovani

2014 ◽  
Vol 58 (10) ◽  
pp. 6172-6184 ◽  
Author(s):  
Ashish Kumar ◽  
Sushmita Das ◽  
Bidyut Purkait ◽  
Abul Hasan Sardar ◽  
Ayan Kumar Ghosh ◽  
...  
Keyword(s):  
Cell Death ◽  
Amphotericin B ◽  
Ascorbate Peroxidase ◽  
Resistant Strain ◽  
Leishmania Donovani ◽  
Cytosolic Calcium ◽  
Resistant Isolate ◽  
Secondary Resistance ◽  
Content Type ◽  
Nuclear Alterations

ABSTRACTAmphotericin B (AmB), a polyene macrolide, is now a first-line treatment of visceral leishmaniasis cases refractory to antimonials in India. AmB relapse cases and the emergence of secondary resistance have now been reported. To understand the mechanism of AmB, differentially expressed genes in AmB resistance strains were identified by a DNA microarray and real-time reverse transcriptase PCR (RT-PCR) approach. Of the many genes functionally overexpressed in the presence of AmB, the ascorbate peroxidase gene from a resistantLeishmania donovanistrain (LdAPx gene) was selected because the gene is present only inLeishmania, not in humans. Apoptosis-like cell death after exposure to AmB was investigated in a wild-type (WT) strain in which the LdAPx gene was overexpressed and in AmB-sensitive and -resistant strains. A higher percentage of apoptosis-like cell death after AmB treatment was noticed in the sensitive strain than in both the resistant isolate and the strain sensitive to LdAPx overexpression. This event is preceded by AmB-induced formation of reactive oxygen species and elevation of the cytosolic calcium level. Enhanced cytosolic calcium was found to be responsible for depolarization of the mitochondrial membrane potential and the release of cytochromec(Cytc) into the cytosol. The redox behavior of Cytcshowed that it has a role in the regulation of apoptosis-like cell death by activating metacaspase- and caspase-like proteins and causing concomitant nuclear alterations, as determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and DNA fragmentation in the resistant strain. The present study suggests that constitutive overexpression of LdAPx in theL. donovaniAmB-resistant strain prevents cells from the deleterious effect of oxidative stress, i.e., mitochondrial dysfunction and cellular death induced by AmB.

scholarly journals In Vitro Evaluation of Antileishmanial Activity of Computationally Screened Compounds against Ascorbate Peroxidase To Combat Amphotericin B Drug Resistance

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Rani Mansuri ◽  
Ashish Kumar ◽  
Sindhuprava Rana ◽  
Bhavana Panthi ◽  
M. Yousuf Ansari ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Ascorbate Peroxidase ◽  
Leishmania Donovani ◽  
Harmful Effect ◽  
Annexin V ◽  
Antileishmanial Activity ◽  
New Drugs ◽  
Content Type ◽  
Lipinski’S Rule

ABSTRACT In visceral leishmaniasis (VL), the host macrophages generate oxidative stress to destroy the pathogen, while Leishmania combats the harmful effect of radicals by redox homeostasis through its unique trypanothione cascade. Leishmania donovani ascorbate peroxidase (LdAPx) is a redox enzyme that regulates the trypanothione cascade and detoxifies the effect of H2O2. The absence of an LdAPx homologue in humans makes it an excellent drug target. In this study, the homology model of LdAPx was built, including heme, and diverse compounds were prefiltered (PAINS, ADMET, and Lipinski's rule of five) and thereafter screened against the LdAPx model. Compounds having good affinity in terms of the Glide XP (extra precision) score were clustered to select diverse compounds for experimental validation. A total of 26 cluster representatives were procured and tested on promastigote culture, yielding 12 compounds with good antileishmanial activity. Out of them, six compounds were safer on the BALB/c peritoneal macrophages and were also effective against disease-causing intracellular amastigotes. Three out of six compounds inhibited recombinant LdAPx in a noncompetitive manner and also demonstrated partial reversion of the resistance property in an amphotericin B (AmB)-resistant strain, which may be due to an increased level of reactive oxygen species (ROS) and decrease of glutathione (GSH) content. However, inhibition of LdAPx in resistant parasites enhanced annexin V staining and activation of metacaspase-like protease activity, which may help in DNA fragmentation and apoptosis-like cell death. Thus, the present study will help in the search for specific hits and templates of potential therapeutic interest and therefore may facilitate the development of new drugs for combination therapy against VL.

