Effect of lysosomotropic molecules on cellular homeostasis

Accepted Manuscript Title: Effect of lysosomotropic molecules on cellular homeostasis Author: Omer F. Kuzu Mesut Toprak M. Anwar Noory Gavin P. Robertson PII: DOI: Reference:

S1043-6618(16)30861-1 http://dx.doi.org/doi:10.1016/j.phrs.2016.12.021 YPHRS 3444

To appear in:

Pharmacological Research

Received date: Accepted date:

27-9-2016 13-12-2016

Please cite this article as: Kuzu Omer F, Toprak Mesut, Noory M Anwar, Robertson Gavin P.Effect of lysosomotropic molecules on cellular homeostasis.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2016.12.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical abstract

Effect of lysosomotropic molecules on cellular homeostasis Omer F. Kuzu 1,6, Mesut Toprak 5, M. Anwar Noory 1,6 and Gavin P. Robertson 1,2,3,4,6,7,8

Departments of 1 Pharmacology, 2 Pathology, 3 Dermatology, 4 Surgery, 5 Psychiatry, 6 Penn State Hershey Melanoma Center, 7 Penn State Melanoma Therapeutics Program, 8

The Foreman Foundation for Melanoma Research, The Pennsylvania State University

College of Medicine, Hershey, PA 17033.

Request for reprints: Gavin P. Robertson, Department of Pharmacology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033. Phone: (717) 531-8098; Fax: (717) 531-5013; E-mail: [email protected].

Running title: Lysosomotropic molecules.

Conflict of Interest: None Key words: Lysosomotropism, lysosomotropic compounds, inhibition of intracellular cholesterol transport, functional inhibitors of acid sphingomyelinase, tricyclic antidepressants, antipsychotics. Chemical compounds studied in this article: Perphenazine (PubChem CID: 4748); Leelamine (PubChem CID: 118215); U18666A (PubChem CID: 9954082); Fluphenazine (PubChem CID: 3372); Desipramine (PubChem CID:

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2995); Dimethylamine (PubChem CID: 674); Butylamine (PubChem CID: 8007); Imidazole (PubChem CID: 795); Hexylamine (PubChem CID: 8102); Propylamine (PubChem CID: 7852)

ABSTRACT Weak bases that readily penetrate through the lipid bilayer and accumulate inside the acidic organelles are known as lysosomotropic molecules. Many lysosomotropic compounds exhibit therapeutic activity and are commonly used as antidepressant, antipsychotic, antihistamine, or antimalarial agents. Interestingly, studies also have shown increased sensitivity of cancer cells to certain lysosomotropic agents and suggested their mechanism of action as a promising approach for selective destruction of cancer cells. However, their chemotherapeutic utility may be limited due to various side effects. Hence, understanding the homeostatic alterations mediated by lysosomotropic compounds has significant importance for revealing their true therapeutic potential as well as toxicity. In this review, after briefly introducing the concept of lysosomotropism and classifying the lysosomotropic compounds into two major groups according to their cytotoxicity on cancer cells, we focused on the subcellular alterations mediated by class-II lysosomotropic compounds. Briefly, their effect on intracellular cholesterol homeostasis, autophagy and lysosomal sphingolipid metabolism was discussed. Accordingly, class-II lysosomotropic molecules inhibit intracellular cholesterol transport, leading to the accumulation of cholesterol inside the late endosomal-lysosomal cell compartments. However, the accumulated lysosomal cholesterol is invisible to the cellular homeostatic circuits, hence class-II lysosomotropic molecules also upregulate cholesterol synthesis pathway as a downstream event. Considering the fact that Niemann-Pick disease, a lysosomal cholesterol storage disorder, also triggers similar pathologic abnormalities, this review combines the knowledge obtained from the Niemann-Pick studies and lysosomotropic compounds. Taken together, this review is aimed at allowing readers a better understanding of subcellular alterations mediated by lysosomotropic drugs, as well as their potential therapeutic and/or toxic activities. 2

