Characterization of the phospholipidogenic potential of 4(1H)-pyridone antimalarial derivatives

Almela, María Jesús; Torres, P.A.; Lozano, Sonia; Herreros, Esperanza

doi:10.1016/j.tiv.2009.06.015

Cited by 4 publications

(2 citation statements)

References 19 publications

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“…In vitro screens with a high‐throughput for the detection of phospholipidosis are highly desirable to screen drugs for their phospholipidosis inducing potential. Several methods for the detection of phospholipids have been reported (29–37). They are summarized in Table 1.…”

Section: Staining Agents For the Detection Of Neutral Lipid Droplets mentioning

confidence: 99%

Fluorescent high‐content imaging allows the discrimination and quantitation of E‐LDL‐induced lipid droplets and Ox‐LDL‐generated phospholipidosis in human macrophages

Grandl

Schmitz

2009

Cytometry Pt A

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Macrophage foam cells formed during uptake of atherogenic lipoproteins are a hallmark of atherosclerotic lesion development. In this study, human macrophages were incubated with two prototypic atherogenic LDL modifications enzymatically degraded LDL (E-LDL) and oxidized LDL (Ox-LDL) prepared from the same donor LDL. To detect differences in macrophage lipid storage, fluorescent high-content imaging was used. Lipid droplets were stained using Bodipy 493/503, and the fluorescent phospholipid probe NBD-PE was used to detect endolysosomal phospholipidosis in high-content imaging assays. The phospholipidosis assay was validated using phospholipidosis-inducing cationic amphiphilic drugs. In addition, neutral lipids and phospholipidosis were determined using LipidTOX. Images of 96-well cell culture microtiter plates were captured with multichannel laser-based high-content confocal microscopy, and subsequently cell-and well-based data were analyzed. E-LDL-loaded macrophages show increased intensity of Bodipy 493/503 and LipidTOX TM -Green neutral lipid droplet staining and a greater mean area and number of lipid droplets per cell compared to Ox-LDL-loaded and M-CSF-differentiated control macrophages. In contrast, Ox-LDLloaded macrophages show increased intensity of NBD-PE and LipidTOX TM -Red detectable phospholipidosis in the endolysosomal compartment compared to E-LDL-loaded and M-CSF-differentiated macrophages. Treatment with the peroxisome proliferatoractivated receptor-c agonist pioglitazone leads to lipid droplet induction depending on the lipid loading state of the macrophages. These results indicate that E-LDL preferentially induces lipid droplets, while Ox-LDL provokes endolysosomal phospholipidosis in human macrophages representing two different lipid storage principles. Therefore, fluorescent high-content imaging is a useful tool to discriminate between and quantify lipid storage compartments in macrophages also in response to drugs affecting cellular lipid metabolism. ' International Society for Advancement of CytometryKey terms E-LDL; Ox-LDL; human macrophages; lipid droplets; phospholipidosis MONOCYTE derived macrophages and lipid-loaded macrophage foam cells play a central role in the initiation and progression of atherosclerotic lesions. Human blood monocytes are a disease relevant tool to unravel lipid storage and release mechanisms, and differential processing of atherogenic lipoproteins like enzymatically modified LDL (E-LDL) or oxidized LDL (Ox-LDL). In macrophages treated in vitro with Ac-LDL or E-LDL, much of the internalized cholesterol accumulates in cytoplasmic cholesterylester containing lipid droplets (1,2) as is the case also in atherosclerotic lesions (3,4). Lipid droplets form the major lipid store in mammalian cells and serve as a reservoir of cholesterol and acyl-glycerols for the synthesis and maintenance of membranes. Lipid droplets consist of a core of neutral lipids, predominantly triacylglycerols or cholesterylesters, that are surrounded by a monolayer of phospholipids

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Section: Staining Agents For the Detection Of Neutral Lipid Droplets mentioning

confidence: 99%

Fluorescent high‐content imaging allows the discrimination and quantitation of E‐LDL‐induced lipid droplets and Ox‐LDL‐generated phospholipidosis in human macrophages

Grandl

Schmitz

2009

Cytometry Pt A

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“…Next to the failure to cross the plasma membrane, lack of activity may also be due to interactions with membranes, sequestration in vesicular compartments, binding to DNA in the cell nucleus and binding to cytoskeletal structures and other proteins. For example, numerous cationic amphiphilic drugs are enriched in the lysosomal compartment (Bareford and Swaan, 2007; Almela et al ., 2009).…”

