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. 2026 Mar 17;11(12):19727–19739. doi: 10.1021/acsomega.6c00013

Drug-Induced Phospholipidosis as an Artifact in Antiviral Drug Repurposing

Isabella S Glenn †, Lu Paris †, Alex D White †, Virginia G Garda ‡,§, Mauricio Montano ∥, Mir M Khalid ∥,⊥, Aimee W Kao ‡, Melanie Ott ∥,⊥,#, Brian K Shoichet †,*
PMCID: PMC13044659  PMID: 41939306

Abstract

Drug repurposing, in principle, can speed antiviral drug discovery. Among the molecules most frequently advanced in such repurposing efforts is a group of structurally diverse cationic amphiphilic drugs (CADs). While CADs have shown micromolar to mid-nanomolar antiviral activity in cell-based assays, they can induce phospholipidosis, confounding repurposing efforts. A barrier to the identification of phospholipidosis inducers has been the involved nature of the microscopy assays used to characterize them. To ease the identification of these artifacts, we describe a rapid microplate-based assay to detect phospholipidosis. Leveraging this assay, we quantified the prevalence of phospholipidosis-inducers across several cell-based antiviral repurposing screens. We selected 40 drugs reported to have micromolar antiviral activities and found that 26 of them (65%) induced phospholipidosis within the same concentration range as their reported antiviral activities. Intriguingly, we identified four non-CADs that also induce phospholipidosis, revealing a non-cationic class of drugs that can lead to this toxic event and highlighting the importance of facile experimental assays to detect it. Understanding how phospholipidosis can confound antiviral drug discovery and its rapid detection will help prevent what is an apparently general artifact, active across viruses, from distracting investigators from potentially more useful candidates.

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Introduction

Drug repurposing is a popular strategy to identify new indications for approved or investigational drugs. Developed drugs are derisked molecules, and advancing them eliminates many of the costly and time-consuming preclinical steps in drug discovery and development. With the Covid-19 pandemic, drug repurposing took on greater urgency, given the need to rapidly discover new antiviral therapeutics. Indeed, two of the triumphs of Covid-19 drug discovery, nirmatrelvir and remdesivir, both reflect some aspect of drug repurposingthe first drawing on extensive efforts in developing Mpro inhibitors for SARS-Cov, , and the second emerging from a drug discovery effort against ebola virus. , Still, these drugs and their antecedents were themselves antivirals with a defined mechanism of action. During the pandemic, many investigators cast a wider net, testing large drug libraries, most of which targeted human proteins, seeking molecules that would be fortuitously − or by mechanism − active against the virus. While several promising molecules emerged from this effort, many of the repurposed molecules, despite having low μM to even mid-nM activities in cell culture, were ultimately shown to be false positives acting via artifactual mechanisms, , and this recently has been seen for drugs repurposed against other viruses as well. Among the most prominent of these is drug-induced phospholipidosis (DIPL).

Phospholipidosis is a lysosomal storage disorder characterized by the intracellular accumulation of phospholipids, as occurs in some pathologies such as Niemann–Pick disease. A morphological hallmark of phospholipidosis is the formation of lamellar bodies and “foamy” and “whorled” membranes that can be captured by electron microscopy. The effect is of wide concern in pharmaceutical development, where drug-induced phospholipidosis (DIPL) can cause idiopathic toxicities, often emerging late in development, in sensitive organs including the liver and kidney. , This has motivated the development of fluorescently conjugated phospholipids, such as NBD-PE, to monitor phospholipid accumulation by microscopy. ,

Chemically, phospholipidosis inducers are diverse and belong to drug classes including antibiotics, antidepressants, antipsychotics, and antiarrhythmics. Physically, they share several properties: most are cationic at physiological pH and are relatively hydrophobic, with clogP (calculated log of the octanol:water partition ratio) values often >3 (and so are called “cationic amphiphilic drugs” (CADs)). − CADs become further ionized in the acidic lysosomal environment, where they accumulate. This lysosomal accumulation is thought to be a driving force for DIPL, though a full mechanism has yet to be elucidated.