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INTRODUCTION Weakly basic amine compounds that induce rapid vacuolization of cells are known as lysosomotropic molecules (1-3). “Lysosomotropism” was first introduced by de Duve et al. to define compounds that accumulate up to several hundred-fold higher concentrations within the late endosomal/lysosomal (LE/L) cell compartments (1). Weakly basic compounds, depending on their lipophilicity, can readily diffuse through the limiting membrane of acidic organelles in their unionized form and get protonated (ionized) in the acidic lumen (Figure 1). Due to their decreased membrane permeability, protonated molecules cannot cross-back to the cytosol, hence get trapped and accumulate inside the acidic lumen (also referred as ion trapping) (4). Although they have diverse structures, most of the lysosomotropic compounds harbor an amine group that is responsible for their weakly basic properties. The degree of ion trapping depends on pH of the cellular compartment as well as physicochemical properties of the compound such as pKa (acid dissociation constant) and membrane permeability. However, in addition to ion trapping, other mechanisms may also be playing a role in the lysosomotropism, as it was reported that experimentally observed accumulation was several fold higher in contrast to theoretically predicted amount (5, 6). As a matter of fact, some weakly basic molecules fail to show lysosomotropism even though their biochemical properties are favorable (7). Therefore, further studies are required to identify other mechanisms that contribute to lysosomotropism.

Lysosomes, endosomes and golgi apparatus are the major acidic organelles of the mammalian cells. As a digestion and recycling center, lysosomes contain over fifty different hydrolytic proteases such as glycosidases, sulfatases, nucleases and lipases, 4

which have optimal activity in acidic lumen of the organelle (8). On the other hand, endosomes are compartments of the endocytic transport system that is primarily involved in internalization of material from the plasma membrane (9). They function as a sorting compartment where ingested material is sorted prior to reaching lysosomes for degradation. Both lysosomes and endosomes have a unique membrane structure that consists of intraluminal membranes and a layer of external limiting membrane. The acidic lumen of both organelles is established by the activity of the vacuolar H+ ATPase (v-ATPase) protein that transports protons (H+) across the external membrane (8). The difference between the pHs of the lumen and cytosol positively correlates with accumulation of protonated amine in the acidic lumen. Therefore, v-ATPase inhibitors such as Bafilomycin A1 suppress lysosomotropism by increasing the pH of acidic organelles (10).

Lysosomotropic compounds trigger cellular vacuolization following their treatment (4, 11). Even though the details of this vacuolar response is not well understood, late endosomal, lysosomal and trans-Golgi specific proteins co-localize with these vacuoles (4, 12). It has been hypothesized that these vacuoles could be greatly expanded hybrid organelles that were formed by fusion of lysosomes with late endosomes through trafficking in a retrograde manner (3). Although there is still a need for further experiments to confirm the identity and origin of these vacuoles, induction of vacuolization is the most notable feature of lysosomotropic agents.

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1. Classification of lysosomotropic compounds Lysosomotropic compounds can be classified into two major groups based on their cytotoxic activity. Hydrophilic lysosomotropic molecules such as ammonium (NH3), methylamine (CH3NH2), ethylamine (CH3CH2NH2) can be classified as class-I compounds, while hydrophobic or amphiphilic lysosomotropic molecules, with at least one or more hydrophobic rings and a hydrophilic tail (generally harbors a polarizable amine group) can be classified as class-II compounds (Table 1 and Figure 2). Many tricyclic antidepressants (e.g.,imipramine, nortriptyline, amitriptyline), phenothiazine antipsychotics (e.g., promazine, fluphenazine, perphenazine), antihistamines (e.g., desloratadine, promethazine), SSRIs (Selective Serotonin Reuptake Inhibitors) (e.g., sertraline, fluvoxamine, fluoxetine), U18666A, leelamine, monodansylcadaverine, acridine orange and chloroquine can be given as the examples of class-II lysosomotropic compounds.

Class-I lysosomotropic compounds are generally well-tolerated by cells. Although they induce massive vacuolization, they do not trigger cell death up to millimolar concentrations (Figure 2). At these high concentrations, hydrophilic lysosomotropic compounds accumulate inside the lumen of the acidic organelles and raise the luminal pH leading to disruption of the lysosomal pathway of protein degradation (13-15).