scholarly journals Characterization of the Proliferating Cell Nuclear Antigen of Leishmania donovani Clinical Isolates and Its Association with Antimony Resistance

2014 ◽  
Vol 58 (6) ◽  
pp. 2997-3007 ◽  
Author(s):  
Rati Tandon ◽  
Sharat Chandra ◽  
Rajendra Kumar Baharia ◽  
Sanchita Das ◽  
Pragya Misra ◽  
...  
Keyword(s):  
Clinical Isolate ◽  
Proliferating Cell Nuclear Antigen ◽  
Leishmania Donovani ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Nuclear Antigen ◽  
Wild Type ◽  
Content Type ◽  
Sensitive Isolate ◽  
Proliferating Cell

ABSTRACTPreviously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate ofLeishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance inL. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by ≥3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ∼5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate.L. donovaniPCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate ofL. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (SbV) and trivalent antimonial (SbIII) drugs was assessedin vitroagainst promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of SbV(from 41.2 ± 0.6 μg/ml to 66.5 ± 3.9 μg/ml) and SbIII(from 24.0 ± 0.3 μg/ml to 43.4 ± 1.8 μg/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon SbIIItreatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance inL. donovaniclinical isolates, which warrants detailed investigations regarding its mechanism.

scholarly journals Disuccinyl Betulin Triggers Metacaspase-Dependent Endonuclease G-Mediated Cell Death in Unicellular Protozoan Parasite Leishmania donovani

2014 ◽  
Vol 58 (4) ◽  
pp. 2186-2201 ◽  
Author(s):  
Sayan Chowdhury ◽  
Tulika Mukherjee ◽  
Somenath Roy Chowdhury ◽  
Souvik Sengupta ◽  
Sibabrata Mukhopadhyay ◽  
...  
Keyword(s):  
Cell Death ◽  
Leishmania Donovani ◽  
Protozoan Parasite ◽  
Degradation Process ◽  
Dna Lesions ◽  
Death Process ◽  
Unicellular Organism ◽  
Content Type ◽  
Atp Production ◽  
Endonuclease G

ABSTRACTThe unicellular organismLeishmaniaundergoes apoptosis-like cell death in response to external stress or exposure to antileishmanial agents. Here, we showed that 3-O,28-O-disuccinyl betulin (DiSB), a potent topoisomerase type IB inhibitor, induced parasitic cell death by generating oxidative stress. The characteristic feature of the death process resembled the programmed cell death (PCD) seen in higher eukaryotes. In the current study, the generation of reactive oxygen species (ROS), followed by the depolarization of mitochondrial membrane potential (ΔΨm), caused a loss in ATP production inLeishmaniaparasites. This further gave positive feedback to produce a large amount of ROS, which in turn caused oxidative DNA lesions and genomic DNA fragmentation. The treatment of promastigotes with DiSB induced high expression levels of metacaspase protein that led to cell death in this unicellular organism. The PCD was insensitive to benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), suggesting that the death process was not associated with the activation of caspases. DiSB treatment translocatedLeishmania donovaniendonuclease G (LdEndoG) from mitochondria to the nucleus, which was responsible for the DNA degradation process. Conditional antisense knockdown ofL. donovanimetacaspase (LdMC), as well as EndoG, -subverted death of the parasite and rescued cell cycle arrest in G1phase. The present study on the effector molecules associated with the PCD pathway of the parasite should help to manifest the mechanisms of PCD and also might be exploited in antileishmanial chemotherapy.