Many of the drugs repurposed for activity against SARS-CoV-2 in cell culture were CADs, and their antiviral activity was subsequently found to correlate with the phospholipidosis they induced. In the same study, drugs that were cationic but not amphiphilic did not induce phospholipidosis and were not antiviral, while known CADs that were not previously known to be antiviral could be shown to be such against SARS-CoV-2 in cell culture. Whereas the CADs could have antiviral effects down to the 100 nM range, they could not be optimized beyond that, consistent with their phospholipidosis range of activities. Exactly how drug-induced phospholipidosis translated into apparent antiviral activity in cell culture remains to be fully characterized, though it is thought that the disruption of phospholipid homeostasis affects the ability of SARS-CoV-2 to replicate, perhaps via disruption of the double-membrane vesicles on which it depends. Meanwhile, the antiviral activity of the CAD phospholipidosis inducers did not translate into animal efficacy but only into the toxicity with which phospholipidosis inducers are associated at high concentrations. ,,

Here, we investigate two further impacts of phospholipidosis on antiviral repurposing: its role on drugs repurposed against viruses different from SARS-CoV-2, and its role in drugs outside of the traditional cationic amphiphiles with which it has been most associated. To do so, we adopt a new high-throughput assay to quantify DIPL, using it to investigate 40 drugs reported as candidates for antiviral repurposing across seven viruses, − including several that were not cationic and so were not expected to induce phospholipidosis by the usual metrics. , Implications for drug repurposing, the range of molecules that might induce phospholipidosis in antiviral drug repurposing, and the ability to rapidly test for this effect will be considered.

Results

A Rapid Assay to Detect Drug-Induced Phospholipidosis

A rapid method to detect phospholipidosis is crucial to ruling out its confounding effects. Electron and confocal microscopy are the gold standard for measuring phospholipid accumulation, but they are slow and require access to imaging facilities. In the initial study of drug-induced phospholipidosis (DIPL) among SARS-CoV-2 inhibitors, a microscopy-based NBD-PE assay was used that required solubilizing NBD-PE in ethanol, followed by extended sonication to ensure proper dispersion in solution. Cells were typically fixed before imaging, although the experiments were conducted by high-content microscopy. Processing the images was time-consuming and required both specialized software and expertise. Though widely used in pharmaceutical research to identify DIPL, this NBD-PE assay is unsuited for rapid detection. Accordingly, we adapted an assay first introduced by a group at the US National Center for Advancing Translational Sciences. While this assay still utilizes high-content microscopy, LipidToxRed is used in place of the traditional NBD-PE reagent. LipidTOX Red is a proprietary probe consisting of phospholipids conjugated to a fluorescent dye. Cells are incubated with LipidTOX Red in the presence of a test compound and after 24 h fluorescence is measured to detect the accumulation of phospholipids with which this probe associates. LipidTox Red offers several advantages: it comes as a ready-mix solution bypassing solubility challenges of traditional PLD reagent, is suitable for live-cell imaging, and is more sensitive for PLD detection compared to NBD-PE. Further, we describe a modified version of this assay that can be performed using a microplate reader, reducing image processing time and improving accessibility to nonspecialists (Figure A).

1.

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Overview of the phospholipidosis assay. (A) Schematic of the three-day experiment (created in BioRender. Glenn, I. (2026) https://BioRender.com/b4tuk1c). (B) Results for the non-PLD-inducing cationic drug melperone and (C) the well-characterized CAD PLD inducer amiodarone. All data shown was measured in Hep G2 cells using LipidTox Red. Error bars represent SD for three independent experiments performed in triplicate.