On the other hand, class-II lysosomotropic compounds are significantly more toxic to the cells and able to induce cell death in micromolar concentrations. 6

Hydrophobic portion of these amphiphilic compounds allows their accumulation in the internal membranes of the lysosomes, causing perturbation of the activity of lysosomal membrane proteins such as acid sphingomyelinase, NPC1, NPC2, acid ceramidase and phospholipases (16, 17). As a consequence, lysosomal lipid homeostasis become disrupted and lipids such as cholesterol, sphingomyelin and certain phospholipids accumulate inside the late endosomal/lysosomal (LE/L) cell compartments, which eventually leads to loss of cell viability (17). Furthermore, increased lipophilicity of class-II lysosomotropic compounds could also enhance their sequestration kinetics contributing to the induction of cell death at low concentrations compared to the class-I lysosomotropic compounds. Since most of the lysosomotropic compounds that exhibit therapeutic activities are class-II lysosomotropic compounds, hereafter the review will focus on this second group of lysosomotropic agents.

2. Effect of class-II lysosomotropic compounds on cellular cholesterol homeostasis. 2.1 Lysosomal cholesterol homeostasis and NPC disease Lysosomes play a crucial role in intracellular cholesterol homeostasis. Mammalian cells acquire cholesterol as low-density-lipoprotein (LDL) bound cholesteryl esters through receptor-mediated endocytosis (18). Following their uptake, LDLs are transported to the lysosomes through early and late endosomes. Lysosomal acid cholesterol esterase (Lipase A, LIPA) hydrolysis cholesteryl esters to unesterified free cholesterol molecules which subsequently are distributed to the endoplasmic reticulum 7

(ER) and the plasma membrane. Two lysosomal proteins, NPC1 and NPC2 regulate lysosomal cholesterol egress. NPC2 is a soluble protein that is localized inside the lumen of lysosomes and late endosomes. It extracts and transfers cholesterol from internal membranes to the NPC1 protein which is localized at the limiting membrane of the organelle. Therefore, NPC1 and NPC2 cooperate to export cholesterol out of lysosomes. Loss of function mutations in NPC1 (95 % of the cases) and NPC2 (5% of the cases) leads to type-C form of Niemann-Pick (NPC) disease, a fatal neurodegenerative disorder (19). In consistent with the function of these two proteins, the hallmark of NPC disease is elevated levels of cholesterol inside the lysosomes and late endosomes. It has been shown that in steady-state conditions, lysosomes of the cultured human fibroblasts accommodate 6% of the total cell cholesterol, and in NPC fibroblasts this can elevate up to 10-fold (20, 21).

Recently, it has been discovered that dysfunctional NPC1 protein causes an increase in lysosomal sphingosine levels leading to calcium depletion inside the organelle; and that is subsequently followed by lysosomal cholesterol as well as sphingomyelin accumulation (21). In concordance with this observation, treatment of RAW macrophages with sphingosine dose-dependently decreased the lysosomal calcium levels and triggered lysosomal cholesterol accumulation (21). Inhibition of sphingosine synthesis with myriocin (an inhibitor of serine palmitoyl transferase 1) treatment was able to overcome the cellular abnormalities that were observed in NPC1mutant cells. The role of sphingosine was also evidenced with the findings that lysosomal sphingosine levels were increased up to 24-fold in liver and spleen of NPC patients (22). Although sphingosine was suggested as an initiating factor in NPC 8

disease, the link between NPC1/2 deficiency and sphingosine has not been truly elucidated yet (21).

In NP disease, altered sphingolipid metabolism and lysosomal cholesterol accumulation are tightly linked to each other. Indeed, types A and B forms of NiemannPick disease are caused by deficiency of acid sphingomyelinase enzyme (ASM), hence also known as Acid Sphingomyelinase Deficiency (ASMD) disorders. In steady-state conditions, ASM is localized to the inner membranes of late endosomes/lysosomes and hydrolysis sphingomyelin to generate phosphocholine and ceramide (8). Deficiency of ASM renders sphingomyelin accumulation in LE/L cell compartments. Abdul-Hammed and colleagues studied the effects of various lipid-binding proteins and lysosomal lipids on the transfer of cholesterol between liposomes (23). In this study, they discovered that NPC2 was the only lipid-binding protein capable of transferring cholesterol between different liposomes, which was greatly inhibited by the addition of sphingomyelin. Moreover, ASM-mediated degradation of liposomal sphingomyelin was able to release the inhibition of cholesterol transport (24). These observations could explain the accumulation of cholesterol in type A and B forms of the NP disease. As a note, cholesterol accumulation could create a vicious cycle by suppressing sphingolipid activator proteins (saposins) that are essential for sphingolipid degrading enzymes (Figure 3a) (25). Indeed, in cultured cells, cholesterol accumulation was reported to hinder lysosomal acid sphingomyelinase activity without affecting total ASM protein levels (26).

