Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2021:62:100089.
doi: 10.1016/j.jlr.2021.100089. Epub 2021 Jun 1.

Inhibition of lysosomal phospholipase A2 predicts drug-induced phospholipidosis

Vania Hinkovska-Galcheva  1 , Taylour Treadwell  1 , Jonathan M Shillingford  1 , Angela Lee  1 , Akira Abe  1 , John J G Tesmer  2 , James A Shayman  3
Affiliations
Review

Inhibition of lysosomal phospholipase A2 predicts drug-induced phospholipidosis

Vania Hinkovska-Galcheva et al. J Lipid Res. 2021.

Abstract

Phospholipidosis, the excessive accumulation of phospholipids within lysosomes, is a pathological response observed following exposure to many drugs across multiple therapeutic groups. A clear mechanistic understanding of the causes and implications of this form of drug toxicity has remained elusive. We previously reported the discovery and characterization of a lysosome-specific phospholipase A2 (PLA2G15) and later reported that amiodarone, a known cause of drug-induced phospholipidosis, inhibits this enzyme. Here, we assayed a library of 163 drugs for inhibition of PLA2G15 to determine whether this phospholipase was the cellular target for therapeutics other than amiodarone that cause phospholipidosis. We observed that 144 compounds inhibited PLA2G15 activity. Thirty-six compounds not previously reported to cause phospholipidosis inhibited PLA2G15 with IC50 values less than 1 mM and were confirmed to cause phospholipidosis in an in vitro assay. Within this group, fosinopril was the most potent inhibitor (IC50 0.18 μM). Additional characterization of the inhibition of PLA2G15 by fosinopril was consistent with interference of PLA2G15 binding to liposomes. PLA2G15 inhibition was more accurate in predicting phospholipidosis compared with in silico models based on pKa and ClogP, measures of protonation, and transport-independent distribution in the lysosome, respectively. In summary, PLA2G15 is a primary target for cationic amphiphilic drugs that cause phospholipidosis, and PLA2G15 inhibition by cationic amphiphilic compounds provides a potentially robust screening platform for potential toxicity during drug development.

Keywords: 1-O-acylceramide; Acyltransferase; amiodarone; cationic amphiphilic drugs; drug development; drug toxicity; drug-induced phospholipidosis; high-throughput screening; lysosome; phospholipase A2 group XV.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. Recombinant LPLA(2) and anti-LPLA(2) monoclonal antibodies are licensed to Echelon Biosciences by the University of Michigan.

Figures

Fig. 1
Fig. 1
The range of LPLA2 inhibition by all compounds studied. The measured IC50s for inhibition of LPLA2 in the cell-free assay are plotted. Compounds from Table 1 in which phospholipidosis has been reported are denoted by in vitro studies (red circles), in vivo studies (blue circles), or both in vitro and in vivo studies (yellow circles). Compounds studied in which no prior reports of phospholipidosis are denoted by black circles.
Fig. 2
Fig. 2
LPLA2 inhibition in relation to the physical properties of compounds previously associated with phospholipidosis. The test compounds listed in Table 1 are graphed in relation to pKa (basic) and ClogP. The exclusion limits of the Ploemen and modified Ploemen models are delineated by the red lines. The IC50s for LPLA2-dependent 1-O-acyl N-acetylsphingosine synthase activity are indicated as follows: greater than 1 mM (red circles), less than 100 μM (green circles), greater than 100 μM and less than 1 μM (blue circles).
Fig. 3
Fig. 3
LPLA2 inhibition in relation to the physical properties of compounds not previously associated with phospholipidosis. The test compounds listed in Table 2 are graphed in relation to pKa (basic) and ClogP. The exclusion limits of the Ploemen and modified Ploemen models are delineated by the red lines. LPLA2 IC50s for LPLA2-dependent 1-O-acyl N-acetylsphingosine synthase activity are indicated as follows: greater than 1 mM (red circles), less than 100 μM (green circles), greater than 100 μM and less than 1 μM blue circles.
Fig. 4
Fig. 4
Inhibition of LPLA2 by fosinopril. A: Thin layer chromatography from the LPLA2 assay in the presence of fosinopril. The reaction products include free fatty acid and 1-O-acyl-N-acetyl-ceramide (1-O-acyl-NAS). B: LPLA2 activity in the presence of fosinopril as a percent of the control assay run in the absence of fosinopril. C: Cosedimentation of liposomes and LPLA2 in the presence or absence of fosinopril. Liposomes consisting of DOPC/ sulfatide (10:1, molar ratio, 127 μM total) were incubated with 5 μg of LPLA2 and different concentrations of fosinopril in 500 μl of 50 mM sodium citrate pH 4.5 for 30 min on ice. The reaction mixture was then centrifuged for 1 h at 150,000 g at 4°C. The resulting precipitate was rinsed with cold 50 mM sodium citrate pH 4.5 and dissolved with 40 μl of SDS-PAGE sample buffer. The sample was separated by using 10% SDS-PAGE. Following electrophoresis, LPLA2 was detected with Simply Blue. Band quantification was performed with the Image J software I1.651j8. D: LPLA2 transacylase activity against comparing DOPC to p-NPB as substrates. Liposomes containing DOPC-sulfatide (10:1 M ratio) were incubated with recombinant LPLA2 (30 ng/ml) with or without p-NPB (200 μM) in the presence or absence of 10 μM NAS at 37 degrees C in 500 μl Na-citrate buffer (50 mM, pH 4.5). E: LPLA2 transacylation activity toward p-NPB comparing liposomes to a monodispersed substrate. Fosinopril (250 nM) was present in lanes 5 and 6. The reactions as detailed in panels E and F were terminated by the addition of 3 ml chloroform/methanol (2/1, v/v), followed by 0.3 ml of 9% (w/v) NaCl. After centrifugation for 7 min at 1,800 g, the resulting lower layer was transferred to new tube and dried under stream of nitrogen gas. The dried lipid was dissolved in 40 μl of chloroform/methanol (2/1, v/v) and applied to HPTLC plates. HPTLC plates were run in chloroform/acetic acid (9/1, v/v). The plates were dried and soaked in 8% (w/v) CuSO4.5H2O, 6.8% (v/v) H3PO4, and 32% (v/v) methanol and then charred for 15 min in an oven at 150 ˚C. Scanned plates were analyzed by NIH ImageJ 1.651j8 (National Institutes of Health).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Anderson N., Borlak J. Drug-induced phospholipidosis. FEBS Lett. 2006;580:5533–5540. - PubMed
    1. Shayman J.A., Abe A. Drug induced phospholipidosis: an acquired lysosomal storage disorder. Biochim. Biophys. Acta. 2013;1831:602–611. - PMC - PubMed
    1. Chen G.L., Sutrina S.L., Frayer K.L., Chen W.W. Effects of lysosomotropic agents on lipogenesis. Arch. Biochem. Biophys. 1986;245:66–75. - PubMed
    1. Kubo M., Hostetler K.Y. Mechanism of cationic amphiphilic drug inhibition of purified lysosomal phospholipase A1. Biochemistry. 1985;24:6515–6520. - PubMed
    1. Ikeda K., Hirayama M., Hirota Y., Asa E., Seki J., Tanaka Y. Drug-induced phospholipidosis is caused by blockade of mannose 6-phosphate receptor-mediated targeting of lysosomal enzymes. Biochem. Biophys. Res. Commun. 2008;377:268–274. - PubMed

Publication types

LinkOut - more resources