scholarly journals A ReplicativeIn VitroAssay for Drug Discovery against Leishmania donovani

2016 ◽  
Vol 60 (6) ◽  
pp. 3524-3532 ◽  
Author(s):  
Diana Tegazzini ◽  
Rosario Díaz ◽  
Fernando Aguilar ◽  
Imanol Peña ◽  
Jesús L. Presa ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Leishmania Donovani ◽  
Horse Serum ◽  
Protozoan Parasite ◽  
Infection Process ◽  
Infection Model ◽  
Content Type ◽  
Fetal Bovine

The protozoan parasiteLeishmania donovaniis the causative agent of visceral leishmaniasis, a disease potentially fatal if not treated. Current available treatments have major limitations, and new and safer drugs are urgently needed. In recent years, advances in high-throughput screening technologies have enabled the screening of millions of compounds to identify new antileishmanial agents. However, most of the compounds identifiedin vitrodid not translate their activities when tested inin vivomodels, highlighting the need to develop more predictivein vitroassays. In the present work, we describe the development of a robust replicative, high-content,in vitrointracellularL. donovaniassay. Horse serum was included in the assay media to replace standard fetal bovine serum, to completely eliminate the extracellular parasites derived from the infection process. A novel phenotypicin vitroinfection model has been developed, complemented with the identification of the proliferation of intracellular amastigotes measured by EdU incorporation.In vitroandin vivoresults for miltefosine, amphotericin B, and the selected compound 1 have been included to validate the assay.

scholarly journals Leishmania donovani Parasites Are Inhibited by the Benzoxaborole AN2690 Targeting Leucyl-tRNA Synthetase

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Reetika Manhas ◽  
Smriti Tandon ◽  
Shib Sankar Sen ◽  
Neha Tiwari ◽  
Manoj Munde ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Drug Targets ◽  
Fungal Pathogens ◽  
Leishmania Donovani ◽  
Protozoan Parasite ◽  
Gene Replacement ◽  
Trna Synthetase ◽  
Content Type

ABSTRACT Visceral leishmaniasis is an important public health threat in parts of India. It is caused by a protozoan parasite, Leishmania donovani. Currently available drugs manifest severe side effects. Hence, there is a need to identify new drug targets and drugs. Aminoacyl-tRNA synthetases, required for protein synthesis, are known drug targets for bacterial and fungal pathogens. The aim of the present study was to obtain essentiality data for Leishmania donovani leucyl-tRNA synthetase (LdLRS) by gene replacement. Gene replacement studies indicate that this enzyme plays an essential role in the viability of this pathogenic organism and appears to be indispensable for its survival in vitro. The heterozygous mutant parasites demonstrated a growth deficit and reduced infectivity in mouse macrophages compared to the wild-type cells. We also report that Leishmania donovani recombinant LRS displayed aminoacylation activity and that the protein localized to both the cytosol and the mitochondrion. A broad-spectrum antifungal, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), was found to inhibit parasite growth in both the promastigote and amastigote stages in vitro as well as in vivo in BALB/c mice. This compound exhibited low toxicity to mammalian cells. AN2690 was effective in inhibiting the aminoacylation activity of the recombinant LdLRS. We provide preliminary chemical validation of LdLRS as a drug target by showing that AN2690 is an inhibitor both of L. donovani LRS and of L. donovani cell growth.

scholarly journals In VivoEfficacy of Anuran Trypsin Inhibitory Peptides against Staphylococcal Skin Infection and the Impact of Peptide Cyclization

2015 ◽  
Vol 59 (4) ◽  
pp. 2113-2121 ◽  
Author(s):  
U. Malik ◽  
O. N. Silva ◽  
I. C. M. Fensterseifer ◽  
L. Y. Chan ◽  
R. J. Clark ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Cyclic Peptide ◽  
Sequence Similarity ◽  
Infection Model ◽  
Content Type ◽  
Wide Range ◽  
Inhibitory Activities ◽  
The Impact

ABSTRACTStaphylococcus aureusis a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weakin vitroinhibitory activities againstS. aureus, but several had strong antibacterial activities againstS. aureusin anin vivomurine wound infection model. pYR, an immunomodulatory peptide fromRana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg−1. Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

scholarly journals Reversal of Azole Resistance inCandida albicansby Sulfa Antibacterial Drugs

