Abstract
To identify novel inhibitors of sphingomyelin (SM) metabolism, a new and selective high throughput microscopy-based screening based on the toxicity of the SM-specific toxin, lysenin, was developed. Out of a library of 2011 natural compounds, the limonoid, 3-chloro-8β-hydroxycarapin-3,8-hemiacetal (CHC), rendered cells resistant to lysenin by decreasing cell surface SM. CHC treatment selectively inhibited the de novo biosynthesis of SM without affecting glycolipid and glycerophospholipid biosynthesis. Pretreatment with brefeldin A abolished the limonoid-induced inhibition of SM synthesis suggesting that the transport of ceramide (Cer) from the endoplasmic reticulum to the Golgi apparatus is affected. Unlike the Cer transporter (CERT) inhibitor HPA-12, CHC did not change the transport of a fluorescent short chain Cer analog to the Golgi apparatus or the formation of fluorescent and short chain SM from the corresponding Cer. Nevertheless, CHC inhibited the conversion of de novo synthesized Cer to SM. We show that CHC specifically inhibited the CERT-mediated extraction of Cer from the endoplasmic reticulum membranes in vitro. Subsequent biochemical screening of 21 limonoids revealed that some of them, such as 8β-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. Model membrane studies suggest that 8β-hydroxycarapin-3,8-hemiacetal reduced the miscibility of Cer with membrane lipids and thus induced the formation of Cer-rich membrane domains. Our study shows that certain limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these limonoids are related to their effect on the ceramide metabolism.
Highlights
Pharmacological inhibitors of sphingolipid metabolism and transport are useful for both biological and therapeutic research
Our study shows that certain limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these limonoids are related to their effect on the ceramide metabolism
We examined the biosynthesis of phosphatidylcholine (PC) because SM is synthesized by the transfer of phosphocholine from PC to Cer
Summary
Pharmacological inhibitors of sphingolipid metabolism and transport are useful for both biological and therapeutic research. Subsequent biochemical screening of 21 limonoids revealed that some of them, such as 8-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. To find new types of inhibitors that affect Cer metabolism and transport as well as SM metabolism, we used an original microscopy-based automated assay to screen a chemical library of natural compounds This type of lipid-specific probebased cell screening appears to be a very efficient technique for high throughput analysis of small compounds that affect lipid metabolism. We recently developed this visual technique coupled to biochemical analysis to successfully identify small molecules that interfere with cholesterol metabolism and transport [18] using the nontoxic cholesterol-binding protein toxin domain 4 [19]. The results indicate that limonoid compounds are novel inhibitors of SL metabolism and suggest that some of their biological activities are partially explained by their inhibition of Cer metabolism and transport
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