scholarly journals Perifosine Mechanisms of Action in Leishmania Species

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Atteneri López-Arencibia ◽  
Carmen Martín-Navarro ◽  
Ines Sifaoui ◽  
María Reyes-Batlle ◽  
Carolina Wagner ◽  
...  
Keyword(s):  
Cell Death ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Leishmania Donovani ◽  
Leishmania Species ◽  
Mechanisms Of Action ◽  
Content Type ◽  
Phosphatidylserine Externalization ◽  
Potential Use

ABSTRACT Here the mechanism by which perifosine induced cell death in Leishmania donovani and Leishmania amazonensis is described. The drug reduced Leishmania mitochondrial membrane potential and decreased cellular ATP levels while increasing phosphatidylserine externalization. Perifosine did not increase membrane permeabilization. We also found that the drug inhibited the phosphorylation of Akt in the parasites. These results highlight the potential use of perifosine as an alternative to miltefosine against Leishmania.

scholarly journals Enhanced Efflux Pump Activity in OldCandida glabrataCells

2018 ◽  
Vol 62 (3) ◽  
Author(s):  
Somanon Bhattacharya ◽  
Bettina C. Fries
Keyword(s):  
Amphotericin B ◽  
Abc Transporters ◽  
Resistant Strain ◽  
Candida Glabrata ◽  
Efflux Pump ◽  
Antifungal Resistance ◽  
Replicative Aging ◽  
Glucan Synthase ◽  
Content Type ◽  
Pump Activity

ABSTRACTWe investigated the effect of replicative aging on antifungal resistance inCandida glabrata. Our studies demonstrate significantly increased transcription of ABC transporters and efflux pump activity in old versus youngC. glabratacells of a fluconazole-sensitive and -resistant strain. In addition, higher tolerance to killing by micafungin and amphotericin B was noted and is associated with higher transcription of glucan synthase geneFKS1and lower ergosterol content in older cells.

scholarly journals Immunoadjuvant Chemotherapy of Visceral Leishmaniasis in Hamsters Using Amphotericin B-Encapsulated Nanoemulsion Template-Based Chitosan Nanocapsules

2013 ◽  
Vol 57 (4) ◽  
pp. 1714-1722 ◽  
Author(s):  
Shalini Asthana ◽  
Anil K. Jaiswal ◽  
Pramod K. Gupta ◽  
Vivek K. Pawar ◽  
Anuradha Dube ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Amphotericin B ◽  
Leishmania Donovani ◽  
Transforming Growth Factor ◽  
Treatment Options ◽  
Interleukin 12 ◽  
Necrosis Factor Alpha ◽  
Content Type

ABSTRACTThe accessible treatment options for life-threatening neglected visceral leishmaniasis (VL) disease have problems with efficacy, stability, adverse effects, and cost, making treatment a complex issue. Here we formulated nanometric amphotericin B (AmB)-encapsulated chitosan nanocapsules (CNC-AmB) using a polymer deposition technique mediated by nanoemulsion template fabrication. CNC-AmB exhibited good steric stabilityin vitro, where the chitosan content was found to be efficient at preventing destabilization in the presence of protein and Ca2+. A toxicity study on the model cell line J774A and erythrocytes revealed that CNC-AmB was less toxic than commercialized AmB formulations such as Fungizone and AmBisome. The results ofin vitro(macrophage-amastigote system; 50% inhibitory concentration [IC50], 0.19 ± 0.04 μg AmB/ml) andin vivo(Leishmania donovani-infected hamsters; 86.1% ± 2.08% parasite inhibition) experiments in conjunction with effective internalization by macrophages illustrated the efficacy of CNC-AmB at augmenting antileishmanial properties. Quantitative mRNA analysis by real-time PCR (RT-PCR) showed that the improved effect was synergized with the upregulation of tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and inducible nitric oxide synthase and with the downregulation of transforming growth factor β (TGF-β), IL-10, and IL-4. These research findings suggest that a cost-effective CNC-AmB immunoadjuvant chemotherapeutic delivery system could be a viable alternative to the current high-cost commercial lipid-based formulations.

