Graduate School of Global Food Resources, Hokkaido University, Kita-9, Nishi-9, Kita-Ku, Sapporo 060-0809, Japan.
Graduate School of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo 060-0812, Japan.
Analyst. 2024 Jun 10;149(12):3293-3301. doi: 10.1039/d4an00304g.
Sphingomyelin synthase (SMS) is a sphingolipid-metabolizing enzyme involved in the 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. 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 support for the experimental findings.
鞘氨醇合酶（SMS）是一种参与鞘氨醇（SM）从神经酰胺（Cer）合成的鞘脂代谢酶。最近的研究表明，SMS 是代谢疾病如脂肪肝、2 型糖尿病、动脉粥样硬化和结直肠癌的关键治疗靶点。然而，由于当前基于荧光的筛选测定法的灵敏度和选择性有限，很少有 SMS 抑制剂被鉴定出来。在这项研究中，我们开发了一种简单的基于细胞的测定法，与液相色谱/串联质谱（LC-MS/MS）相结合，用于筛选 SMS 抑制剂。使用稳定表达 SMS1 或 SMS2 的 HeLa 细胞进行筛选。非荧光非天然 C6-Cer 被用作 SMS 的底物，以产生 C6-SM。使用 LC-MS/MS 监测和定量细胞中的 C6-Cer 和 C6-SM 水平。测量了已知的 SMS 抑制剂白果醇 C15:1（GA）的活性。GA 对 SMS1 和 SMS2 的半最大抑制浓度分别为 5.5 μM 和 3.6 μM。为了验证这些发现，对 hSMS1 和 hSMS2 蛋白进行了优化，以进行分子对接研究。进行了分析以评估 GA 与 SMS1 和 SMS2 的相互作用及其结合亲和力。这项研究提供了一种筛选新型 SMS 抑制剂的分析方法，并为实验结果提供了支持。