Consistent with earlier studies, the plate-based assay performed well against positive and negative controls, accurately distinguishing a non-PLD-inducing cationic drug melperone (Figure B) from the well-characterized PLD-inducer amiodarone (Figure C). Encouragingly, the potency of amiodarone was consistent in the plate-based assay to previously reported values from orthogonal assays, ,, including our earlier microscopy-based study. This assay can rapidly detect and quantify drug-induced phospholipidosis without lengthy microscopy experiments and analyses.

Screening for Phospholipidosis among Drugs Repurposed as Antivirals

We searched the literature for drugs reported as cell-based antivirals with an unclear mechanism of action. We filtered these for those with similar physiochemical properties to known phospholipidosis inducers: clogP ≥2 and calculated pK a ≥7.4. Forty compounds met these criteria (Table , Table S1), including four that did not pass both property cutoffs but that had multiple reports of antiviral activity. All of the compounds were tested for PLD induction using the assay introduced above. Of the 40, 26 (65%) induced PLD in the same potency range as their reported antiviral activity (Figures A, S2). Twenty-two were stereotypical CADs with the top ten most potent PLD inducers having EC50 values from 0.4 to 5 μM in Hep G2 cells (Figure B). Although these cells are widely used to study PLD, they are uncommon in antiviral screens. Thus, we also screened our most potent CADs in A549 cells, which are routinely used for antiviral drug discovery. Of the 10 screened, 9 induced PLD in the A549 cells, with several being even more potent and efficacious than in the Hep G2 cells.

1. Repurposed Drugs That Induce Phospholipidosis.

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a

Literature reported EC50 values in viral replication assays; see citations in Table S1.

b

Chemical properties, including clogP and pK a (basic), were calculated using RDkit-2018.09 and JChem-21.13, respectively.

c

Phospholipidosis EC50 using LipidTox Red stain. Experiments were performed in HepG2 cells with standard deviation determined from three independent experiments each performed in triplicate.

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Repurposed drugs that induce phospholipidosis. (A) Percentage fluorescence with treatment of drug at a single-point concentration at approximately PLD E max in Hep G2 cells. Itraconazole and U18666A were screened at 3.16 μM and Daptomycin, Nelfinavir, Tilorone, and Verapamil at 31.6 μM. All other repurposed drugs were screened at 17.8 μM. Error bars represent SD for three independent experiments performed in triplicate. (B) Ten most potent phospholipidosis inducers that are prototypical CADs screened in Hep G2 (black) and A549 (red) cells with their corresponding literature reported antiviral EC50 to E max boundaries colored in gray. Error bars for Hep G2 cells represent SD for three independent experiments performed in triplicate. Error bars for A549 cells represent SD for three technical replicates. All PLD data shown was performed using the LipidTox Red phospholipid reagent. Data was normalized to positive control compound, amiodarone, and cell count using Hoechst nucleus stain.

Intriguingly, four compounds that did not meet either the clogP or pK a cutoffs and would not ordinarily be classified as CADs also induce phospholipidosis (Figure ). These included two that are neutral (noncationic) even at lysosomal pH values, itraconazole and ivermectin (Figure C), and two highly soluble, nonamphiphilic cationic drugs that had clogP values of −0.9 and −5.6: azithromycin and daptomycin, respectively (Figure B). The four compounds were included in our screen owing to their frequent appearance in the antiviral repurposing literature. While azithromycin is well-known to be a PLD inducer, ,, the other three molecules are not, as far as we know. Thus, while status as a CAD remains the best predictor of being a phospholipidosis-inducing drug, it is neither necessary nor sufficient to predict such activity, a point also made by other studies and to which we will return.

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Non-CAD phospholipidosis inducers. (A) Chemical structures of non-CAD inducers. (B) More polar phospholipidosis inducers clogP<2 and (C) uncharged phospholipidosis inducers. All data shown was performed in HepG2 cells using LipidTox Red phospholipid stain. Error bars represent SD for three independent experiments performed in triplicate. Data was normalized to positive control compound, amiodarone, and cell count using Hoechst nucleus stain.

