Inhibition of lysosomal phospholipase A2 predicts drug-induced phospholipidosis.

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.

Voluntary wheel running activates Akt/AMPK/eNOS signaling cascades without improving profound endothelial dysfunction in mice deficient in α-galactosidase A.

Fabry disease is caused by loss of activity of the lysosomal hydrolase α-galactosidase A (GLA). Premature life-threatening complications in Fabry patients arise from cardiovascular disease, including stroke and myocardial infarction. Exercise training has been shown to improve endothelial dysfunction in various settings including coronary artery disease. However, the effects of exercise training on endothelial dysfunction in Fabry disease have not been investigated. Gla knockout mice were single-housed in a cage equipped with a voluntary wheel (EX) or no wheel (SED) for 12 weeks. Exercised mice ran 10 km/day on average during the voluntary running intervention (VR) period. Despite significantly higher food intake in EX than SED, body weights of EX and SED remained stable during the VR period. After the completion of VR, citrate synthase activity in gastrocnemius muscle was significantly higher in EX than SED. VR resulted in greater phosphorylation of Akt (S473) and AMPK (T172) in the aorta of EX compared to SED measured by western blot. Furthermore, VR significantly enhanced eNOS protein expression and phosphorylation at S1177 by 20% and 50% in the aorta of EX when compared with SED. Similarly, plasma nitrate and nitrite levels were 77% higher in EX than SED. In contrast, measures of anti- and pro-oxidative enzymes (superoxide dismutase and p67phox subunit of NADPH oxidase) and overall oxidative stress (plasma oxidized glutathione) were not different between groups. Although the aortic endothelial relaxation to acetylcholine was slightly increased in EX, it did not reach statistical significance. This study provides the first evidence that VR improves Akt/AMPK/eNOS signaling cascades, but not endothelial function in the aorta of aged Gla deficient mice.