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. 2020 Apr 27;9(5):1082.
doi: 10.3390/cells9051082.

The Lysosomotropic Activity of Hydrophobic Weak Base Drugs is Mediated via Their Intercalation into the Lysosomal Membrane

Michal Stark  1 , Tomás F D Silva  2 , Guy Levin  1 , Miguel Machuqueiro  2 , Yehuda G Assaraf  1
Affiliations

The Lysosomotropic Activity of Hydrophobic Weak Base Drugs is Mediated via Their Intercalation into the Lysosomal Membrane

Michal Stark et al. Cells. .

Abstract

Lipophilic weak base therapeutic agents, termed lysosomotropic drugs (LDs), undergo marked sequestration and concentration within lysosomes, hence altering lysosomal functions. This lysosomal drug entrapment has been described as luminal drug compartmentalization. Consistent with our recent finding that LDs inflict a pH-dependent membrane fluidization, we herein demonstrate that LDs undergo intercalation and concentration within lysosomal membranes. The latter was revealed experimentally and computationally by (a) confocal microscopy of fluorescent compounds and drugs within lysosomal membranes, and (b) molecular dynamics modeling of the pH-dependent membrane insertion and accumulation of an assortment of LDs, including anticancer drugs. Based on the multiple functions of the lysosome as a central nutrient sensory hub and a degradation center, we discuss the molecular mechanisms underlying the alteration of morphology and impairment of lysosomal functions as consequences of LDs' intercalation into lysosomes. Our findings bear important implications for drug design, drug induced lysosomal damage, diseases and pertaining therapeutics.

Keywords: drug sequestration; lysosomes; lysosomotropic drugs; membrane intercalation; molecular dynamics.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
The effect of central nervous system acting drugs (CNSDs) on the number and size of lysosomes. U2OS cells were seeded in black glass bottom plates and treated for 72 h with either 10 µM Clomp, 10 µM Ethop, 30 µM Pimo or 0.1% DMSO for the drug free control. In addition, ClompR cells continuously grown in 10 µM Clomp were used, as well as U2OS cells treated for 48 h with 20 µM CHQ as a positive lysosomotropic control. Nuclei were stained with the viable DNA dye Hoechst 33342 (blue fluorescence), and lysosomes with the lysosomal probe LysoTracker Red DND-99 (LTR) (red fluorescence). (a–f): Representative images captured using a confocal Zeiss LSM 710 microscope (×63 magnification). Insets: (a) show the hardly visible drug free control lysosomes (upper inset) and over-enhanced lysosomes for better visualization (bottom inset), while (b–f) show examples of ring-like structures indicated by white arrows. All LTR images are representative of data collected from at least three independent experiments. (g,h) Cells were captured using an InCell Analyzer 2000 fluorescence microscope, and lysosomes were analyzed using the InCell Investigator software. Histograms depict the average median values obtained from three independent experiments ± S.D. All p values < 0.045 except for the ones indicated by an asterisk. See also Figure S3.
Figure 2
Figure 2
Z-Stack analysis of LTR-loaded lysosomes. ClompR cells (a–f) or vacuolin-1-treated U2OS cells (g–n) were labeled with LTR and scanned with a confocal Zeiss LSM 710 microscope (×63 magnification) using focus stacking with 0.2 µm slices. White arrows indicate ring-like structures as they first appear. The yellow arrow points to a lysosome harboring internal vesicles.
Figure 3
Figure 3
Lysosomal membrane staining with naturally fluorescent lysosomotropic compounds. Vacuolin-1-treated U2OS cells were loaded for 45 min with LysoTracker Green DND-26 (LTG) (a,b), daunorubicin (DNR) (c–e) or nintedanib (NTD) (f–h) and imaged with a confocal Zeiss LSM 710 microscope (×63 magnification). All images are representative of data collected from at least three independent experiments. See also Figure S4.
Figure 4
Figure 4
Lysosomal DNR fluorescence analysis. Vacuolin-1-treated U2OS cells were loaded for 45 min with DNR and imaged with a confocal Zeiss LSM 710 microscope (×63 magnification). Individual lysosomes were analyzed for fluorescence intensity along their diameter using the ImageJ software. Each graph corresponds to a lysosome numbered in the photo.
Figure 5
Figure 5
CpHMD simulations of the membrane insertion of lysosomotropic compounds. Average protonation (left subplots) and abundance histograms (right subplots) are depicted along the membrane insertion axis for Ethop (a), Pimo (b), Clomp (c), NTD (d) and DNR (e). The gray-shaded areas represent the membrane internal regions below the phosphate groups, which are used as insertion references (see Materials and Methods).
Figure 6
Figure 6
Predicted shifts in pKa values during membrane insertion. pKa profiles along the membrane normal for the CNSDs (a) and anticancer drugs (b). The unfilled points represent the experimental solution pKa values (Figure S1). The gray-shaded areas represent the membrane internal regions below the phosphate atoms, which are used as insertion references (see Materials and Methods).
Figure 7
Figure 7
Model representation of lysosomotropic drug accumulation within the lysosomal membranes and its lysosomotropic effects. (a) A lysosomotropic drug (LD) reaches the lysosomal outer leaflet surface in its protonated form. The lysosomal limiting membrane (LLM) is illustrated with neutral phospholipids (e.g., phosphatidylcholine and sphingomyelin) and the transmembrane protein Niemann-Pick C1 (NPC1). For simplicity, the lysosomal glycocalyx is not shown. (b) The hydrophobic nature of the LD promotes its insertion into the bilayer, during which it undergoes complete deprotonation. Under physiological conditions, NPC1 exports cholesterol to the cytosol. (c) LD molecules diffuse through the LLM until their amine residues encounter the acidic lysosomal lumen, where they undergo protonation. As more LD molecules accumulate, they induce enhanced bilayer fluidity, thus enhancing NPC1 activity and competing with cholesterol for binding to NPC1. While most LD molecules concentrate within the inner leaflet of the LLM above the phospholipid head groups, some detach and move through the aqueous lumen. (d) The LD reaches the intra-lysosomal vesicle (ILV) surface, where various lipases (e.g., acid sphingomyelinase (ASM), acid ceramidase (AC) and phospholipase A (PLA)) are electrostatically bound to the membrane’s negatively charged lipids, while degrading their lipid substrates. (e) The LD inserts into the ILV’s outer leaflet and electrostatically interacts with negatively charged lipids, thereby abolishing the binding of lipases. (f) Lipases, in their closed-inactive form, are more rapidly degraded by luminal proteases. As a result of decreased lipase activity, phospholipids accumulate within the lumen of lysosomes, leading to lipidosis. The latter, along with the accumulation of cholesterol, is the hallmark of drug-induced and hereditary LSD.
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