To ensure that our non-CAD compounds were indeed inducing phospholipidosis and not merely confounding the assay, we cross-validated some of our prior hits (Figure , Figure ) and the non-CAD phospholipidosis inducers (Figure ) using traditional NBD-PE dye and microscopy (Figure ). We first validated that the negative control compound melperone and the positive control amiodarone agreed with our results and literature reports (Figure A,B). Next, we screened terfenadine (a prototypical CAD), itraconazole (noncationic), and azithromycin (nonlipophilic) for phospholipidosis and found all three were inducers by the traditional assay (Figure B). The efficacy and potencies achieved after quantifying NBD-PE intensities (Figure C,D) aligned with the data from our LipidTox red assay, demonstrating that the assay retains accuracy despite being higher-throughput.

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NBD-PE phospholipidosis assay. (A) Non-PLD-inducing CAD melperone (31.6 μM) and DMSO control. (B) Top panel traditional CAD amiodarone (17.8 μM) and terfenadine (17.8 μM). Bottom panel non-CAD Itraconazole (pK a ≤7.4) (1.78 μM) and azithromycin (clogP<2) (17.8 μM). (C,D) Quantification of mean fluorescence intensity as a concentration–response. All data shown was performed in HepG2 cells using NBD-PE phospholipid stain in triplicate with error bars representing SD. Images were taken from 9 fields with representative image shown for each condition with a scale bar (20 μm). Mean NBD-PE intensity values were normalized to positive control compound, amiodarone, and cell count using Hoechst nucleus stain.

Investigating the Role of Lysosomal Phospholipases in Drug-Induced Phospholipidosis

To examine potential mechanisms underlying drug-induced phospholipidosis and its in vitro antiviral effect, we assessed whether inducers could alter lysosomal pH or directly inhibit lysosomal phospholipases. One proposed mechanism of drug-induced phospholipidosis involves CADs becoming protonated at an ionizable amine in the lysosome’s acidic environment, leading to ion trapping. In this view, once trapped in the lysosome, these “lysosomotropic” drugs accumulate and potentially raise lysosomal pH, inactivating acidic hydrolases including phospholipases. − Such lysosomal alkalinization has been implicated in antiviral activity, with several studies reporting that the lysosomal V-ATPase inhibitor, bafilomycin A1, acts as a potent antiviral through this mechanism. − Given these precedents and that some of the PLD inducers characterized here do not contain an amine ionizable at accessible pH values, we investigated whether these PLD inducers elevate the lysosomal pH and thereby disrupt lysosomal homeostasis.

To do so, we used the FIRE-pHLy (Fluorescence Indicator REporting pH in Lysosomes) , biosensor to screen our non-CAD PLD inducers (azithromycin, daptomycin, itraconazole, and ivermectin), a panel of PLD inducers ranging in potency, a non-PLD inducing cationic drug (melperone), and bafilomycin A1 as a positive control (Figure A). At concentrations where they led to strong phospholipidosis, many of the PLD inducers had neither substantial nor statistically significant effects on the Fire-pHLy ratio fold change, relative to the positive control bafilomycin, indicating that the pH of the lysosomes remained relatively unchanged after the 24 h drug treatment. For instance, while bafilomycin changed the FIRE-pHLy ratio by 2.46-fold versus DMSO, potent PLD inducers like amiodarone and U18666A had little detectability and no significant effect on the Fire-pHLy ratio. There were other PLD inducers, like azithromycin, emetine, and terfenadine, that did show statically significant fold changes versus baseline (Table S2), but these values only ranged from 0.33 to 0.57 and were far below the change observed for positive control bafilomycin A (Figure A). These results suggest that while some PLD inducers may raise lysosomal pH, the extent of alkalinization is modest, and in most cases, the lysosomal pH remains unchanged.

