A 96-well flow cytometric screening assay for detecting in vitro phospholipidosis-induction in the drug discovery phase

Mesens, Natalie; Steemans, Margino; Hansen, Poul Erik; Annelieke, Peters; Geert, Verheyen; Vanparys, Philippe

doi:10.1016/j.tiv.2008.11.010

Cited by 31 publications

(7 citation statements)

References 33 publications

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“…[3a], [44] The synthesis of phospholipids that are stably labelled with a fluorescent tag and the development of automated fluorescence readouts enabled the design of a reliable high-throughput assay (HTA) to identify PLD-inducing compounds. [29b] This HTA is suitable for determining the lysosomal aggregation of phospholipids upon application of chemical compounds in a fast, reproducible, and quantitative manner. In contrast to previously described HTAs for determining drug-induced PLD,[23], [29b] we employed the adherent, rapidly proliferating, human neuroglioma cell line H4, as this neuroglioma cell line is perfectly compatible with the LipidTOX assay.…”

Section: Discussionmentioning

confidence: 99%

“…[29b] This HTA is suitable for determining the lysosomal aggregation of phospholipids upon application of chemical compounds in a fast, reproducible, and quantitative manner. In contrast to previously described HTAs for determining drug-induced PLD,[23], [29b] we employed the adherent, rapidly proliferating, human neuroglioma cell line H4, as this neuroglioma cell line is perfectly compatible with the LipidTOX assay. The H4 neuroglioma cell line that was used here exhibits a high proliferation rate (24 h per cell cycle).…”

Section: Discussionmentioning

confidence: 99%

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Identification of Drugs Inducing Phospholipidosis by Novel in vitro Data

et al. 2012

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Drug-induced phospholipidosis (PLD) is a lysosomal storage disorder characterized by the accumulation of phospholipids within the lysosome. This adverse drug effect can occur in various tissues and is suspected to impact cellular viability. Therefore, it is important to test chemical compounds for their potential to induce PLD during the drug design process. PLD has been reported to be a side effect of many commonly used drugs, especially those with cationic amphiphilic properties. To predict drug-induced PLD in silico, we established a high-throughput cell-culture-based method to quantitatively determine the induction of PLD by chemical compounds. Using this assay, we tested 297 drug-like compounds at two different concentrations (2.5 μm and 5.0 μm). We were able to identify 28 previously unknown PLD-inducing agents. Furthermore, our experimental results enabled the development of a binary classification model to predict PLD-inducing agents based on their molecular properties. This random forest prediction system yields a bootstrapped validated accuracy of 86 %. PLD-inducing agents overlap with those that target similar biological processes; a high degree of concordance with PLD-inducing agents was identified for cationic amphiphilic compounds, small molecules that inhibit acid sphingomyelinase, compounds that cross the blood–brain barrier, and compounds that violate Lipinski’s rule of five. Furthermore, we were able to show that PLD-inducing compounds applied in combination additively induce PLD.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Identification of Drugs Inducing Phospholipidosis by Novel in vitro Data

et al. 2012

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“…Recently, in vitro assay for phospholipidosis-induced CADs using fluorescent phospholipid accumulation in a human monocyte cell line and HepG2 cell cultured in a 96-well plate has been reported [13, 14]. For CAD-induced phospholipidosis associated with LPLA2, the present assay system consisting of recombinant LPLA2 and liposomes containing 1-FAM-2-DABCYL-PG may provide a more effective and facile means to determine whether newly or already developed CADs evoke a LPLA2 mediated phospholipidosis.…”

Section: Discussionmentioning

confidence: 99%

A fluorogenic phospholipid for the detection of lysosomal phospholipase A2 activity

Abe

Rzepecki

Shayman

2013

Analytical Biochemistry

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Lysosomal phospholipase A2 (group XV PLA2, LPLA2) is a lysosomal enzyme linked to drug induced phospholipidosis. We developed phospholipid “smart probes” based on the conversion of a quenched fluorogenic substrate to a fluorescent product. Due to the preference of LPLA2 for phosphatidylglycerol, three fluorogenic phosphatidylglycerols were synthesized. Two fluorogenic phosphatidylglycerols were conjugated with one fluorescein amidite (FAM) group and one 4-(4-dimethylaminophenylazo)-benzoyl (DABCYL) group; the third substrate consisted of two FAM groups conjugated at the sn-1 and sn-2 positions. The sn-1 ester linkage was replaced with an amide linkage. 1-FAM-2-DABCYL-PG was degraded by recombinant LPLA2 and mouse serum but not by the serum obtained from LPLA2-deficient mice when 1,2-dioleoyl-PG/1-FAM-2-DABCYL-PG liposomes were used. The formation of 1-FAM-lyso-PG generated from 1-FAM-2-DABCYL-PG in the presence of LPLA2 was quantitatively determined by fluorescent measurements. The 1-FAM-2-DABCYL-PG incorporated into 1,2-dioleoyl-phosphpatidylcholine/sulfatide liposomes was used to evaluate the effect of the cationic amphiphilic drugs amiodarone and fluoxetine on LPLA2 activity. The IC50s of amiodarone and fluoxetine estimated by fluorescent measurement were 10 and 19 µM. These results indicate that 1-FAM-2-DABCYL-PG is a specific substrate for LPLA2 and a useful reagent for the detection of LPLA2 activity from multiple sources.

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“…38 The disorder is considered to be mild and can oen self-resolve. However, drugs that cause phospholipidosis can also produce organ damage, and thus this disorder is a concern to the regulatory agencies.…”

Section: Phospholipidosismentioning

confidence: 99%

Human in Vitro ADMET and Prediction of Human Pharmacokinetics and Toxicity Liabilities at the Discovery Stage

Tsaioun

2014

Human-Based Systems for Translational Research

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The drug development process has undergone a rapid evolution due to an expanding biological and chemical toolbox that allows novel target identification and rapid synthesis of a large number of diverse chemical libraries. The discovery of novel therapeutics is an inherently complex and interdisciplinary process, which requires close integration of scientists from several disciplines in an environment in which lessons are shared and taught across an organisation. However, traditionally the industry suffered from the lack of integration between chemists and biologists. Each discipline produced results that were scientifically valid, but frequently had little relevance to the likelihood of launching a commercial product. ADMET is an area that has emerged over the past 15 years and has created a unique interdisciplinary interface between medicinal chemists, biologists, formulators, toxicologists, clinicians and regulators. The implementation of ADMET profiling of drug candidates in conjunction with biological efficacy optimisation has dramatically reduced drug failures in clinical trials for pharmacokinetic reasons and has become a lingua franca between disciplines that are involved in drug development. The goal of an ADMET programme is to guide candidate selection by identifying molecules with optimal potency and drug-like properties. The purpose of this chapter is to briefly review the current state-of-the-art of ADMET and its scientific principles and describe some of the most prevalent ADMET strategies used to de-risk drug discovery programmes.

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A 96-well flow cytometric screening assay for detecting in vitro phospholipidosis-induction in the drug discovery phase

Cited by 31 publications

References 33 publications

Identification of Drugs Inducing Phospholipidosis by Novel in vitro Data

Identification of Drugs Inducing Phospholipidosis by Novel in vitro Data

A fluorogenic phospholipid for the detection of lysosomal phospholipase A2 activity

Human in Vitro ADMET and Prediction of Human Pharmacokinetics and Toxicity Liabilities at the Discovery Stage

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