2017 ◽  
Vol 62 (3) ◽  
Author(s):  
Hassan E. Eldesouky ◽  
Abdelrahman Mayhoub ◽  
Tony R. Hazbun ◽  
Mohamed N. Seleem
Keyword(s):  
Treatment Options ◽  
Antifungal Drugs ◽  
Infection Model ◽  
Azole Resistance ◽  
Global Public Health ◽  
Sulfa Drugs ◽  
Antibacterial Drugs ◽  
Content Type

ABSTRACTInvasive candidiasis presents an emerging global public health challenge due to the emergence of resistance to the frontline treatment options, such as fluconazole. Hence, the identification of other compounds capable of pairing with fluconazole and averting azole resistance would potentially prolong the clinical utility of this important group. In an effort to repurpose drugs in the field of antifungal drug discovery, we explored sulfa antibacterial drugs for the purpose of reversing azole resistance inCandida. In this study, we assembled and investigated a library of 21 sulfa antibacterial drugs for their ability to restore fluconazole sensitivity inCandida albicans. Surprisingly, the majority of assayed sulfa drugs (15 of 21) were found to exhibit synergistic relationships with fluconazole by checkerboard assay with fractional inhibitory concentration index (ΣFIC) values ranging from <0.0312 to 0.25. Remarkably, five sulfa drugs were able to reverse azole resistance in a clinically achievable range. The structure-activity relationships (SARs) of the amino benzene sulfonamide scaffold as antifungal agents were studied. We also identified the possible mechanism of the synergistic interaction of sulfa antibacterial drugs with azole antifungal drugs. Furthermore, the ability of sulfa antibacterial drugs to inhibitCandidabiofilm by 40%in vitrowas confirmed. In addition, the effects of sulfa-fluconazole combinations onCandidagrowth kinetics and efflux machinery were explored. Finally, using aCaenorhabditis elegansinfection model, we demonstrated that the sulfa-fluconazole combination does possess potent antifungal activityin vivo, reducingCandidain infected worms by ∼50% compared to the control.

scholarly journals Role of BrnQ1 and BrnQ2 in Branched-Chain Amino Acid Transport and Virulence in Staphylococcus aureus

2014 ◽  
Vol 83 (3) ◽  
pp. 1019-1029 ◽  
Author(s):  
Julienne C. Kaiser ◽  
Sameha Omer ◽  
Jessica R. Sheldon ◽  
Ian Welch ◽  
David E. Heinrichs
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Gene ◽  
Defined Medium ◽  
Infection Model ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Branched Chain Amino Acid ◽  
Branched Chain

The branched-chain amino acids (BCAAs; Ile, Leu, and Val) not only are important nutrients for the growth ofStaphylococcus aureusbut also are corepressors for CodY, which regulates virulence gene expression, implicating BCAAs as an important link between the metabolic state of the cell and virulence. BCAAs are either synthesized intracellularly or acquired from the environment.S. aureusencodes three putative BCAA transporters, designated BrnQ1, BrnQ2, and BrnQ3; their functions have not yet been formally tested. In this study, we mutated all threebrnQparalogs so as to characterize their substrate specificities and their roles in growthin vitroandin vivo. We demonstrated that in the community-associated, methicillin-resistantS. aureus(CA-MRSA) strain USA300, BrnQ1 is involved in uptake of all three BCAAs, BrnQ2 transports Ile, and BrnQ3 does not have a significant role in BCAA transport under the conditions tested. Of the three, only BrnQ1 is essential for USA300 to grow in a chemically defined medium that is limited for Leu or Val. Interestingly, we observed that abrnQ2mutant grew better than USA300 in media limited for Leu and Val, owing to the fact that this mutation leads to overexpression ofbrnQ1. In a murine infection model, thebrnQ1mutant was attenuated, but in contrast,brnQ2mutants had significantly increased virulence compared to that of USA300, a phenotype we suggest is at least partially linked to enhancedin vivoscavenging of Leu and Val through BrnQ1. These data uncover a hitherto-undiscovered connection between nutrient acquisition and virulence in CA-MRSA.

scholarly journals Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
Jonathan L. Portman ◽  
Qiongying Huang ◽  
Michelle L. Reniere ◽  
Anthony T. Iavarone ◽  
Daniel A. Portnoy
Keyword(s):  
Protein Function ◽  
Inhibitory Effect ◽  
Cysteine Residue ◽  
Infection Model ◽  
Listeriolysin O ◽  
Content Type ◽  
Pore Forming Proteins

