Differential cytotoxicity of iron chelators on malaria-infected cells versus mammalian cells

Iron chelators of the hydroxamate class arrest in vitro proliferation of malaria parasites end of mammalian cells. The factors determining the biological activity of the chelators have classically been attributed to the chelators' capacity for binding iron and to their ability to traverse membranes as free chelators and as chelator-iron complexes. We show in this work that the nature of the chelatable pool of cell iron also contributes to the susceptibility of cells to iron chelators. A class of N-terminal (Nt derivatives of desferrioxamine (DFO), (Nt-DFO), is shown here to differentially affect growth and replication of intraerythrocytic parasites (Plasmodium falciparum). Methyl-anthranilic DFO (MADFO), the relatively less hydrophilic member of the Nt-DFOs series, reduced parasite proliferation (48 hour test) with an IC50 of 4 +/- 1 micromol/L and mammalian cell (K562 and HepG2) proliferation with an IC50 > 100 micromol/L. On the other hand, the more hydrophilic Nt-free DFO, displayed IC50 values of 21 +/- 5 micromol/L for parasites and 7 +/- 1 micromol/L for mammalian cells. The selective antiparasitic activity of MA-DFO, as reflected in the speed of action and IC50 values on cell proliferation, is attributed primarily to membrane permeation and iron (III) binding properties of the drug. In contrast, the relatively low antiproliferative activity of the more permeant MA-DFO on mammalian cells, resulted from MA-DFO's reduced capacity for scavenging intracellular iron. This is apparent from MA-DFO reduced effects on: (1) the chelatable iron (II) pool that is associated with the cell cytosol; (2) the cell chelator-extractable iron, and (3) cell ferritin levels. The potent antimalarial efficacy and biological selectivity of MA-DFO relative to the parent DFO, is of importance for improved design of chemotherapeutic agents.

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Discovery of 3-Cinnamamido-N-Substituted Benzamides as Potential Antimalarial Agents

Medicinal Chemistry ◽

10.2174/1573406416666200817160708 ◽

2020 ◽

Vol 16 ◽

Author(s):

Haicheng Liu ◽

Yushi Futamura ◽

Honghai Wu ◽

Aki Ishiyama ◽

Taotao Zhang ◽

...

Keyword(s):

Mammalian Cells ◽

Antimalarial Activity ◽

In Vitro Assays ◽

Compound Library ◽

Antimalarial Agents ◽

Phenotypic Screen ◽

Ic50 Values ◽

Substituted Benzamides

Background: Malaria is one of the most devastating parasitic diseases, yet the discovery of antimalarial agents remains profoundly challenging. Very few new antimalarials have been developed in the past 50 years, while the emergence of drug-resistance continues to appear. Objective: This study focuses on the discovery, design, synthesis, and antimalarial evaluation of 3-cinnamamido-N-substituted benzamides. Method: In this study, a screening of our compound library was carried out against the multidrug-sensitive Plasmodium falciparum 3D7 strain. Derivatives of the hit were designed, synthesized and tested against P. falciparum 3D7 and the in vivo antimalarial activity of the most active compounds was evaluated using the method of Peters’ 4-day suppressive test. Results: The retrieved hit compound 1 containing a 3-cinnamamido-N-substituted benzamide skeleton showed moderate antimalarial activity (IC50 = 1.20 µM) for the first time. A series of derivatives were then synthesized through a simple four-step workflow, and half of them exhibited slightly better antimalarial effect than the precursor 1 during the subsequent in vitro assays. Additionally, compounds 11, 23, 30 and 31 displayed potent activity with IC50 values of approximately 0.1 µM, and weak cytotoxicity against mammalian cells. However, in vivo antimalarial activity is not effective which might be ascribed to the poor solubility of these compounds. Conclusion: In this study, phenotypic screen of our compound library resulted in the first report of 3-cinnamamide framework with antimalarial activity and 40 derivatives were then designed and synthesized. Subsequent structure-activity studies showed that compounds 11, 23, 30 and 31 exhibited the most potent and selective activity against P. falciparum 3D7 strain with IC50 values around 0.1 µM. Our work herein sets another example of phenotypic screen-based drug discovery, leading to potentially promising candidates of novel antimalarial agents once given further optimization.

