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| | Measurement of DGAT1 Inhibitory Activity |
| Description: | As a buffer used for the enzymatic reaction of DGAT1, 100 mM Tris-HCl (pH7.4), 200 mM Sucrose, and 20 mM MgCl2, 0.125% Bovin Serum Albumin (BSA) were used. To this buffer, a test compound with predetermined concentration of test compound, 15 μM dioleylglycerol, 5 μM [14C]-palmitoyl-CoA, 100 μg-protein/mL, highly DGAT1-expressing expresSF+ microsome, 0.75% acetone, and 1% dimethylsulfoxide were added, and a triglyceride (TG) synthesis reaction in a volume of 100 μL was carried out at 30° C. for 20 minutes. 90 μL of the reaction solution was added to 810 μL of methanol to cease the reaction. The reaction solution was added to Oasis μ Elution plate (Waters) and eluted with 150 μL of mixture of acetonitrile:isopropanol (=2:3). 150 μL of MicroScinti-40 (Perkin-Elmer Corp.) were added to the eluted solution and the mixture was sufficiently stirred, and an amount of [14C]-TG produced in the reaction was determined by measuring using TopCount-NXT (Perkin-Elmer Corp.).The inhibitory ratio was calculated by the following equation.Inhibitory ratio (%)=(1−(TG amount when the test compound was added−blank TG amount)/(control TG amount−blank TG amount))×100Here, a count of [14C]-TG in the solution where the reaction was carried out without adding the test compound was regarded control TG amount, and a count of [14C]-TG in the solution to which the test compound and highly DGAT1 expressing expresSF+ microsome were not added was regarded as blank TG amount. Further, a concentration of test compound required to inhibit the synthesis of [14C]-TG by 50% (IC50 value) was calculated by Prism 5.01 (GraphPad Softwear). |
| Affinity data for this assay | |
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