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2.2 Class-II lysosomotropic compounds inhibit lysosomal cholesterol egress A variety of class-II lysosomotropic compounds such as imipramine, stearylamine and U18666A have long been reported to induce NPC-like cellular phenotype. Indeed, U18666A is a widely used agent to study NP disease. U18666A causes several fold increase in LE/L cholesterol without affecting the pH of these organelles (20, 21). Treatment of RAW macrophages with U18666A have been shown to elevate the lysosomal sphingosine levels within 10 minutes. 30 minutes after the treatment, calcium levels in the lysosomes were reduced by 50% and continued to decrease up to 20%. 2 and 8 hours after the treatment, sphingolipid and cholesterol levels were elevated respectively (21). These observations were consistent with the cellular alterations mediated by the NPC1 deficiency, as discussed in previous section.

Using a structure-property-activity relation model, Kornhuber et al. identified class-II lysosomotropic compounds as inhibitors of acid sphingomyelinase and coined the term “FIASMA” (Functional Inhibitors of Acid Sphingomyelinase) (27, 28). Accordingly, class-II lysosomotropic agents inhibit lysosomal acid sphingomyelinase activity without altering ASM protein itself (29). ASM is a water-soluble glycoprotein harboring positively charged regions that promote its attachment to the intraluminal membranes enriched in negatively charged lipids such as Bis(Monoacylglycero) Phosphate (BMP). This attachment not only brings the enzyme to the close proximity of its substrate but also protects it from the degradation by cathepsins (30, 31). Protonation of lysosomotropic molecules trigger their interaction with negatively charged intraluminal membranes in LE/L compartments. This interaction disrupts electrostatic attraction between ASM and intraluminal membranes leading to loss of ASM activity as 10

well as its degradation by cathepsins (32, 33). Decreased ASM activity renders sphingomyelin and cholesterol accumulation. Taken together, class-II lysosomotropic compounds inhibit lysosomal cholesterol egress leading to a phenotype that mimics NP disease.

2.3 Chemotherapeutic potential of class-II lysosomotropic compounds Recently, in Cancer Cell, Peterson et al. have reported ASM inhibitors as a triggerer of cancer-specific lysosomal cell death (32). Although many class-II lysosomotropic agents have been previously reported to exhibit anti-cancer activity, reduce xenografted tumor growth, and revert multidrug resistance, the study of Peterson and colleagues was the first to link anti-ASM activity of lysosomotropic compounds to the observed sensitivity of cancer cells (8, 34-43). According to this study, ASM levels are low in certain malignancies and loss of this residual activity provides a molecular explanation for the increased sensitivity of cancer cells to class-II lysosomotropic agents (32). This is evidenced by the fact that both ectopic expression of ASM1 and treatment with bacterial acid sphingomyelinase protect transformed cells from cell death mediated by these agents. However, we should note that class-II lysosomotropic compounds do not only interfere with the activity of the ASM but also potentially with several other lysosomal enzymes (44, 45). Hence, disruption of other enzymes could also play a role in the enhanced cytotoxicity against cancer cells. Indeed, very recently U18666A was identified as a direct inhibitor of NPC1 protein (46). Moreover, many downstream alterations such as inhibition of AKT and MAP Kinase signaling cascades, induction of ER or oxidative stress could also provide rationale for

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sensitivity of cancer cells to class-II lysosomotropic compounds. However, there is still a need for further studies to reveal the true chemotherapeutic mechanism and potential of lysosomotropic compounds.