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Characterization and selective inhibition of myristoyl-CoA:protein N-myristoyltransferase from Trypanosoma brucei and Leishmania major

Biochemical Journal ◽

10.1042/bj20051886 ◽

2006 ◽

Vol 396 (2) ◽

pp. 277-285 ◽

Cited By ~ 46

Author(s):

Chrysoula Panethymitaki ◽

Paul W. Bowyer ◽

Helen P. Price ◽

Robin J. Leatherbarrow ◽

Katherine A. Brown ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Leishmania Major ◽

Homo Sapiens ◽

Selective Inhibition ◽

Fungal Species ◽

Chemotherapeutic Agents ◽

Human Pathogens ◽

Ic50 Values

The eukaryotic enzyme NMT (myristoyl-CoA:protein N-myristoyltransferase) has been characterized in a range of species from Saccharomyces cerevisiae to Homo sapiens. NMT is essential for viability in a number of human pathogens, including the fungi Candida albicans and Cryptococcus neoformans, and the parasitic protozoa Leishmania major and Trypanosoma brucei. We have purified the Leishmania and T. brucei NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and specific peptide substrates. A number of inhibitory compounds that target NMT in fungal species have been tested against the parasite enzymes in vitro and against live parasites in vivo. Two of these compounds inhibit TbNMT with IC50 values of <1 μM and are also active against mammalian parasite stages, with ED50 (the effective dose that allows 50% cell growth) values of 16–66 μM and low toxicity to murine macrophages. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against infectious diseases including African sleeping sickness and Nagana.

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Mode of action of iron (III) chelators as antimalarials: II. Evidence for differential effects on parasite iron-dependent nucleic acid synthesis

Blood ◽

10.1182/blood.v84.3.910.910 ◽

1994 ◽

Vol 84 (3) ◽

pp. 910-915 ◽

Cited By ~ 22

Author(s):

SD Lytton ◽

B Mester ◽

J Libman ◽

A Shanzer ◽

ZI Cabantchik

Keyword(s):

Drug Treatment ◽

Dna Synthesis ◽

Mammalian Cells ◽

Iron Chelation ◽

Acid Synthesis ◽

Iron Chelators ◽

Intracellular Iron ◽

Trophozoite Stage ◽

Drug Removal ◽

Irreversible Effects

Abstract Iron chelation treatment of red blood cells infected with Plasmodium falciparum selectively intervenes with iron-dependent metabolism of malaria parasites and inhibits their development. Highly permeant hydroxamate iron chelator RSFileum2 affects all parasite stages when cultures are continuously exposed to drug, but affects primarily ring stages when assessed for irreversible effects, ie, sustained inhibition remaining after drug removal. On the other hand, the hydrophilic and poorly permeant desferrioxamine (DFO) affects primarily trophozoite/schizont stages when tested either in the continuous mode or irreversible mode. Unlike parasites, mammalian cells subjected to similar drug treatment show complete growth recovery once drugs are removed. Our studies indicate that parasites display a limited capacity to recover from intracellular iron depletion evoked by iron chelators. Based on these findings we provide a working model in which the irreversible effects of RSFs on rings are explained by the absence of pathways for iron acquisition/utilization by early forms of parasites. Trophozoite/schizonts can partially recover from RSFileum2 treatments, but show no DNA synthesis following DFO treatment even after drug removal and iron replenishment by permeant iron carriers. At trophozoite stage, the parasite uses a limited pathway for refurnishing its iron-containing enzymes, thus overcoming iron deprivation caused by permeant RSFileum2, but not by DFO because this latter drug is not easily removable from parasites. Their DNA synthesis is blocked by the hydroxamate iron chelators probably by affecting synthesis of ribonucleotide reductase (RNRase). Presumably in parasites, prolonged repression of the enzyme leads also to irreversible loss of activity. The action profiles of RSFileum2 and DFO presented in this study have implications for improved chemotherapeutic performance by combined drug treatment and future drug design based on specific intervention at parasite DNA synthesis.