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Phospholipidosis inducer effect on lysosomal pH and LPLA2 activity. (A) Bar graph quantification of FIRE-pHLy ratio fold change in HepG2 cells after 24 h drug treatment. Baseline control matched for vehicle concentration (DMSO or water) is shown to the left of each compound group. (B) LPLA2 activity assay. All compounds screened at 31.6 μM. Data points are presented as mean ± SD from three technical replicates. (C) Dynamic light scattering performed on colloidal aggregator candidates by a scattering intensity threshold of 1 × 107. The point of intersection serves as the critical aggregation concentration (CAC) value for each compound. All measurements were performed as three technical replicates in 50 mM KPi buffer. (D) LPLA2 activity before (blue) and after (purple) centrifugation screened at 31.6 μM. Data points are presented as mean ± SD from three technical replicates.

Next, we investigated whether PLD inducers could directly bind to and inhibit lysosomal phospholipases, as represented by lysosomal phospholipase A2 (LPLA2), a previously described target of PLD inducers. , We tested several canonical CAD PLD inducers and our non-CAD PLD inducers and the characterized LPLA2 inhibitor, fosinopril, as a positive control (Figure B; we note that fosinopril is an anionic phosphonate and itself far from a CAD). While fosinopril potently inhibited LPLA2, as expected, amiodarone and itraconazole substantially inhibited LPLA2, but this was only observed at 31.6 μM, well above the concentration that they induce PLD.

The relatively high concentrations necessary to inhibit LPLA2, and the high lipophilicity of amiodarone and itraconazole, prompted us to control for nonspecific inhibition via colloidal aggregation, a common artifact among enzyme inhibitors to which itraconazole was prone. Colloidal drug aggregates act by sequestering enzymes on their surface, partly denaturing them. Of the nine PLD inducers, three (amiodarone, itraconazole, and ivermectin) scattered light intensely by dynamic light scattering (DLS), as did the positive control aggregator, sorafenib , (Figure S3). All four molecules also underwent a phase transition to the colloidal form, with critical aggregation concentrations (CACs) in the low-μM range (Figure C), which we note is below their inhibitory concentrations in the LPLA2 assay. This is consistent with a colloidal (nonspecific) mechanism for LPLA2 inhibition. To further test for this mechanism, we spun down solutions of the four colloid-forming molecules in LPLA2 assay buffer on a benchtop microfuge for 30 min. This will pellet out the ∼200 nM radius colloidal drug particles but leave well-behaved soluble inhibitors in solution. ,, After the spin, we collected the supernatant, added the LPLA2 enzyme and its substrate, and measured the activity (Figure D). If the drug colloids are the active inhibitory species rather than the soluble monomer, the supernatant should have much reduced inhibition. For instance, the well-characterized colloidal aggregator sorafenib, which is not a CAD or a PLD inducer, potently inhibited LPLA2 before but not after spin-down (Figure D). Amiodarone and itraconazole saw similar behavior, but ivermectin saw no detectable change in inhibition before and after spin-down. Taken together, these observations demonstrate that seven out of the nine PLD -inducers screened do not inhibit LPLA2 at concentrations where they robustly induce phospholipidosis. The two that do inhibit the enzyme in a relevant concentration range, amiodarone and itraconazole, appear to act via a nonspecific mechanism, colloidal aggregation, and are unlikely to be relevant in vivo. These results suggest that inhibition of LPLA2 may be disconnected from PLD induction and perhaps should not be used as a proxy assay for it.