ABSTRACT Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogen Listeria monocytogenes, was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein in vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain of L. monocytogenes expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild type in vitro, yet it was attenuated 4- to 6-fold in a competitive murine infection model in vivo. This study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.

scholarly journals TCP1γ Subunit Is Indispensable for Growth and Infectivity of Leishmania donovani

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Shailendra Yadav ◽  
Jitendra Kuldeep ◽  
Mohammad I. Siddiqi ◽  
Neena Goyal
Keyword(s):  
Cell Cycle Progression ◽  
Leishmania Donovani ◽  
Gene Replacement ◽  
Titration Calorimetry ◽  
Content Type ◽  
T Complex ◽  
Complex Protein ◽  
Oligomeric Complex

ABSTRACT T-complex protein-1 (TCP1) is a ubiquitous group II chaperonin and is known to fold various proteins, such as actin and tubulin. In Leishmania donovani, the γ subunit of TCP1 (LdTCP1γ) has been cloned and characterized. It forms a high-molecular-weight homo-oligomeric complex that performs ATP-dependent protein folding. In the present study, we evaluated the essentiality of the LdTCP1γ gene. Gene replacement studies indicated that LdTCP1γ is essential for parasite survival. The LdTCP1γ single-allele-replacement mutants exhibited slowed growth and decreased infectivity in mouse macrophages compared to the growth and infectivity of the wild-type parasites. Modulation of LdTCP1γ expression in promastigotes also modulated cell cycle progression. Suramin, an antitrypanosomal drug, not only inhibited the luciferase refolding activity of the recombinant LdTCP1γ (rLdTCP1γ) homo-oligomeric complex but also exhibited potential antileishmanial efficacy both in vitro and in vivo. The interaction of suramin and LdTCP1γ was further validated by isothermal titration calorimetry. The study suggests LdTCP1γ as a potential drug target and also provides a framework for the development of a new class of drugs.

scholarly journals In VivoEfficacy of Human Simulated Regimens of Carbapenems and Comparator Agents against NDM-1-Producing Enterobacteriaceae

2013 ◽  
Vol 58 (3) ◽  
pp. 1671-1677 ◽  
Author(s):  
Dora E. Wiskirchen ◽  
Patrice Nordmann ◽  
Jared L. Crandon ◽  
David P. Nicolau
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Similar Efficacy ◽  
Infection Model ◽  
High Dose ◽  
Wild Type ◽  
Prolonged Infusion ◽  
Content Type

ABSTRACTDoripenem and ertapenem have demonstrated efficacy against several NDM-1-producing isolatesin vivo, despite having high MICs. In this study, we sought to further characterize the efficacy profiles of humanized regimens of standard (500 mg given every 8 h) and high-dose, prolonged infusion of doripenem (2 g given every 8 h, 4-h infusion) and 1 g of ertapenem given intravenously every 24 h and the comparator regimens of ceftazidime at 2 g given every 8 h (2-h infusion), levofloxacin at 500 mg every 24 h, and aztreonam at 2 g every 6 h (1-h infusion) against a wider range of isolates in a murine thigh infection model. An isogenic wild-type strain and NDM-1-producingKlebsiella pneumoniaeand eight clinical NDM-1-producing members of the familyEnterobacteriaceaewere tested in immunocompetent- and neutropenic-mouse models. The wild-type strain was susceptible to all of the agents, while the isogenic NDM-1-producing strain was resistant to ceftazidime, doripenem, and ertapenem. Clinical NDM-1-producing strains were resistant to nearly all five of the agents (two were susceptible to levofloxacin). In immunocompetent mice, all of the agents produced ≥1-log10CFU reductions of the isogenic wild-type and NDM-1-producing strains after 24 h. Minimal efficacy of ceftazidime, aztreonam, and levofloxacin against the clinical NDM-1-producing strains was observed. However, despitein vitroresistance, ≥1-log10CFU reductions of six of eight clinical strains were achieved with high-dose, prolonged infusion of doripenem and ertapenem. Slight enhancements of doripenem activity over the standard doses were obtained with high-dose, prolonged infusion for three of the four isolates tested. Similar efficacy observations were noted in neutropenic mice. These data suggest that carbapenems are a viable treatment option for infections caused by NDM-1-producingEnterobacteriaceae.

