Issue 12, 2024

A facile method for monitoring sphingomyelin synthase activity in HeLa cells using liquid chromatography/mass spectrometry

Abstract

Sphingomyelin synthase (SMS) is a sphingolipid-metabolizing enzyme involved in the de novo synthesis of sphingomyelin (SM) from ceramide (Cer). Recent studies have indicated that SMS is a key therapeutic target for metabolic diseases such as fatty liver, type 2 diabetes, atherosclerosis, and colorectal cancer. However, very few SMS inhibitors have been identified because of the limited sensitivity and selectivity of the current fluorescence-based screening assay. In this study, we developed a simple cell-based assay coupled with liquid chromatography/tandem mass spectrometry (LC-MS/MS) to screen for SMS inhibitors. HeLa cells stably expressing SMS1 or SMS2 were used for the screening. A non-fluorescent unnatural C6-Cer was used as a substrate for SMS to produce C6-SM. C6-Cer and C6-SM levels in the cells were monitored and quantified using LC-MS/MS. The activity of ginkgolic acid C15:1 (GA), a known SMS inhibitor, was measured. GA had half-maximal inhibitory concentrations of 5.5 μM and 3.6 μM for SMS1 and SMS2, respectively. To validate these findings, hSMS1 and hSMS2 proteins were optimized for molecular docking studies. In silico analyses were conducted to assess the interaction of GA with SMS1 and SMS2, and its binding affinity. This study offers an analytical approach for screening novel SMS inhibitors and provides in silico support for the experimental findings.

Graphical abstract: A facile method for monitoring sphingomyelin synthase activity in HeLa cells using liquid chromatography/mass spectrometry

Supplementary files

Article information

Article type
Paper
Submitted
26 ก.พ. 2567
Accepted
29 เม.ย. 2567
First published
02 พ.ค. 2567
This article is Open Access
Creative Commons BY-NC license

Analyst, 2024,149, 3293-3301

A facile method for monitoring sphingomyelin synthase activity in HeLa cells using liquid chromatography/mass spectrometry

P. M. Sundaraswamy, Y. Minami, J. Jayaprakash, S. G. B. Gowda, H. Takatsu, D. Gowda, H. Shin and S. Hui, Analyst, 2024, 149, 3293 DOI: 10.1039/D4AN00304G

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