Quantification of Intracellular Accumulation and Retention of Lysosomotropic Macrocyclic Compounds by High-Throughput Imaging of Lysosomal Changes |
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Authors: | Arrabi Easwaranathan Beril Inci Sam Ulrich Lars Brunken Violetta Nikiforova Ulf Norinder Stephen Swanson Vesna Munic Kos |
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Affiliation: | 1. Swetox, Karolinska Institutet, Unit of Toxicology Sciences, Forskargatan 20, SE-151 36 Södertälje, Sweden;2. GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK |
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Abstract: | Many marketed pharmaceuticals reach extremely high tissue concentrations due to accumulation in lysosomes (lysosomotropism). Quantitative prediction of intracellular concentrations of accumulating drugs is challenging, especially for macrocyclic compounds that mainly do not fit in current in silico models. We tested a unique library of 47 compounds (containing 39 macrocycles) specifically designed to cover the entire range of accumulation intensities observed with pharmaceuticals so far. For the first time, we show that intracellular concentration of compounds measured by liquid chromatography with tandem mass spectrometry correlates with the induction of phospholipidosis and inhibition of autophagy, but the highest correlation was observed with the increase of lysosomal volume (R = 0.95), all measured by high-throughput imaging assays. Based only on imaging data, we developed a 5-class in vitro model for the prediction of compound accumulation with the accuracy of 81%. The measured change of total lysosomal volume can thus be used in high-throughput screening for determination of the actual intensity of intracellular accumulation of new macrocyclic compounds. The models are largely based on macrocycles, greatly improving the screening and prediction of intracellular accumulation of this challenging class. However, all tested nonmacrocyclic compounds fitted well in the models, indicating potential use of the models in broader chemical space. |
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Keywords: | accumulation macrocycle cationic amphiphilic drug (CAD) high-content analysis lysosome phospholipidosis autophagy CAD cationic amphiphilic drug ACC compound accumulation in cells ACE acetaminophen AMIO amiodarone AMIT amitriptyline AZAG azithromycin-aglycon AZI azithromycin CHL chloroquine CLA clarithromycin DMSO dimethylsulphoxide ERY erythromycin FLU fluoxetine I/E intracellular to extracellular concentration ratio IMI imipramine IND indomethacin LTR assay an assay of lysosome volume increase measured with Lysotracker Red dye LTR RET assay compound retention assay measured by the recovery of lysosomes after the washout OFL ofloxacin PLD assay phospholipidosis induction assay RET compound retention in cells after washout ROX roxithromycin TEL telithromycin |
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