A High-throughput two-phase partition assay to measure the activity of lipid-metabolizing enzymes

Kristie, James; Toth, Joshuaine; Silverstrim, Christine; Pickett, Walter C.; Landro, James A.

doi:10.1016/j.ab.2006.07.036

Cited by 8 publications

(3 citation statements)

References 37 publications

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“…After phase partitioning by the addition of a phase partition scintillation fluid (MicroScint-E, PerkinElmer), which serves as both a scintillation fluid and a phase partition agent, the generated product [ 14 C]tridecanoylglycerol ([ 14 C]TAG) was quantified from the upper organic phase. 27 An initial experiment comparing DGAT2 versus mock membranes (1.5 μg for each), which was analyzed via TLC, showed that the mock membrane generated significant levels of [ 14 C]decanoic acid (1.26 μM, 13% hydrolysis of [ 14 C]decanoyl-CoA) (Figure 2A, lane 1) while the DGAT2 membrane generated 1.73 μM [ 14 C]TAG as well as 0.44 μM [ 14 C]decanoic acid (Figure 2A, lane 6) under the DGAT2 assay conditions described in Materials and Methods, except 10 μM [ 14 C]decanoyl-CoA and 30 μM 1,2-didecanoyl-sn-glycerol were used as substrates in this assay. Because the background activity ([ 14 C]decanoic acid generated by mock membrane) is significant (∼25% of [ 14 C]TAG) in the DGAT2 reaction, this system would not allow an optimal DGAT2 assay window without separation of [ 14 C]TAG from [ 14 C]decanoic acid.…”

Section: ■ Resultsmentioning

confidence: 99%

“…We have utilized the DGAT2 membrane fraction prepared from the Sf9 insect cell expression system as a source of DGAT2 enzyme, which has been widely used in the literature. ,− ,,,,, DGAT2 activity was determined by measuring the incorporation of the [1- 14 C]decanoyl moiety into triacylglycerol using [1- 14 C]decanoyl-CoA and 1,2-didecanoyl- sn -glycerol as substrates. After phase partitioning by the addition of a phase partition scintillation fluid (MicroScint-E, PerkinElmer), which serves as both a scintillation fluid and a phase partition agent, the generated product [ 14 C]tridecanoylglycerol ([ 14 C]TAG) was quantified from the upper organic phase . An initial experiment comparing DGAT2 versus mock membranes (1.5 μg for each), which was analyzed via TLC, showed that the mock membrane generated significant levels of [ 14 C]decanoic acid (1.26 μM, 13% hydrolysis of [ 14 C]decanoyl-CoA) (Figure A, lane 1) while the DGAT2 membrane generated 1.73 μM [ 14 C]TAG as well as 0.44 μM [ 14 C]decanoic acid (Figure A, lane 6) under the DGAT2 assay conditions described in Materials and Methods, except 10 μM [ 14 C]decanoyl-CoA and 30 μM 1,2-didecanoyl- sn -glycerol were used as substrates in this assay.…”

Section: Resultsmentioning

confidence: 99%

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Mechanistic Characterization of Long Residence Time Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2)

Pabst

Futatsugi

et al. 2018

Biochemistry

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Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triacylglycerol (TAG) synthesis. Genetic knockdown or pharmacological inhibition of DGAT2 leads to a decrease in very-low-density lipoprotein TAG secretion and hepatic lipid levels in rodents, indicating DGAT2 may represent an attractive therapeutic target for treatment of hyperlipidemia and hepatic steatosis. We have previously described potent and selective imidazopyridine DGAT2 inhibitors with high oral bioavailability. However, the detailed mechanism of DGAT2 inhibition has not been reported. Herein, we describe imidazopyridines represented by PF-06424439 (1) and 2 as long residence time inhibitors of DGAT2. We demonstrate that 1 and 2 are slowly reversible, time-dependent inhibitors, which inhibit DGAT2 in a noncompetitive mode with respect to the acyl-CoA substrate. Detailed kinetic analysis demonstrated that 1 and 2 inhibit DGAT2 in a two-step binding mechanism, in which the initial enzyme–inhibitor complex (EI) undergoes an isomerization step resulting in a much higher affinity complex (EI*) with overall apparent inhibition constants (K i*app values) of 16.7 and 16.0 nM for 1 and 2, respectively. The EI* complex dissociates with dissociation half-lives of 1.2 and 1.0 h for 1 and 2, respectively. A binding assay utilizing 125I-labeled imidazopyridine demonstrated that the level of imidazopyridine binding to DGAT2 mutant enzymes, H161A and H163A, dramatically decreased to 11–17% of that of the wild-type enzyme, indicating that these residues are critical for imidazopyridines to bind to DGAT2. Taken together, imidazopyridines may thus represent a promising lead series for the development of DGAT2 inhibitors that display an unprecedented combination of potency, selectivity, and in vivo efficacy.

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Section: ■ Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Mechanistic Characterization of Long Residence Time Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2)

Pabst

Futatsugi

et al. 2018

Biochemistry

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“…one published 2-phase partition assay relies on a viscous liquid scintillation fluid that can be difficult to rapidly and accurately dispense in low microliter volumes. 7 in addition, the light emission from the scintillation fluid is blue-shifted, a less favorable emission profile for compound screening compared to red light emission. another assay using the scintillation format for fatty acids requires the phospholipid coating of a scintillating microplate to capture labeled fatty acids.…”

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confidence: 99%

A Simplified Scintillation Proximity Assay for Fatty Acid Synthase Activity: Development and Comparison with Other FAS Activity Assays

Bays¹,

Hill²,

Kariv³

2009

SLAS Discovery

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Fatty acid synthase (FaS), an essential enzyme for de novo lipogenesis, has been implicated in a number of disease states, including obesity, dyslipidemia, and cancer. to identify small-molecule inhibitors of FaS, the authors developed a beadbased scintillation proximity assay (SPa) to detect the fatty acid products of FaS enzymatic activity. this homogeneous SPa assay discriminates between a radiolabeled hydrophilic substrate of FaS (acetyl-coenzyme a) and the labeled lipophilic products of FaS (fatty acids), generating signal only when labeled fatty acids are present. the assay requires a single addition of unmodified polystyrene imaging SPa beads and can be miniaturized to 384-or 1536-well density with appropriate assay statistics for high-throughput screening. high-potency FaS inhibitors were used to compare the sensitivity of the SPa bead assay with previously described assays that measure FaS reaction intermediates (coa-Sh and nadP + ). the advantages and disadvantages of these different FaS assays in small-molecule inhibitor discovery are discussed. (Journal of Biomolecular Screening 2009:636-642)

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Lead discovery for mammalian elongation of long chain fatty acids family 6 using a combination of high-throughput fluorescent-based assay and RapidFire mass spectrometry assay

Takamiya

Sakurai²,

Teranishi³

et al. 2016

Biochemical and Biophysical Research Communications

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A High-throughput two-phase partition assay to measure the activity of lipid-metabolizing enzymes

Cited by 8 publications

References 37 publications

Mechanistic Characterization of Long Residence Time Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2)

Mechanistic Characterization of Long Residence Time Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2)

A Simplified Scintillation Proximity Assay for Fatty Acid Synthase Activity: Development and Comparison with Other FAS Activity Assays

Lead discovery for mammalian elongation of long chain fatty acids family 6 using a combination of high-throughput fluorescent-based assay and RapidFire mass spectrometry assay

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