Identification of drugs inducing phospholipidosis by novel in vitro data

Abstract

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.

Keywords: cationic amphiphilic drugs; lysosomal storage disorders; phospholipidosis; phospholipids; toxicology.

Figure 1

Histogram of measured cellular phospholipid fluorescence at both compound concentrations (green: 2.5 μm; blue: 5.0 μm) was used to determine the threshold for PLD-inducing agents. The results are given as a percentage (x-axis) of the corresponding control values. Assuming a normal distribution of the values resulting from inactive compounds with a mean of approximately 100 %, this plot suggests a limit of 200 % for active compounds, which is equal to a doubling of the LipidTox content.

Figure 2

Scatterplot showing the experimentally determined cellular LipidTox fluorescence values given as a percentage of the respective control at 5.0 μm (y-axis) and 2.5 μm (x-axis). Most test compounds show an increase in phospholipid fluorescence with increasing concentration.

Figure 3

PLD is assumed to be caused by pharmacological interactions in an additive manner. Loperamide (94), desloratadine (248), sertindole (255), trifluoperazine (163), and raloxifene (146) at moderate concentrations (0.5 μm) only slightly affect cellular phospholipid levels, while combinations of these agents clearly induced PLD. Mean values are given ±SD.

Figure 4

Associations between drug-induced PLD and properties related to membrane permeability (inhibition of ASM, BBB permeability, CAD characteristic, and Lo5 violations) were analyzed using contingency tables. The classification “+” represents PLD-inducing agents, ASM inhibitors, CAD (according to their molecular properties), and compounds that violate Lo5.

Figure 5

Cellular LipidTox content increases upon incubation of cells with fluoxetine (79). Human neuroglioma cells (H4) were treated with the well-known PLD inducer fluoxetine in the presence of LipidTox. Each concentration was applied in quadruplicate, and bars represent the mean values of LipidTox fluorescence ±SD. To further validate our results (c), representative pictures obtained with a fluorescence microscope are shown (b). The enlarged fluorescence microscope images highlight the punctual localization of the fluorescent phospholipid mixture (a).

Figure 6

The optimal time to examine drug-induced PLD in our cell-culture-based assay was 24 h after addition of LipidTox. Experiments performed at different incubation times for four compounds with slow lysosomal accumulation (dicyclomine (59): ; bepridil (25): ; lofepramine (93): ; solasodine (259): ) suggest that this is the optimal incubation time. LipidTox was added 24 h before the indicated time points. Results are given as mean values of quadruplicate measurements compared with the respective control.

Figure 7

DAPI staining is a valid method for demonstrating the presence of cells in a high-throughput manner. H4 cells were seeded at different cell densities (n) as indicated. Quantified DAPI fluorescence clearly shows a correlation between measured fluorescence and the number of cells seeded up to 20 000 cells per cm². This linear relationship enables confirmation of the presence of cells in high-throughput PLD assays. Mean values are given ±SD.