Discussion

Three observations from this study merit particular emphasis. First, PLD inducers are prevalent in antiviral drug repurposing screens, as also seen in other recent studies. Out of 40 putatively antiviral drugs tested here, 26 (65%) induced phospholipidosis in the low- to sub-micromolar range, overlapping with their reported antiviral EC50s. This supports the idea that drug-induced phospholipidosis is a general artifact in antiviral drug repurposing, affecting multiple viruses. Second, four of the phospholipidosis inducers were not classical cationic amphiphilic drugs (CADs). Two of the drugs, daptomycin and the previously characterized PLD inducer azithromycin, ,, are cationic but are not amphiphilic (Figure C). Another two, itraconazole and ivermectin, are not cationic, even at lysosomal pH values. Meanwhile, there are both cationic and cationic amphiphilic drugs that do not induce phospholipidosis, such as melperone and elacridar. While most known phospholipidosis inducers are cationic amphiphiles, being a cationic amphiphile seems neither necessary nor sufficient to induce phopholipidosis. Although surprising, this observation aligns with emerging reports from other groups identifying non-CAD compounds as phospholipidosis inducers. These nonionizable compounds suggest that proposed mechanisms for drug-induced phospholipidosis that depend solely on the titration of ionizable groups into cations ,, are incomplete. Third, our results indicate that the mechanism responsible for PLD induction and its associated antiviral activity differs from previously proposed models involving lysosomal alkalinization or LPLA2 inhibition, something also recently shown in a fascinating study of phospholiposis induced by putative ebola virus inhibitors. A wide range of PLD inducers had little or often no measurable effect on lysosomal pH suggesting that strong lysosomal alkalinization may not be necessary for phospholipid accumulation or for an antiviral effect. Nor did these PLD inducers inhibit the lysosomal enzyme LPLA2 except via nonspecific colloidal aggregation.

These results highlight a broader challenge in drug discovery: lipophilic, bioactive compounds often act through off-target or artifactual mechanisms, such as phospholipidosis or colloidal aggregation. While drug-induced phospholipidosis cannot be fully predicted based on drug physical properties or mechanism of action, we describe a facile assay, optimized from an earlier study, that allows drug-induced phospholipidosis to be rapidly characterized in dose response, ruling out this artifact and allowing investigators to focus on more promising candidates. This assay and strategy joins others that have been developed since the advent of high-throughput screening − and drug repurposing , to rapidly rule out artifacts in early discovery.

Certain caveats should be mentioned. We investigated only the correlation between phospholipidosis and antiviral activity. We do not pretend to fully understand how phospholipidosis leads to an antiviral effect, and while several commonly suggested mechanisms for drug-induced phospholipidosis, such as neutralization of lysosomal pH and inhibition of LPLA2 enzyme, are not supported by our results (Figure ), there are other proposed mechanisms that we have not investigated. , Meanwhile, drugs such as nelfinavir are validated antivirals, even though they are also phospholipidosis inducers. For nelfinavir, the antiviral potency is at least 10-fold lower than its EC50 for phospholipidosis induction. We also acknowledge that PLD induction can be cell line dependent as shown here with thioridazine and by others, including through differences in drug metabolism. For example, a recent study reports that ivermectin does not induce PLD against SARS-CoV-2 in A549 cells, but in HepG2 cells, it does so. As a result, the apparent antiviral activity of a compound may vary depending on the cell line in which the viral assay is performed. We encourage investigators to screen for phospholipidosis in the cell line most relevant to their system. Finally, we cannot rule out the possibility that phospholipidosis may be optimized into a mechanism that is not simply artifactual but can lead to antiviral activity with meaningful selective toxicity, though until now apparently antiviral phospholipidosis inducers have had little effect in vivo, other than toxic ones.

Returning to our main theme, a pessimistic outcome of this study is that many drugs repurposed against multiple viruses have cell-based activities that overlap with phospholipidosis induction and likely have their antiviral actions via this mechanism; the search for broad acting antivirals, especially from repurposing libraries, may compound this problem. Such molecules are unlikely to progress therapeutically as antivirals. More optimistically, phospholipidosis can be rapidly and quantitatively detected by the LipidTox assay used here; counter screening with this assay in early antiviral discovery, perhaps combined with new computational prediction methods, may save much time and resources. The potent induction of phospholipidosis by drugs such as itraconazole and ivermectin, which are distinctly non-CADs, points to a more widespread prevalence of this effect among bioactive molecules. Finally, while we have focused on antiviral drug discovery, the general disruption of cellular lipid homeostasis by phospholipidosis may make it an artifact in drug screens for other diseases as well, not least those affecting the lysosome and mechanisms associated with it.