scholarly journals Mechanism of Amphotericin B Resistance in Clinical Isolates of Leishmania donovani

2011 ◽  
Vol 56 (2) ◽  
pp. 1031-1041 ◽  
Author(s):  
Bidyut Purkait ◽  
Ashish Kumar ◽  
Nilay Nandi ◽  
Abul Hasan Sardar ◽  
Sushmita Das ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Metabolic Pathway ◽  
Resistant Strain ◽  
Leishmania Donovani ◽  
Expression Patterns ◽  
Sensitive Strain ◽  
Clinical Isolates ◽  
Membrane Composition ◽  
Clinical Value ◽  
Resistant Parasite

ABSTRACTThe clinical value of amphotericin B, the mainstay therapy for visceral leishmaniasis in sodium antimony gluconate-nonresponsive zones of Bihar, India, is now threatened by the emergence of acquired drug resistance, and a comprehensive understanding of the underlying mechanisms is the need of the hour. We have selected an amphotericin B-resistant clinical isolate which demonstrated 8-fold-higher 50% lethal doses (LD50) than an amphotericin B-sensitive strain to explore the mechanism of amphotericin B resistance. Fluorimetric analysis demonstrated lower anisotropy in the motion of the diphenylhexatriene fluorescent probe in the resistant strain, which indicated a higher fluidity of the membrane for the resistant strain than for the sensitive strain. The expression patterns of the two transcripts ofS-adenosyl-l-methionine:C-24-Δ-sterol methyltransferase and the absence of ergosterol, replaced by cholesta-5,7,24-trien-3β-ol in the membrane of the resistant parasite, indicate a decreased amphotericin B affinity, which is evidenced by decreased amphotericin B uptake. The expression level of MDR1 is found to be higher in the resistant strain, suggesting a higher rate of efflux of amphotericin B. The resistant parasite also possesses an upregulated tryparedoxin cascade and a more-reduced intracellular thiol level, which helps in better scavenging of reactive oxygen species produced by amphotericin B. The resistance to amphotericin B was partially reverted by the thiol metabolic pathway and ABC transporter inhibitors. Thus, it can be concluded that altered membrane composition, ATP-binding cassette transporters, and an upregulated thiol metabolic pathway have a role in conferring amphotericin B resistance in clinical isolates ofLeishmania donovani.

scholarly journals Antileishmanial Activity, Uptake, and Biodistribution of an Amphotericin B and Poly(α-Glutamic Acid) Complex

2013 ◽  
Vol 57 (10) ◽  
pp. 4608-4614 ◽  
Author(s):  
Abeer H. A. Mohamed-Ahmed ◽  
Karin Seifert ◽  
Vanessa Yardley ◽  
Hollie Burrell-Saward ◽  
Stephen Brocchini ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Amphotericin B ◽  
Leishmania Donovani ◽  
Water Soluble ◽  
Molecular Weight Range ◽  
Acid Complex ◽  
Content Type ◽  
New Treatment

ABSTRACTA noncovalent, water-soluble complex of amphotericin B (AMB) and poly(α-glutamic acid) (PGA), with AMB loadings ranging from 25 to 55% (wt/wt) using PGA with a molecular weight range of 50,000 to 70,000, was prepared as a potential new treatment for visceral leishmaniasis (VL). The AMB-PGA complex was shown to be as active as Fungizone (AMB deoxycholate) against intracellularLeishmania donovaniamastigotes in differentiated THP-1 cells. Thein vitrouptake of the AMB-PGA complex by differentiated THP-1 cells was similar to that of Fungizone and higher than that of AmBisome (liposomal AMB). The AMB-PGA complex also displayed a dose-response profile similar to that of AmBisomein vivoin BALB/c mice againstL. donovani, with 50% effective doses (ED50s) of 0.24 ± 0.03 mg/kg of body weight for the AMB-PGA complex and 0.24 ± 0.06 mg/kg for AmBisome. A biodistribution study with mice indicated that the AMB-PGA complex cleared more rapidly from plasma than AmBisome, with a comparable low level of distribution to the kidneys.