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Zinc(II)-Dipicolylamine Coordination Complexes as Targeting and Chemotherapeutic Agents for Leishmania major

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00410-16 ◽

2016 ◽

Vol 60 (5) ◽

pp. 2932-2940 ◽

Cited By ~ 14

Author(s):

Douglas R. Rice ◽

Paola Vacchina ◽

Brianna Norris-Mullins ◽

Miguel A. Morales ◽

Bradley D. Smith

Keyword(s):

Cutaneous Leishmaniasis ◽

Mammalian Cells ◽

Leishmania Major ◽

Parasite Burden ◽

Coordination Complexes ◽

Chemotherapeutic Agents ◽

Selective Toxicity ◽

Content Type

ABSTRACTCutaneous leishmaniasis is a neglected tropical disease that causes painful lesions and severe disfigurement. Modern treatment relies on a few chemotherapeutics with serious limitations, and there is a need for more effective alternatives. This study describes the selective targeting of zinc(II)-dipicolylamine (ZnDPA) coordination complexes towardLeishmania major, one of the species responsible for cutaneous leishmaniasis. Fluorescence microscopy ofL. majorpromastigotes treated with a fluorescently labeled ZnDPA probe indicated rapid accumulation of the probe within the axenic promastigote cytosol. The antileishmanial activities of eight ZnDPA complexes were measured using anin vitroassay. All tested complexes exhibited selective toxicity againstL. majoraxenic promastigotes, with 50% effective concentration values in the range of 12.7 to 0.3 μM. Similar toxicity was observed against intracellular amastigotes, but there was almost no effect on the viability of mammalian cells, including mouse peritoneal macrophages.In vivotreatment efficacy studies used fluorescence imaging to noninvasively monitor changes in the red fluorescence produced by an infection of mCherry-L. majorin a mouse model. A ZnDPA treatment regimen reduced the parasite burden nearly as well as the reference care agent, potassium antimony(III) tartrate, and with less necrosis in the local host tissue. The results demonstrate that ZnDPA coordination complexes are a promising new class of antileishmanial agents with potential for clinical translation.

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A subpopulation of arenavirus nucleoprotein localizes to mitochondria

Scientific Reports ◽

10.1038/s41598-021-99887-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Francesca Baggio ◽

Udo Hetzel ◽

Lisbeth Nufer ◽

Anja Kipar ◽

Jussi Hepojoki

Keyword(s):

Mammalian Cells ◽

Cytoplasmic Inclusion ◽

Mitochondrial Morphology ◽

Cellular Functions ◽

Cellular Defense ◽

Mitochondrial Functions ◽

Infected Cells ◽

Cytoplasmic Inclusion Bodies ◽

Targeting Signal

AbstractViruses need cells for their replication and, therefore, ways to hijack cellular functions. Mitochondria play fundamental roles within the cell in metabolism, immunity and regulation of homeostasis due to which some viruses aim to alter mitochondrial functions. Herein we show that the nucleoprotein (NP) of arenaviruses enters the mitochondria of infected cells, affecting the mitochondrial morphology. Reptarenaviruses cause boid inclusion body disease (BIBD) that is characterized, especially in boas, by the formation of cytoplasmic inclusion bodies (IBs) comprising reptarenavirus NP within the infected cells. We initiated this study after observing electron-dense material reminiscent of IBs within the mitochondria of reptarenavirus infected boid cell cultures in an ultrastructural study. We employed immuno-electron microscopy to confirm that the mitochondrial inclusions indeed contain reptarenavirus NP. Mutations to a putative N-terminal mitochondrial targeting signal (MTS), identified via software predictions in both mamm- and reptarenavirus NPs, did not affect the mitochondrial localization of NP, suggesting that it occurs independently of MTS. In support of MTS-independent translocation, we did not detect cleavage of the putative MTSs of arenavirus NPs in reptilian or mammalian cells. Furthermore, in vitro translated NPs could not enter isolated mitochondria, suggesting that the translocation requires cellular factors or conditions. Our findings suggest that MTS-independent mitochondrial translocation of NP is a shared feature among arenaviruses. We speculate that by targeting the mitochondria arenaviruses aim to alter mitochondrial metabolism and homeostasis or affect the cellular defense.