Experimental Section

Literature Search

To identify drug candidates to screen for phospholipidosis, we searched Pubmed and Google Scholar with the keywords: “FDA-approved drug”, “antiviral”, “drug repositioning”, and “drug repurposing”. To filter out Covid-19 results, we added common viral names (e.g., ebola, zika, and influenza) for which investigators are still pursuing therapeutics. Identified compounds were active in the midnanomolar to micromolar range in cell-based assays. Drug SMILES data were generated using ChemDraw version 22.2.0.3348. Using these SMILES, a drug’s clogP (protonated SMILES pH 7.4) and most basic pK a was calculated using the Chem.Descriptors module from RDkit-2018.09 and JChem-21.13, respectively. Drugs that met the following criteria: clogP≥2, calculated pK a >7.4, were added to the testing list. If a drug was already reported as a phospholipidosis inducer, it was also added to the drug candidate list.

Compounds

All compounds were supplied as >95% pure by HPLC analysis as reported by the vendors and were used as supplied without further purification. Compounds were ordered from Sigma-Aldrich, SelleckChem, Cayman Chemical, TargetMol, or Medchem Express.

Cell Lines

Hep G2 cells (ATCC, HB-8065) were maintained in Eagle’s Minimum Essential Medium (EMEM, Corning, 10–009-CV) supplemented with 10% Fetal Bovine Serum (FBS, Caisson Laboratories, FBL01) and 1X Penicillin–Streptomycin (Sigma-Aldrich, P4333) and were grown at 37 °C and 5% CO2. A549 cells (ATCC, CCL-185) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Quality Biological, 112-319-101) supplemented with 10% Fetal Bovine Serum and 1X Penicillin–Streptomycin. All cells and reagents were guaranteed mycoplasma free by the supplier with no additional testing carried out.

Phospholipidosis Assay (LipidTox Red)

Hep G2 cells were seeded at a density of 1 × 104 in a black 96-well plate (Corning, 3340) in EMEM supplemented with 10% FBS and 1× Penicillin–Streptomycin. The following day, the medium was removed and replaced with fresh media containing a test compound with final DMSO concentration 1% (v/v) and 1× LipidTox red dye (Fisher Scientific, H34351). After 24 h, fluorescence emission was measured at 618 nm using a CLARIOstar plate reader (BMG Labtech). Following fluorescence reading, nucleus stain (Thermo Fisher Scientific, 62249) was added to cells, incubated for 15 min, and fluorescence was measured at an excitation/emission of 360/480. Phospholipidosis fluorescence was normalized to cell count by dividing phospholipidosis values by nucleus stain values. All compounds screened were normalized to amiodarone, a well-characterized phospholipidosis inducer. Each compound was tested in triplicate for a total of three independent experiments. Data was analyzed using GraphPad Prism software version 10.1.1 (San Diego, CA). PLD screening in A549 cells was slightly modified to accommodate the faster growth rate of the cells as compared to Hep G2 cells. Briefly, cells were seeded at a density of 8 × 104. The medium was removed 4 h after seeding and was replaced with fresh DMEM containing a test compound with final DMSO concentration 1% (v/v) and 1X LipidTox red dye. After 24 h, fluorescence was measured and normalized as described above with the Hep G2 cells. Each compound was tested in triplicate in A549 cells.

Phospholipidosis Assay (NBD-PE)

Similar to the LipidTox Red assay, Hep G2 cells were seeded at a density of 1 × 104 in a black 96-well plate in EMEM supplemented with 10% FBS and 1X Penicillin–Streptomycin. The following day, the medium was removed and replaced with fresh media containing a test compound with final DMSO concentration 0.2% (v/v) and 7.5 μM NBD-PE (Thermo Fisher, N360). Cells were incubated for another 24 h before nucleus staining and fixed with 4% (v/v) paraformaldehyde. Images were taken on a CellInsight CX7 (ThermoFisher) equipped with a 20× objective. Drug conditions were performed in triplicate with 9 fields taken from each well. Images were analyzed using the HCS Studio software (Thermo Fisher).