2.4 The effects of inhibition of lysosomal cholesterol transport on cellular cholesterol homeostasis The disruption of lysosomal cholesterol transport not only alters lysosomal cholesterol levels but also disrupts cholesterol homeostasis in the whole cell. In steadystate conditions, free cholesterol is transported from lysosomes to plasma membrane, and then to endoplasmic reticulum (Figure 3b). An increase in ER cholesterol triggers a signaling cascade that induces acyl-CoA cholesterol acyltransferase (ACAT) for esterification and storage of free cholesterol molecules as cholesterol esters (47). Free cholesterol molecules in ER also inhibit sterol regulatory element-binding protein (SREBP) cleavage (48). When it is cleaved, SREBP translocates to nucleus and triggers transcription of genes involved in cholesterol synthesis and import, such as HMG-CoA reductase and LDL receptors, respectively. Therefore, excess cholesterol not only triggers its own esterification but also inhibits new cholesterol synthesis and import. In contrast, in NP disease, inhibition of lysosomal cholesterol export rapidly diminishes ER cholesterol levels, which in turn inhibits ACAT activity and triggers cholesterol synthesis (49, 50).

Similar to NP disease, inhibition of lysosomal cholesterol egress by lysosomotropic compounds inhibits ACAT activity while inducing cholesterol synthesis.

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Indeed, 5 uM U18666A has been shown to inhibit ACAT activity up to 65% in fibroblast cells while upregulating genes involved in cholesterol synthesis (51, 52). Activation of cholesterol synthesis by class-II lysosomotropic compounds was also evidenced by analysis of gene expression data from Connectivity Map (CMap) database. This database contains more than 7000 microarray based transcriptional expression data from cultured human cells treated with biologically active small compounds. Using ArrayStar software, comparison of the expression profile of the MCF7 cell lines treated with 20 different class-II lysosomotropic agents or DMSO vehicle identified the impact of lysosomotropic agents on cholesterol homeostasis (Table 2). At a 99% confidence level, moderated T-test identified 272 significantly altered probes representing 213 individual genes (Supplementary Table 2). Gene set enrichment analysis showed highly significant alteration in cholesterol biosynthetic process with a p-value of 1.81X10-30 and a z-score of 30 (Table 2). 26 of the 59 proteins (~44%) of the cholesterol biosynthetic process were dysregulated by class-II lysosomotropic compounds. This data as well as several other reports strongly suggest aberrant cholesterol homeostasis induced by the class-II lysosomotropic agents (53, 54).

It is important to note that exogenous LDL-cholesterol is not the only source of lysosomal cholesterol. In fact, U18666A was able to trigger lysosomal cholesterol accumulation in the absence of exogenous LDL-cholesterol (20). There is significant evidence suggesting that cholesterol flows between different compartments (e.g plasma membrane, lysosome, ER, Golgi, mitochondria) of the cell (55). Around 65 to 80% of the cellular cholesterol is located in the plasma membrane and this cholesterol pool constantly moves between the lumen and cell surface. For instance, cholesterol-rich 13

lipid rafts are important signaling domains located in the plasma membrane. Induction of various membrane receptors triggers lipid raft internalization to lysosomes through the endocytic pathway. Hence, during the lipid-raft-involved endocytosis significant amount of cholesterol is transported to the lysosomes.

Autophagy has also been reported as another important source of lysosomal cholesterol. Ouimet and colleagues discovered that autophagy plays an essential role in delivering cholesteryl esters to lysosomes for their hydrolyzation into free cholesterol molecules prior to their efflux (56). As a matter of fact, cholesterol efflux from ATG5-null autophagy-deficient macrophages was significantly diminished in contrast to wild-type counterparts. These findings were in concordance with the previous observations, in which, in contrast to wild-type counterparts, ATG5-null-MEF cells were shown to accumulate less cholesterol following U18666A treatment (57). Furthermore, we have observed that ATG5-null-MEF cells were also more resistant to cell death mediated by inhibition of lysosomal cholesterol transport by lysosomotropic compounds (10).

