Data Sheet 1_Potential lipid-lowering effects of preussin B on inhibition of intestinal cholesterol absorption: integrative mechanisms of action and proteomic analysis.docx

IntroductionHyperlipidemia remains a significant risk factor for cardiovascular diseases and is a leading cause of death, driving the need for novel and effective cholesterol-lowering agents. Preussin (1) has recently demonstrated lipid-lowering effects by inhibiting intestinal cholesterol absorption in human colorectal adenocarcinoma (Caco-2) cells and in an ex vivo intestinal loop in rats, comparable to those of ezetimibe. Ezetimibe is the only drug that targets reducing intestinal cholesterol absorption. Recently, two natural (preussin C, 2, and preussin B, 4) and three unnatural (3, 5, and 6) analogues of preussin have been synthesized and also displayed interesting lipid-lowering effects in human hepatocellular carcinoma (HepG2) cells. However, the underlying mechanisms and the potential lipid-lowering effects of preussin and its synthetic analogues in inhibiting cholesterol absorption are not yet fully understood.
MethodsThis study aims to evaluate the inhibitory effect of cholesterol absorption by preussin and its analogues using a fluorescent-micelle cholesterol transport in intestinal Caco-2 cells, which further confirmed by an in vivo cholesterol absorption assay. The most potent analogue was further investigated for its cellular and molecular mechanisms in reducing lipid levels and identifying possible target proteins.
ResultsAll synthetic derivatives markedly inhibited cholesterol absorption in the intestinal Caco-2 cells to a similar extent as preussin and ezetimibe. However, only compound 4 (preussin B) displayed the most significant reduction in plasma cholesterol, identical to preussin and ezetimibe, with similar potency in rats. The precise mechanisms and potential targets of this potent compound were additionally identified using protein binding assay and label-free quantification via proteomics analysis. The results revealed substantial differential expression in four proteins associated with lipid metabolism. Notably, glutamic-oxaloacetic transaminase 2 (GOT2), one of the altered proteins, was shown to interact with compound 4 in a protein binding assay. Molecular dynamics simulation analysis indicated that compound 4 binds to a pocket on GOT2 comparable to that of its natural cofactor. This interaction, combined with the observed downregulation of GOT2 expression, contributed to the inhibition of cholesterol absorption.
ConclusionThese findings suggest that synthetic compound 4 (preussin B) is a promising candidate for inhibiting cholesterol absorption in the treatment of hyperlipidemia.

引言：高脂血症（Hyperlipidemia）仍是心血管疾病（cardiovascular diseases）的重要危险因素，也是致死的主要原因，推动了新型高效降胆固醇药物的研发需求。普鲁辛（Preussin，1）近期被证实可通过抑制人结肠腺癌细胞（Caco-2细胞）及大鼠离体肠袢的肠道胆固醇吸收发挥降脂作用，其效果与依折麦布（ezetimibe）相当。依折麦布是目前唯一一款靶向抑制肠道胆固醇吸收的药物。近期，研究者已合成2种普鲁辛天然类似物（普鲁辛C，2；普鲁辛B，4）及3种非天然类似物（3、5、6），并在人肝细胞癌（HepG2）细胞中证实其具有良好的降脂活性。然而，普鲁辛及其合成类似物抑制胆固醇吸收的具体分子机制与潜在降脂效果尚未完全阐明。
方法：本研究旨在通过肠道Caco-2细胞的荧光胶束胆固醇转运实验，评估普鲁辛及其类似物对胆固醇吸收的抑制作用，并进一步通过体内胆固醇吸收实验验证该效果。选取活性最强的类似物，深入研究其降低脂质水平的细胞与分子机制，并筛选潜在的作用靶蛋白。
结果：所有合成衍生物均可显著抑制肠道Caco-2细胞的胆固醇吸收，其效果与普鲁辛及依折麦布相当。但仅化合物4（普鲁辛B）可显著降低大鼠血浆胆固醇水平，效果与普鲁辛及依折麦布一致，且活性相近。本研究进一步通过蛋白结合实验与蛋白质组学分析的无标记定量技术，明确了该强效化合物的精准作用机制与潜在靶点。结果显示，共有4种与脂质代谢相关的蛋白质出现显著差异表达。值得注意的是，差异表达蛋白之一的谷草转氨酶2（glutamic-oxaloacetic transaminase 2, GOT2）在蛋白结合实验中被证实可与化合物4相互结合。分子动力学模拟分析表明，化合物4可结合至GOT2的口袋位点，该位点与其天然辅因子的结合位点一致。这种相互作用，加上观测到的GOT2表达下调，共同促成了胆固醇吸收的抑制效应。
结论：本研究结果表明，合成化合物4（普鲁辛B）是一款极具潜力的胆固醇吸收抑制剂，有望用于高脂血症的治疗。