Dynamic Light Scattering (DLS)

Compounds were diluted in filtered 50 mM KPi buffer, pH 7, at a final concentration of 1% DMSO (v/v). All compounds were initially screened at a top concentration of 100 μM using a Wyatt DynaPro Plate Reader II (Waters Corporation). Samples that had a scattering intensity >1 × 107 cnts/s or 1-fold over baseline scattering were considered to be forming colloidal-like particles (50–1000 nm diameter) and were rescreened as a concentration–response in eight-point half-log dilutions. During data analysis, data were separated into two groups: aggregating concentrations (scattering >1 × 107 cnts/s) and nonaggregating concentrations (scattering <1 × 107 cnts/s). A line was generated for each group, and the point of intersection of the two lines serves as the critical aggregation concentration (CAC).

LPLA2 Enzyme Inhibition Assay

Compounds were screened using a commercial kit (Echelon Biosciences, K-70001) following the manufacturer’s instruction. Reagent-grade water was added to the LPLA2 substrate, followed by vortexing for 5 min to form liposomes. LPLA2 substrate and test compounds were preincubated for 1 h RT. After preincubation, the LPLA2 enzyme and reaction buffer (1x final) were added and incubated for an additional hour at RT with shaking. All compounds were screened at a final concentration of 31.6 μM with a final DMSO concentration of 6.25% (v/v). A DMSO-only, no-compound control was added to measure baseline enzyme activity, and a no-enzyme sample was used to measure the background. After a 1 h incubation, the reaction was stopped by adding stop buffer (1X final) and measured using a CLARIOStar Plus Plate Reader (BMG Biotech). Fluorescence was recorded at 490 nm excitation/540 nm emission, and background was subtracted from raw values. All data were then normalized with the DMSO-only control sample, indicating 100% enzyme activity.

To rule out the effects of aggregation, a centrifugation step was added. In this modified procedure, the compound is first added to reaction buffer (1X final) and centrifuged using a Sorvall Legend Micro 21R centrifuge (Thermo Scientific) at 15,700 xg for 30 min to remove colloidal particles. For method validation, this modified procedure with and without the centrifugation steps was compared to samples prepared using the original kit instructions. The data for each procedure were normalized to those of their respective DMSO-only control sample. After method validation, all compounds displaying greater than 30% LPLA2 inhibition using the original protocol were rescreened with the centrifugation procedure to mitigate the effects of aggregation.

Supplementary Material

ao6c00013_si_001.pdf (202.2KB, pdf)

Glossary

Abbreviations Used

(CAD)

cationic amphiphilic drug

(DIPL)

drug-induced phospholipidosis

(NBD-PE)

N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt

(PLD)

phospholipidosis

(SD)

standard deviation

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c00013.

  • Table showing all compounds tested, concentration–response for additional repurposed drug candidates that induce phospholipidosis, and single-point concentration of repurposed drug candidates that did not induce phospholipidosis (PDF)

Conceived by B.K.S., I.S.G., and A.D.W. Proof of concept assays done by A.D.W.; all results reported here by I.S.G. and L.P. L.P.: screening compounds for phospholipidosis. M.M., M.M.K., and M.O.: intellectual contributions to virology.

This work is supported by the National Institute of Health grant R35GM122481 (to BKS).

The authors declare the following competing financial interest(s): BKS is co-founder of BlueDolphin LLC, Epiodyne Inc, and Deep Apple Therapeutics, Inc., and serves on the SRB of Genentech, the SAB of Schrodinger LLC, and the SAB of Vilya Therapeutics, and consults for Frontier Discovery. No other authors declare competing interests.

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