scholarly journals Leishmanicidal Activity of an In Silico-Screened Novel Inhibitor against Ascorbate Peroxidase of Leishmania donovani

2020 ◽  
Vol 64 (7) ◽  
Author(s):  
Mohammad Kashif ◽  
Ankush Paladhi ◽  
Ranjeet Singh ◽  
Sankar Bhattacharyya ◽  
Sumit Kumar Hira ◽  
...  
Keyword(s):  
Ascorbate Peroxidase ◽  
In Silico ◽  
Leishmania Donovani ◽  
Protein Complexes ◽  
Therapeutic Option ◽  
Cellular Functions ◽  
Redox Enzyme ◽  
Content Type ◽  
Aaa Atpase ◽  
Drug Candidates

ABSTRACT Peroxidases are a heterogeneous family of enzymes that have diverse biological functions. Ascorbate peroxidase is a redox enzyme that is reduced by trypanothione, which plays a central role in the redox defense system of Leishmania. In view of developing new and novel therapeutics, we performed in silico studies in order to search for a ligand library and identify new drug candidates and their physiological roles against promastigotes and intracellular amastigotes of Leishmania donovani. Our results demonstrated that the selected inhibitor ZINC96021026 has significant antileishmanial effect and effectively killed both free and intracellular forms of the parasite. ZINC96021026 was found to be identical to ML-240, a selective inhibitor of valosin-containing protein (VCP), or p97, a member of the AAA-ATPase protein family which was derived from the scaffold of N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), targeting the D2-ATPase domain of the enzyme. ZINC96021026 (ML-240) thus has a broad range of cellular functions, thought to be derived from its ability to unfold proteins or disassemble protein complexes, besides inhibiting the ascorbate peroxidase activity. ML-240 may inhibit the parasite’s ascorbate peroxidase, leading to extensive apoptosis and inducing generation of reactive oxygen species. Taken together, our results demonstrated that ML-240 could be an attractive therapeutic option for treatment against leishmaniasis.

scholarly journals Apoptotic Marker Expression in the Absence of Cell Death in Staurosporine-Treated Leishmania donovani

2012 ◽  
Vol 57 (3) ◽  
pp. 1252-1261 ◽  
Author(s):  
Aude L. Foucher ◽  
Najma Rachidi ◽  
Sarah Gharbi ◽  
Thierry Blisnick ◽  
Philippe Bastin ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Kinases ◽  
Leishmania Donovani ◽  
Environmental Changes ◽  
Specific Inhibitor ◽  
Surface Binding ◽  
Casein Kinase 1 ◽  
Content Type ◽  
Apoptotic Marker ◽  
Marker Expression

ABSTRACTThe protozoan parasiteLeishmania donovaniundergoes several developmental transitions in its insect and vertebrate hosts that are induced by environmental changes. The roles of protein kinases in these adaptive differentiation steps and their potential as targets for antiparasitic intervention are only poorly characterized. Here, we used the generic protein kinase inhibitor staurosporine to gain insight into how interference with phosphotransferase activities affects the viability, growth, and motility ofL. donovanipromastigotesin vitro. Unlike the nonkinase drugs miltefosine and amphotericin B, staurosporine strongly reduced parasite biosynthetic activity and had a cytostatic rather than a cytotoxic effect. Despite the induction of a number of classical apoptotic markers, including caspase-like activity and surface binding of annexin V, we determined that, on the basis of cellular integrity, staurosporine did not cause cell death but caused cell cycle arrest and abrogated parasite motility. In contrast, targeted inhibition of the parasite casein kinase 1 (CK1) protein family by use of the CK1-specific inhibitor D4476 resulted in cell death. Thus, pleiotropic inhibition ofL. donovaniprotein kinases and possibly other ATP-binding proteins by staurosporine dissociates apoptotic marker expression from cell death, which underscores the relevance of specific rather than broad kinase inhibitors for antiparasitic drug development.

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