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Mode of action of iron (III) chelators as antimalarials: II. Evidence for differential effects on parasite iron-dependent nucleic acid synthesis

Blood ◽

10.1182/blood.v84.3.910.bloodjournal843910 ◽

1994 ◽

Vol 84 (3) ◽

pp. 910-915

Author(s):

SD Lytton ◽

B Mester ◽

J Libman ◽

A Shanzer ◽

ZI Cabantchik

Keyword(s):

Drug Treatment ◽

Dna Synthesis ◽

Mammalian Cells ◽

Iron Chelation ◽

Acid Synthesis ◽

Iron Chelators ◽

Intracellular Iron ◽

Trophozoite Stage ◽

Drug Removal ◽

Irreversible Effects

Iron chelation treatment of red blood cells infected with Plasmodium falciparum selectively intervenes with iron-dependent metabolism of malaria parasites and inhibits their development. Highly permeant hydroxamate iron chelator RSFileum2 affects all parasite stages when cultures are continuously exposed to drug, but affects primarily ring stages when assessed for irreversible effects, ie, sustained inhibition remaining after drug removal. On the other hand, the hydrophilic and poorly permeant desferrioxamine (DFO) affects primarily trophozoite/schizont stages when tested either in the continuous mode or irreversible mode. Unlike parasites, mammalian cells subjected to similar drug treatment show complete growth recovery once drugs are removed. Our studies indicate that parasites display a limited capacity to recover from intracellular iron depletion evoked by iron chelators. Based on these findings we provide a working model in which the irreversible effects of RSFs on rings are explained by the absence of pathways for iron acquisition/utilization by early forms of parasites. Trophozoite/schizonts can partially recover from RSFileum2 treatments, but show no DNA synthesis following DFO treatment even after drug removal and iron replenishment by permeant iron carriers. At trophozoite stage, the parasite uses a limited pathway for refurnishing its iron-containing enzymes, thus overcoming iron deprivation caused by permeant RSFileum2, but not by DFO because this latter drug is not easily removable from parasites. Their DNA synthesis is blocked by the hydroxamate iron chelators probably by affecting synthesis of ribonucleotide reductase (RNRase). Presumably in parasites, prolonged repression of the enzyme leads also to irreversible loss of activity. The action profiles of RSFileum2 and DFO presented in this study have implications for improved chemotherapeutic performance by combined drug treatment and future drug design based on specific intervention at parasite DNA synthesis.

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Ethanolic extract of gabirobeira (Campomanesia adamantium) leaves reduces proliferation of osteosarcoma cells in vitro

Acta Scientiarum Biological Sciences ◽

10.4025/actascibiolsci.v43i1.52783 ◽

2021 ◽

Vol 43 ◽

pp. e52783

Author(s):

Nara Cristina Silva ◽

Leandro Lopes Nepomuceno ◽

Nayane Peixoto Soares ◽

Vanessa de Souza Vieira ◽

Vanessa de Sousa Cruz ◽

...

Keyword(s):

Ethanolic Extract ◽

Metastatic Potential ◽

Chemotherapeutic Agents ◽

In Vitro Cytotoxicity ◽

Osteosarcoma Cells ◽

Trypan Blue Exclusion ◽

Trypan Blue Exclusion Method ◽

Ic50 Value ◽

Ic50 Values

Osteosarcoma is the most commonly diagnosed malignant bone tumor in humans, with a higher incidence in children and young people. It is highly aggressive and has a high metastatic potential. Its treatment is based on both chemotherapy and surgical intervention. However, currently used chemotherapeutic agents, such as doxorubicin, have several adverse effects on the patient. Therefore, there is a growing demand for new chemotherapeutic agents that stimulate new researches, such as those involving compounds extracted from plants, such as the gabirobeira. In this study, we aimed to evaluate the cytotoxic effects of ethanolic extract, both crude and ethyl acetate, of gabirobeira leaves on osteosarcoma cells in vitro. Cytotoxicity was evaluated using the Trypan blue exclusion method and the IC50 values were calculated using the tetrazolium reduction method. The ethanolic extract of gabirobeira leaves showed a cytotoxic effect on osteosarcoma cells in vitro. The group treated with the crude extract at 1. 0μL mL-1 concentration for 48 hours showed higher cytotoxicity and the lowest IC50 value for this extract was found in the 24 to 48 hours interval. The ethanolic extract of gabirobeira leaves is cytotoxic for osteosarcoma cells.