2.5.1 Inhibition of intracellular cholesterol transport induces autophagy Although studies have suggested autophagy as an important source of lysosomal cholesterol, it has also been shown that lysosomal cholesterol accumulation itself triggers autophagy (Figure 3c). Decreased levels of cholesterol in cellular organelles, such as ER, could trigger autophagy as a nutritional deficiency response (58, 59). In fact, activation of SREBP in NPC1-deficient cells indicates that accumulated cholesterol is invisible to cholesterol regulatory circuits of the cell and thus could trigger autophagic

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pathways (60). In NPC-null mice fibroblasts, autophagy is upregulated as evidenced by increased LC3-II levels and enhanced degradation of long-lived proteins (61). In wildtype fibroblasts, U18666A treatment also increases LC3-II expression while inducing Beclin-1 (BECN1) levels that is required for the initiation and maturation of autophagosomes (62). Similarly, imipramine, an another class-II lysosomotropic compound, has been shown to trigger autophagic cell death, which is suppressed by the knockdown of BECN1 expression (63). Furthermore, autophagy in NPC disease also depends on BECN1 expression; and accumulation of autophagosomes was observed in the brains of NPC mice as well as in the fibroblasts of NPC patients (62). The data obtained from CMap database was also consistent with these observations (Table 2). Both “cellular response to starvation” and “autophagy” processes were significantly altered by class-II lysosomotropic compounds.

It is noteworthy that, autophagic process could create a vicious cycle where its induction exacerbates the lipid storage, as lysosomal cholesterol accumulation triggers autophagy leading to further cholesteryl ester transport to the lysosomes (57). This hypothesis was supported by the finding that inhibition of autophagy decreases cholesterol accumulation in NPC1-deficient cells and restores lysosomal homeostasis (57).

2.5.2 Inhibition of intracellular cholesterol transport blocks autophagic flux Lysosomal cholesterol accumulation not only induces autophagy but also blocks autophagic flux due to the inhibition of lysosomal protease activity by the stored lipids

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(Figure 3c) (57). Many studies have reported the accumulation of LC3B labeled autophagosomes following treatment with various class-II lysosomotropic agents, including U18666A and imipramine (4, 64). In our studies, we have also observed the accumulation of LC3B following treatment of melanoma cells with lysosomotropic agents such as leelamine and perphenazine (10). LC3B accumulation was suggested to be triggered by the inhibition of autophagic flux due to failed fusion of autophagosomes with lysosomes, rather than induction of autophagy (65, 66). Impairment of autophagic flux is potentially lethal to the cells due to the loss of intracellular homeostasis caused by the accumulation of damaged intracellular components. Taken together, these findings suggest that inhibition of lysosomal cholesterol transport leads to both the induction of autophagy and disruption of autophagic flux and especially the latter one has the potential to lead cellular death. 2.5.3 Implication of dysregulated cholesterol homeostasis on cellular endocytosis and viral infection Disruption of intracellular cholesterol homeostasis, especially lysosomal and endosomal cholesterol, could have deleterious effect on Golgi vesiculation, caveolin transport as well as endocytic pathway (67). Analyses of the trafficking profile of dipeptidylpeptidase IV (DPPIV), a lipid raft associated membrane protein, in NPC1 patient fibroblasts showed significantly hindered endocytosis in NPC1 patient fibroblasts (68). In our studies we have also observed that, both leelamine and several other class-II lysosomotropic compounds effectively inhibit endocytosis of Alexa Fluor conjugated transferrin protein. The inhibition of cellular endocytosis can be attributed to altered lipid raft composition as well as to many other factors such as an overall change 16

in membrane elasticity of endosomes or defective calcium homeostasis inside the acidic vesicles which could prevent transportation and budding of endocytic vesicles (21, 69).

Entry of many viruses into the cell depends on the receptor-mediated endocytosis (70, 71). It was discovered that NPC1 protein is an essential host factor for the viral entry especially (72-74). Primary fibroblast cells derived from NPC1 patients were found to be resistant to infection by Ebola and Marburg viruses (72). Both, shRNA mediated knockdown of NPC1 or treatment of cells with U18666A or imipramine was shown to hinder Ebola and Marburg infection. These studies could indicate disrupted lysosomal cholesterol homeostasis and cellular endocytosis as an important factor in the inhibition of viral entry.

