Identification and Validation of a Selective Small Molecule Inhibitor Targeting the Diacylglycerol Acyltransferase 2 Activity

Abstract

Diacylglycerol acyltransferase 2 (DGAT2) is one of two distinct DGAT enzymes that catalyze the last step in triacylglycerol (TG) synthesis. Findings from previous studies suggest that inhibition of DGAT2 is a promising strategy for the treatment of hepatic steatosis and insulin resistance. Here, we identified compound 122 as a potent and selective inhibitor of human DGAT2, which appeared to act competitively against oleoyl-CoA in vitro. The selective inhibition of DGAT2 was also confirmed by the reductions in enzymatic activity and de novo TG synthesis in DGAT2-overexpressing HEK293 cells and hepatic cells HepG2. Compound 122, as a newly identified inhibitor of DGAT2, will be useful for the research on DGAT2-related lipid metabolism as well as the development of therapeutic drug for several metabolic diseases.Key words metabolic disease; triacylglycerol; diacylglycerol acyltransferase 2; small molecule inhibitor; isatin Triacylglycerol (TG), a class of neutral lipids, is the most representative storage form of energy in eukaryotic cells. 1)However, an imbalance between energy intake and expenditure can lead to the excessive accumulation of TG in tissues, which is pathologically associated with metabolic diseases such as obesity, hyperlipidemia, hypertension, hepatic steatosis, and insulin resistance.2-4) Therefore, inhibition of TG biosynthesis has been suggested to be one of therapeutic strategies to treat these diseases.The final and only committed step in the synthesis of TG is catalyzed by diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, which are encoded by two distinct genes and share limited sequence homology. 5) Both genes are ubiquitously expressed, but with the highest levels of expression found in tissues that are active in TG synthesis such as adipose tissue, liver, small intestine, and mammary gland. 6) Studies in mice have shown that DGAT2 is responsible for the majority of TG synthesis. DGAT2 knockout mice are severely deficient in TG (ca. 90% TG reduction) and have impaired skin barrier function, leading to early death, 7) while DGAT1 knockout mice are still viable with a moderate reduction in TG (ca. 50% TG reduction).8) In addition, hepatic suppression of DGAT2 with antisense oligonucleotides reduced hepatic TG content, increased fatty acid oxidation, and thereby reversed diet-induced hepatic steatosis and insulin resistance in rodents.9,10) Conversely, upregulation of DGAT2 expression induced increase of cytoplasmic TG content and lipid droplets (LDs) in rat hepatocytes 11) and adipocytes, 12) and similarly liver-directed DGAT2 overexpression caused hepatic TG accumulation 11) and promoted hepatic insulin resistance 13) in mice. Therefore, inhibition of DGAT2 enzyme, particularly by small molecule, is expected to be a feasible therapeutic strategy for hepatic steatosis and its complications, such as insulin resistance. Even though DGAT1 and DGAT2 take part in the same reaction in TG biosynthetic pathway, their biochemical properties seem to be somewhat different. In order to def...

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

“…In order to overcome this challenge and examine whether H2-003 and -005 selectively inhibit DGAT2 in mammalian cells, we first established HEK293 cell systems that inducibly overexpress DGAT2 or DGAT1 upon the addition of doxycycline. As shown in our previous report, 17) overexpression of either DGAT2 or DGAT1 alone drastically increased the fluorescence signal of LD by more than 20-fold compared with control cells that showed a faint basal signal (Fig. 3A).…”

“…17) Briefly, human DGAT1 and DGAT2 were overexpressed in Sf-9 insect cells using the Bac-to-Bac Baculovirus Expression System according to the manufacturerʼs instructions. The DGAT1 and DGAT2 cDNA clones were amplified by polymerase chain reaction (PCR) and inserted into the pFastBac1 donor vector to obtain recombinant baculoviruses.…”

“…17) Briefly, DGAT activity in total membranes prepared from DGAT2-or DGAT1-overexpressing Sf-9 and HEK293 Tet-on cells was determined by measuring the formation of Plasmid Construction and Establishment of Stable HEK293 Tet-On Cell Lines The pTRE2-hygro (Clontech) was used to tightly regulate the expression of human DGAT1 and DGAT2. Each cDNA was amplified by PCR and inserted into the pTRE2-hygro vector.…”

“…17) In this process, we found out a class of compounds that commonly have a 1H-pyrrolo [2,3-b] pyridine core structure; these molecules were designated as H2-series (Fig. 1A).…”

“…17) However, the pharmacological validation of their use, particularly in animal models, remains to be examined. Still, DGAT2 inhibitors with different modes of action or pharmacophores will be useful to advance our understanding of DGAT2 biology and develop therapeutic drugs for treating several metabolic diseases.…”