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Apoptotic induction induces Leishmania aethiopica and L. mexicana spreading in terminally differentiated THP-1 cells

Parasitology ◽

10.1017/s0031182017001366 ◽

2017 ◽

Vol 144 (14) ◽

pp. 1912-1921 ◽

Cited By ~ 2

Author(s):

RAJEEV RAI ◽

PAUL DYER ◽

SIMON RICHARDSON ◽

LAURENCE HARBIGE ◽

GIULIA GETTI

Keyword(s):

Mammalian Cells ◽

Molecular Mechanisms ◽

Secondary Infection ◽

Human Macrophages ◽

Leishmania Aethiopica ◽

Reliable Model ◽

Infected Cells ◽

Insight Into ◽

Apoptotic Induction

SummaryLeishmaniasis develops after parasites establish themselves as amastigotes inside mammalian cells and start replicating. As relatively few parasites survive the innate immune defence, intracellular amastigotes spreading towards uninfected cells is instrumental to disease progression. Nevertheless the mechanism of Leishmania dissemination remains unclear, mostly due to the lack of a reliable model of infection spreading. Here, an in vitro model representing the dissemination of Leishmania amastigotes between human macrophages has been developed. Differentiated THP-1 macrophages were infected with GFP expressing Leishmania aethiopica and Leishmania mexicana. The percentage of infected cells was enriched via camptothecin treatment to achieve 64·1 ± 3% (L. aethiopica) and 92 ± 1·2% (L. mexicana) at 72 h, compared to 35 ± 4·2% (L. aethiopica) and 36·2 ± 2·4% (L. mexicana) in untreated population. Infected cells were co-cultured with a newly differentiated population of THP-1 macrophages. Spreading was detected after 12 h of co-culture. Live cell imaging showed inter-cellular extrusion of L. aethiopica and L. mexicana to recipient cells took place independently of host cell lysis. Establishment of secondary infection from Leishmania infected cells provided an insight into the cellular phenomena of parasite movement between human macrophages. Moreover, it supports further investigation into the molecular mechanisms of parasites spreading, which forms the basis of disease development.

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Quassinoids from the Roots of Eurycoma longifolia and Their Anti-Proliferation Activities

Molecules ◽

10.3390/molecules26195939 ◽

2021 ◽

Vol 26 (19) ◽

pp. 5939

Author(s):

Wei-Qun Yang ◽

Wei Tang ◽

Xiao-Jun Huang ◽

Jian-Guo Song ◽

Yue-Yue Li ◽

...

Keyword(s):

Spectroscopic Analysis ◽

Hydroxyl Group ◽

Human Leukemia ◽

In Vitro Proliferation ◽

X Ray ◽

Eurycoma Longifolia ◽

Chemical Structures ◽

Phytochemical Investigation ◽

Ic50 Values

A phytochemical investigation on the roots of medicinal plant Eurycoma longifolia resulted in the isolation of 10 new highly oxygenated C20 quassinoids longifolactones G‒P (1–10), along with four known ones (11–14). Their chemical structures and absolute configurations were unambiguously elucidated on the basis of comprehensive spectroscopic analysis and X-ray crystallographic data. Notably, compound 1 is a rare pentacyclic C20 quassinoid featuring a densely functionalized 2,5-dioxatricyclo[5.2.2.04,8]undecane core. Compound 4 represents the first example of quassinoids containing a 14,15-epoxy functionality, and 7 features an unusual α-oriented hydroxyl group at C-14. All isolated compounds were evaluated for their anti-proliferation activities on human leukemia cells. Among the isolates, compounds 5, 12, 13, and 14 potently inhibited the in vitro proliferation of K562 and HL-60 cells with IC50 values ranging from 2.90 to 8.20 μM.

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SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA

PLoS Biology ◽

10.1371/journal.pbio.3001425 ◽

2021 ◽

Vol 19 (10) ◽

pp. e3001425

Author(s):

Amanda Jack ◽

Luke S. Ferro ◽

Michael J. Trnka ◽

Eddie Wehri ◽

Amrut Nadgir ◽

...

Keyword(s):

Small Molecules ◽

Mammalian Cells ◽

Underlying Mechanism ◽

Host Cells ◽

Genomic Rna ◽

N Protein ◽

Infected Cells ◽

Rna Genome ◽

Viral Genomic Rna

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (COVID-19), a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry (CLMS) identified a region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. We also identified small molecules that alter the formation of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.

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