3. Conclusion In this review, we have summarized the cellular alterations mediated by lysosomotropic compounds. Briefly, these compounds accumulate in the lumen of acidic cell compartments leading to disruption of their homeostatic balance. As a consequence, cholesterol and various sphingolipids accumulate in LE/L cell compartments leading to disruption of various vital processes such as autophagy and endocytosis. Inhibition of cholesterol egress from lysosomes by class-II lysosomotropic compounds resemble Niemann-Pick disease, and hence could help to understand the molecular pathology of this deadly disorder. In this respect, the interaction between sphingosine and cholesterol metabolism needs further investigation. Sphingosine 17

accumulation was suggested to pioneer cholesterol accumulation in Niemann-Pick disease and this is resembled by class-II compounds. However, the source of sphingosine accumulation is still not clear and thus hypotheses such as involvement of NPC proteins in sphingosine or amine export need further studies. Identification of a lysosomal membrane protein (probably a solute carrier family protein) that exports sphingosine out of lysosomes, could lead new insights for Niemann-Pick disease as well as mechanism of action of the class-II lysosomotropic compounds.

Alteration of cellular cholesterol homeostasis by class-II lysosomotropic drugs could explain various clinical side effects of these agents. For instance, antidepressants and antipsychotics trigger hypertriglyceridemia and hypercholesterolemia (75-77). Hyperlipidaemia is reported to be an early metabolic response to various antipsychotics. Up to five fold elevated serum lipid levels accompanied by low level of high-density-lipoprotein (HDL) cholesterol were observed in more than half of the antipsychotic consumers (78, 79). These observations could be associated with inhibition of intracellular cholesterol transport by the antipsychotics. As discussed above, inhibition of cholesterol egress from LE/L compartments diminishes cholesterol levels in ER, and triggers a cellular response that includes induction of lipid synthesis by SREBP and inhibition of cholesterol efflux by ABCA1 (an important mediator of cholesterol efflux) downregulation. In consistent with these, acute or subchronic treatments of antipsychotics are found to stimulate SREBP expression and elevate serum lipid levels in rats (80-83). Interestingly, people affected by Tangier disease, a rare inherited disorder caused by defective ABCA1 protein, also have hypertriglyceridemia with a severe reduction in HDL-cholesterol, in the bloodstream 18

(84). Hence, many of the side effects of antipsychotics could be associated with their effect on intracellular cholesterol transport.

Many class-II lysosomotropic drugs have been reported to restore sensitivity of multidrug resistant cancer cells to various chemotherapeutic agents and exhibit antiproliferative, anti-metastatic as well as proapoptotic effects (34-37). They were identified to exhibit several fold selectivity towards cancer cells in contrast to normal ones (32, 85). This observation was linked to the transformation associated changes in lysosomal lipid metabolism and suggested to be a novel approach for killing tumor cells without affecting normal ones (25, 32). Therefore, revealing the details of homeostatic alterations mediated by lysosomotropic compounds could lead to development of novel approaches for the treatment of cancer. In here the effect of lysosomotropic agents on various signaling cascades were briefly summarized. However, inhibition of intracellular cholesterol and disruption of the integrity of lysosomal/endosomal processes could alter many other oncogenic signaling cascades such as receptor tyrosine kinase/MAPK/AKT signaling. Therefore, further studies are required to identify the true chemotherapeutic potential of lysosomotropic agents.

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Abbreviation List AO Acridine orange ATP Adenosine triphosphate ASMD Acid Sphingomyelinase Deficiency CAD Cationic amphiphilic drugs CMap Connectivity map database (www.broadinstitute.org/cmap/) DMSO Dimethyl sulfoxide ER Endoplasmic reticulum HDL High-density-lipoprotein LDL Low-density-lipoprotein MDR Multidrug resistant NPC Niemann-Pick Type-C disease SREBP Sterol regulatory element-binding protein SSRI Selective Seratonine Receptor Inhibitor LE/L Late endosomal/lysosomal

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Tables Table 1. Classification of lysosomotropic compounds. Class

I

II



Name Diethylamine Ammonium chloride (NH4Cl) Dimethylamine Propylamine Hexylamine Tetrabutylammonium chloride Butylamine tert-Butylamine Imidazole n-Octylamine

