Abstract
Recent genome wide association studies have linked tribbles pseudokinase 1 (TRIB1) to the risk of coronary artery disease (CAD). Based on the observations that increased expression of TRIB1 reduces secretion of VLDL and is associated with lower plasma levels of LDL cholesterol and triglycerides, higher plasma levels of HDL cholesterol and reduced risk for myocardial infarction, we carried out a high throughput phenotypic screen based on quantitative RT-PCR assay to identify compounds that induce TRIB1 expression in human HepG2 hepatoma cells. In a screen of a collection of diversity-oriented synthesis (DOS)-derived compounds, we identified a series of benzofuran-based compounds that upregulate TRIB1 expression and phenocopy the effects of TRIB1 cDNA overexpression, as they inhibit triglyceride synthesis and apoB secretion in cells. In addition, the compounds downregulate expression of MTTP and APOC3, key components of the lipoprotein assembly pathway. However, CRISPR-Cas9 induced chromosomal disruption of the TRIB1 locus in HepG2 cells, while confirming its regulatory role in lipoprotein metabolism, demonstrated that the effects of benzofurans persist in TRIB1-null cells indicating that TRIB1 is sufficient but not necessary to transmit the effects of the drug. Remarkably, active benzofurans, as well as natural products capable of TRIB1 upregulation, also modulate hepatic cell cholesterol metabolism by elevating the expression of LDLR transcript and LDL receptor protein, while reducing the levels of PCSK9 transcript and secreted PCSK9 protein and stimulating LDL uptake. The effects of benzofurans are not masked by cholesterol depletion and are independent of the SREBP-2 regulatory circuit, indicating that these compounds represent a novel class of chemically tractable small-molecule modulators that shift cellular lipoprotein metabolism in HepG2 cells from lipogenesis to scavenging.
Highlights
Despite widespread use of cholesterol-lowering drugs, cardiovascular disease remains one of the leading causes of death worldwide and there is a need for novel approaches to improve therapies [1]
Clinical efficacy of statin therapy is limited by the fact that activation of SREBP-2 leads to increased expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), which acts as a negative regulator of LDL uptake by promoting degradation of LDL receptor
After completing the triaging process and reviewing existing structure-activity relationships (SAR) generated from the diversity-oriented synthesis (DOS) collection, we identified a benzofuran scaffold [24] (Fig. 1A), which upregulated tribbles pseudokinase 1 (TRIB1) in both cell lines and was able to reduce apoB secretion in HepG2 cells >30%, mimicking the effects of TRIB1 cDNA overexpression [14]
Summary
Despite widespread use of cholesterol-lowering drugs, cardiovascular disease remains one of the leading causes of death worldwide and there is a need for novel approaches to improve therapies [1]. Because 70% of LDL is removed from the circulation by LDL receptor-mediated uptake in the liver, therapeutic strategies that lead to elevated hepatic expression of the LDL receptor gene, LDLR, have proven to be efficacious in lowering LDL-C and provide protection from cardiovascular disease. Through the inhibition of HMG CoA reductase, deplete cholesterol in the ER of hepatic cells, leading to the activation of the SREBP-2-dependent transcriptional program, which includes increased expression of LDLR.
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