Leishmania donovani infection of bone marrow stromal macrophages selectively enhances myelopoiesis, by a mechanism involving GM-CSF and TNF-α

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1642-1651 ◽  
Author(s):  
Sara E. J. Cotterell ◽  
Christian R. Engwerda ◽  
Paul M. Kaye
Keyword(s):  
Infectious Disease ◽  
Bone Marrow ◽  
Stromal Cell ◽  
Leishmania Donovani ◽  
Protozoan Parasite ◽  
Gm Csf ◽  
Tnf Α ◽  
Macrophage Colony

Alterations in hematopoiesis are common in experimental infectious disease. However, few studies have addressed the mechanisms underlying changes in hematopoietic function or assessed the direct impact of infectious agents on the cells that regulate these processes. In experimental visceral leishmaniasis, caused by infection with the protozoan parasite Leishmania donovani, parasites persist in the spleen and bone marrow, and their expansion in these sites is associated with increases in local hematopoietic activity. The results of this study show that L donovani targets bone marrow stromal macrophages in vivo and can infect and multiply in stromal cell lines of macrophage, but not other lineages in vitro. Infection of stromal macrophages increases their capacity to support myelopoiesis in vitro, an effect mediated mainly through the induction of granulocyte macrophage-colony stimulating factor and tumor necrosis factor-. These data are the first to directly demonstrate that intracellular parasitism of a stromal cell population may modify its capacity to regulate hematopoiesis during infectious disease.

scholarly journals Tridecaptin M, a New Variant Discovered in Mud Bacterium, Shows Activity against Colistin- and Extremely Drug-ResistantEnterobacteriaceae

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Manoj Jangra ◽  
Manpreet Kaur ◽  
Rushikesh Tambat ◽  
Rohit Rana ◽  
Sushil K. Maurya ◽  
...  
Keyword(s):  
Hemolytic Activity ◽  
World Health ◽  
Fourth Generation ◽  
Infection Model ◽  
Drug Resistant ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
New Variant

ABSTRACTThe World Health Organization has categorized the Gram-negative superbugs, which are inherently impervious to many antibiotics, as critical priority pathogens due to the lack of effective treatments. The breach in our last-resort antibiotic (i.e., colistin) by extensively drug-resistant and pan-drug-resistantEnterobacteriaceaestrains demands the immediate development of new therapies. In the present study, we report the discovery of tridecaptin M, a new addition to the family, and its potential against colistin-resistantEnterobacteriaceae in vitroandin vivo. Also, we performed mode-of-action studies using various fluorescent probes and studied the hemolytic activity and mammalian cytotoxicity in two cell lines. Tridecaptin M displayed strong antibacterial activity (MICs of 2 to 8 μg ml−1) against clinical strains ofKlebsiella pneumoniae(which were resistant to colistin, carbapenems, third- and fourth-generation cephalosporins, fluoroquinolones, fosfomycin, and other antibiotics) andmcr-1-positiveEscherichia colistrains. Unlike polymyxins, tridecaptin M did not permeabilize the outer membrane or cytoplasmic membrane. It blocked ATP synthesis in bacteria by dissipating the proton motive force. The compound exhibited negligible acquired resistance, lowin vitrocytotoxicity and hemolytic activity, and no significant acute toxicity in mice. It also showed promising efficacy in a thigh infection model of colistin-resistantK. pneumoniae. Altogether, these results demonstrate the future prospects of this class of antibiotics to address the unmet medical need to circumvent colistin resistance in extensively drug-resistantEnterobacteriaceaeinfections. The work also emphasizes the importance of natural products in our shrunken drug discovery pipeline.

scholarly journals Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway

2020 ◽  
Vol 64 (3) ◽  
Author(s):  
Jixu Li ◽  
Huanping Guo ◽  
Eloiza May Galon ◽  
Yang Gao ◽  
Seung-Hun Lee ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Drug Targets ◽  
Protozoan Parasite ◽  
Cell Types ◽  
Lytic Cycle ◽  
Type I ◽  
Content Type ◽  
Mechanism Of Inhibition

ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum. Therefore, AATs are suggested as drug targets against Plasmodium. The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondii in vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo. Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro. However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro. HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

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