Formula C4H11N NH4Cl C2H7N C3H9N C6H15N C16H36ClN C4H11N C4H11N C3H4N2 C8H19N

IC50 4.1 mM 30.2 mM 10 mM 15.2 mM 3 mM 5 mM 6.8 mM 10.3 mM >40 mM 1.4 mM

Log P* 0.5 -1 -0.2 0.3 1.6 1.3 0.7 0.4 -0.2 2.5

pKa* 10.6 8.9 10.5 10.2 10.2 NA 10.2 10.6 7 10.2

4.4 10.5 Nortriptyline C19H21N 14.9 uM 3.7 8.2 Perphenazine C21H26ClN3OS 9.7 uM 3.7 7.5 Mebhydroline C19H20N2 42.1 uM 3.9 10 Desipramine C18H22N2 20.6 uM 4.8 9.2 Triflupromazine C18H19F3N2S 12.4 uM 4.7 9.4 U18666A C25H41NO2 32 uM 5.2 9.9 Leelamine C20H31N 2.1 uM 7.8 4 Fluphenazine C22H26F3N3OS 10.5 uM 5.4 8.7 Astemizole C28H31FN4O 5.7 uM Calculated Log P and pKa values were obtained from www.chemicalize.com website.

Table 2. Significantly altered biological processes by class-II lysosomotropic compounds. Term Lipid biosynthetic process Cholesterol biosynthetic process Cellular response to sterol depletion Cellular response to starvation Cellular response to nutrient levels Autophagy Autophagosome assembly Vacuole organization

P-Value 1.79E-12 1.81E-30 8.33E-05 4.01E-10 1.35E-08 3.58E-05 5.24E-05 5.64E-03

Z-Score 10.55 30.01 13.47 12.43 10.66 7.62 8.82 5.41

Gene Set 40 26 4 16 16 12 8 8

Total 739 59 7 115 148 149 57 121

% 5.4% 44.1% 57.1% 13.9% 10.8% 8.1% 14.0% 6.6%

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Figure and Table Legends Figure 1. Protonation of weakly basic amines triggers their accumulation in acidic organelles. Lipophilic, weak bases can readily diffuse through membranes in their neutral form (B). However, upon protonation (BH+) in acidic environments, they lose their membrane permeability and become trapped in the lumen of these organelles.

Figure 2. Classification of lysosomotropic compounds. Viability curves of UACC 903 melanoma cell line for lysosomotropic compounds listed in Table 1 (left panel). Distribution of IC50 values of various lysosomotropic compounds in different cancer cell lines (right panel). Lysosomotropic compounds can be classified into two groups based on their cytotoxicity. Class-I lysosomotropic compounds do not induce cell death up to millimolar concentrations. Class-II lysosomotropic compounds are more lipophilic molecules, accumulate in luminal membranes of the acidic organelles, and induce cell death in micromolar concentrations. Please see Supplementary Table 1 for the estimated IC50 values of the tested agents.

Figure 3. Signaling pathways modulated by inhibition of lysosomal/endosomal cholesterol egress. a) In Niemann-Pick disease, mutation of SMPD1, NPC1 or NPC2 genes leads to inhibition of lysosomal cholesterol egress. Lysosomal cholesterol and sphingolipid homeostasis are intraconnected. Lysosomal cholesterol accumulation could create a vicious cycle by suppressing sphingolipid activator proteins that are essential for the activity of sphingolipid degrading enzymes; b) inhibition of lysosomal cholesterol transport leads to deregulation of cellular cholesterol homeostasis; c) inhibition of lysosomal cholesterol egress induces autophagy. Knockdown of ATG5 or 28

BECLIN1 partially protects cells from cell death mediated by inhibition of cholesterol transport indicating role of autophagy in this process. Autophagy could deliver cholesteryl esters to lysosomes leading to formation of a vicious cycle. Interestingly, accumulation of lysosomal cholesterol also inhibits autophagic flux as it depends to degradative capacity of lysosomes.

Table 1. Classification of lysosomotropic compounds. Lysosomotropic compounds can be classified into two groups based on their cytotoxicity. Please note that, class-I lysosomotropic compounds are less hydrophobic and less toxic to the cells. IC50: Approximate half maximal inhibitory concentration for UACC 903 melanoma cells following 24 hours treatment; LogP: partition-coefficient, a measure of lipophilicity; pKa: the acid dissociation constant at logarithmic scale.

Table 2: Significantly altered biological processes by class-II lysosomotropic compounds. Gene set enrichment analysis of the 213 genes that were identified to be altered by class-II lysosomotropic compounds suggests modulation of cholesterol biosynthetic process as well as induction of cellular response to sterol depletion, starvation and autophagy. Please see the supplementary data for the list of 213 genes and list of compounds used in the study.

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