Section: Introductionmentioning

confidence: 99%

Simultaneous detection of intracellular target and off‐target binding of small molecule cancer drugs at nanomolar concentrations

Glauner

Ruttekolk

Hansen³

et al. 2010

British J Pharmacology

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Background and purpose:In vitro assays that determine activities of drug candidates with isolated targets have only limited predictive value for activities in cellular assays. Poor membrane permeability and off-target binding are major reasons for such discrepancies. However, it still difficult to directly analyse off-target binding at the same time as target binding, on a subcellular level. Here, we present a combination of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) as a solution to this problem. Experimental approach: The well-established dihydrofolate reductase inhibitor methotrexate and the kinase inhibitors PD173956 and purvalanol B were conjugated via polyethylene glycol linkers with the fluorophore Cy5. The cellular uptake and subcellular distribution of these compounds in single human cancer-derived cells were investigated by confocal laser scanning microscopy. In addition, molecular interactions inside the cell with the respective target proteins and off-target binding were detected simultaneously in the nanomolar range by FCCS and FCS, respectively, using cells expressing green fluorescent protein fusion proteins of dihydrofolate reductase and Abelson kinase 1. Key results: Large differences in the interaction patterns were found for these compounds. For methotrexate-Cy5, drug-target interactions could be detected and dissociation constants determined. In contrast, PD173956-Cy5 showed strong interactions with intracellular high-molecular weight structures, other than its target. Conclusions and implications:The combination of FCS and FCCS provides a powerful means to assess subcellular pharmacokinetics and dynamics of drug candidates at nanomolar concentrations.British Journal of Pharmacology (2010) 160, 958-970; doi:10.1111/j.1476-5381.2010.00732.x Keywords: fluorescence correlation spectroscopy; fluorescence cross-correlation spectroscopy; anti-cancer drug; cellular pharmacology; drug-target interaction; drug development Abbreviations: Abl 1, Abelson kinase 1; cpm, counts per molecule; DHFR, dihydrofolate reductase; FCS, fluorescence correlation spectroscopy; FCCS, fluorescence cross-correlation spectroscopy; FRET, fluorescence resonance energy transfer; Hek, human embryonic kidney; MTX, methotrexate; RP-HPLC, reversed-phase high-performance liquid chromatography; PBS, phosphate-buffered saline; PD, Park Davis; PEG, polyethylene glycol; Pur, purvalanol; RT, room temperature IntroductionCellular assays are gaining significance in drug development (Perlman et al., 2004;Lang et al., 2006;Korn and Krausz, 2007). While providing less information on the interaction of a drug candidate towards a specific molecular target, such assays reveal off-target effects and cytotoxicity. In addition, many compounds fail to show any biological effect, even if activity was detected towards an isolated target molecule in an in vitro screen.For compounds that exert their activity inside the cell, such failure is typically attributed to poor cellular uptake. Howeve...

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Palladium‐Catalyzed Novel C(formyl)‐C(aryl)/C(aryl)‐N(amine) Coupling Sequence Between 2‐(Methylamino)nicotinaldehyde and 3‐Bromo‐2‐chlorothiophene: An Efficient Construction of 4‐Pyridone Fused Hybrid Scaffolds

2021

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Characterization of the phospholipidogenic potential of 4(1H)-pyridone antimalarial derivatives

Cited by 4 publications

References 19 publications

Fluorescent high‐content imaging allows the discrimination and quantitation of E‐LDL‐induced lipid droplets and Ox‐LDL‐generated phospholipidosis in human macrophages

Fluorescent high‐content imaging allows the discrimination and quantitation of E‐LDL‐induced lipid droplets and Ox‐LDL‐generated phospholipidosis in human macrophages

Simultaneous detection of intracellular target and off‐target binding of small molecule cancer drugs at nanomolar concentrations

Palladium‐Catalyzed Novel C(formyl)‐C(aryl)/C(aryl)‐N(amine) Coupling Sequence Between 2‐(Methylamino)nicotinaldehyde and 3‐Bromo‐2‐chlorothiophene: An Efficient Construction of 4‐Pyridone Fused Hybrid Scaffolds

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