Discovery of a Novel Class of Diacylglycerol Acyltransferase 2 Inhibitors with a 1<i>H</i>-Pyrrolo[2,3-<i>b</i>]pyridine Core

Abstract

Diacylglycerol acyltransferase 2 (DGAT2), which catalyzes the final step in triacylglycerol (TG) synthesis, is a key enzyme associated with hepatic steatosis and insulin resistance. Here, using an in vitro screen of 20000 molecules, we identified a class of compounds with a substituted 1H-pyrrolo[2,3-b]pyridine core which proved to be potent and selective inhibitors of human DGAT2. Of these compounds, H2-003 and -005 exhibited a considerable reduction in TG biosynthesis in HepG2 hepatic cells and 3T3-L1 preadipose cells. These compounds exert DGAT2-specific-inhibitory activity, which was further confirmed in DGAT2-or DGAT1-overexpressing HEK293 cells. In addition, these compounds almost completely abolished lipid droplet formation in 3T3-L1 cells when co-treated with a DGAT1 inhibitor, which was not attained using either a DGAT2 or DGAT1 inhibitor alone. Collectively, we identified two DGAT2 inhibitors, H2-003 and -005. These compounds will aid in DGAT2-related lipid metabolism research as well as in therapeutic development for the treatment of metabolic diseases associated with excessive TG.Key words metabolic disease; triacylglycerol; diacylglycerol acyltransferase 2 (DGAT2); small molecule inhibitor Triacylglycerols (TGs), a major type of neutral lipid, are essential forms of energy storage composed of three fatty acids attached to a glycerol in eukaryotes.1) However, positive energy balance results in excessive accumulation of TG throughout the body and leads to several metabolic diseases, such as obesity, hyperlipidemia, hypertension, hepatic steatosis, and insulin resistance.2-4) Therefore, inhibiting TG biosynthesis has been suggested as one of therapeutic strategies for treating these diseases.Diacylglycerol acyltransferase (DGAT) enzymes, which exist as two isoforms DGAT1 and DGAT2, catalyze the final step in TG biosynthesis. These isozymes are encoded by two distinct genes and share no amino acid sequence homology. 5)Furthermore, DGAT1 is highly expressed in the small intestine, whereas DGAT2 is primarily expressed in the liver. 6)This differential expression pattern suggests a fundamentally different role for each DGAT enzyme in TG metabolism.Several lines of evidence have shown that DGAT2 is more essential than DGAT1 for homeostatic control of TGs in vivo. DGAT2 knockout mice are severely deficient in TGs (approximately 90% TG reduction) and have impaired skin barrier function, leading to early death. 7) In contrast, DGAT1 knockout mice are still viable with a moderate reduction in TG (approximately 50% TG reduction). 8,9) In addition, inhibition of DGAT2 expression by antisense oligonucleotides reduced hepatic TG content and increased fatty acid oxidation, thereby reversing diet-induced hepatic steatosis and insulin resistance in rodents. 10,11) Conversely, upregulation of DGAT2 expression increased cytoplasmic TG content and lipid droplets (LDs) in rat hepatocytes 12) and adipocytes. 13) Similarly, liver-directed DGAT2 overexpression caused hepatic TG accumulation 12) and promoted hepatic insulin r...

Hepatocyte Deletion of Triglyceride‐Synthesis Enzyme Acyl CoA: Diacylglycerol Acyltransferase 2 Reduces Steatosis Without Increasing Inflammation or Fibrosis in Mice

“…Inhibition of DGAT2 is being considered as a therapy for NAFLD/NASH in humans. (12,14,15) In this respect, our findings in mice suggest that DGAT2 inhibition could be effective in lowering hepatic fat content in humans and may do so without increased risk of inflammation and injury. Additionally, we found no adverse effects on insulin sensitivity with DGAT2 deletion in liver.…”

“…It is unclear whether blocking TG synthesis by DGAT2 would be beneficial for treating human NAFLD. This is an important question, given that highly potent and specific inhibitors are available for DGAT2 . To address this question, DGAT2 activity was previously lowered in adult mice by inhibiting enzymatic activity or gene expression.…”

“…This is an important question, given that highly potent and specific inhibitors are available for DGAT2. (12)(13)(14)(15) To address this question, DGAT2 activity was previously lowered in adult mice by inhibiting enzymatic activity or gene expression. In one study, both diet-induced and genetically obese mice were treated with DGAT2 antisense oligonucleotides (ASOs), which reduced Dgat2 expression in liver (and adipose tissue) and decreased hepatic TG content.…”

The Role of Diacylglycerol Acyltransferase (DGAT) 1 and 2 in Cardiac Metabolism and Function

“…Targeting TG synthesis has been proposed as a therapeutic strategy for hyperlipidemia for more than a decade 9 . Multiple small molecular inhibitors of DGAT, especially DGAT2, have been developed recently 18 , 27 , 28 . However, previous studies have suggested that inhibition of TG synthesis and turnover in the heart could contribute to lipotoxicity and hence cautioned the use of pharmacological inhibition of DGAT.…”

Identification of DGAT2 Inhibitors Using Mass Spectrometry

“…13 However, the assay conditions reported produced a small window (~1.5-fold) for DGAT2 compared with mock membranes. Recently, new in vitro approaches have been developed for DGAT2 screening assays, including the use of basic FlashPlate, which is based on scintillation proximity assay, 14,15 and mass spectrometry (HTMS). 16 Here we describe additional attempts to generate a robust DGAT2 assay amenable to small-molecule screening and detailed enzymatic characterization for development of highly potent DGAT2 inhibitors.…”