Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2001-12-19
2003-08-19
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S004000, C435S019000
Reexamination Certificate
active
06607893
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally provides a method of measuring the biological activity of diacylglycerol acyltransferase (DGAT). Specifically, the present invention provides a method for rapid, mass screening of compounds which are able to modulate the biological activity of DGAT. More specifically, the present invention provides an assay system for measuring DGAT activity which allows for greater DGAT activity while eliminating acyl CoA:acyltransferase (ACAT) and ethanol acyltransferase (EAT) activities as well as significantly reducing fatty acylhydrolase (AH) activity.
BACKGROUND OF THE INVENTION
Hypertriglyceridemia is a risk factor for the development of cardiovascular diseases (Gaziano J., Hennekens C., O'Donnell C, Breslow J., Buring J. Fasting triglycerides, high-density lipoprotein, and risk of myocardial infarction. Circulation 1997;96:2520-2525). Triglycerides (TG) also play an important role in the fat loading of adipocytes (Coleman R., Bell R. Triacylglycerol synthesis in isolated fat cells.
J. Biol. Chem.
1976;251 :4537-4543), and therefore play a major role in obesity. Additionally, triglycerides are required for the assembly of apoB-100 containing lipoproteins such as VLDL and LDL (Bostrom K., Boren J., Wettesten M., et al. Studies on the assembly of apo B-100-containing lipoproteins in HepG2 cells.
J. Bio. Chem.
1988;263:4434-4442; Pullinger C., North J., Teng B., Rifici V., Ronhild de Brito A; Scott J. The apolipoprotein B gene is constituitively expressed in HepG2 cells: regulation of secretion by oleic acid, albumin, and insulin, and measurement of the mRNA half-life.
J. Lipid Res.
1989;30:1065-1077). Consequentially, there is considerable interest in developing therapies for lowering triglyceride levels. The enzyme known to catalyze the final/committed step in triglyceride biosynthesis is diacylglycerol acyltransferase (DGAT) (Coleman R. Diacylglycerol acyltransferase and monoacylglycerol acyltransferase from liver and intestine.
Methods in Enzymology
1992;209:98-104). DGAT catalyzes the transfer of coenzymeA activated fatty acids to the 3 position of 1,2-diacylglycerols, forming a triglyceride molecule (Lehner R; Kuksis A. Biosynthesis of triacylglycerols.
Prog. Lipid Res.
1996;35(No. 2):169-201; Bell R. Enzymes of glycerolipid synthesis in eukaryotes.
Ann Rev. Biochem.
1980;49:459-487). Therefore, inhibition of DGAT activity would lead to decreased triglyceride production through this pathway, which would result in the concomitant lowering of plasma VLDL/LDL and possibly increases in HDL. However, a mass screen for the isolation of specific DGAT inhibitors has not been previously established due to technical difficulties associated with establishment of such an assay.
Conventional DGAT assays have low activities on the order of pmoles TG/min/mg microsomal protein and are contaminated by the products of several other enzymatic reactions. Furthermore, the product of the DGAT catalyzed reaction is usually resolved by TLC analysis, which is impractical for use in a mass screen. A method using organic solvents to extract DGAT generated TG from isolated microsomes has been previously described (Coleman R. A. Diacylglycerol acyltransferase and monoacylglycerol acyltransferase from liver and intestine.
Meth. Enzymology
1992;209:98-104). However, several steps are involved in the extraction making this technique very difficult to adapt to a mass screen. Applicants have developed a solvent system comprising of acetone:chloroform or ethanol:chloroform which dramatically boosts DGAT activity while simultaneously suppressing the activity of interfering enzymes. In addition, a 1-step extraction method that specifically extracts TG from the DGAT catalyzed reaction mixture has been developed. To screen for modulators of DGAT activity, applicants have modified the DGAT assay of the present invention and have altered the 1-step extraction method of the present invention into a 96-well format that allows for high throughput screening modulators/compounds. These modifications enable the DGAT assay to be automated, i.e., to be carried out by a robot.
SUMMARY OF THE INVENTION
The present invention provides a method for measuring diacylglycerol acetyltransferase (DGAT) activity which utilizes a novel solvent system to reduce and/or eliminate the activities of related or interfering compounds.
The present invention also discloses a method for determining whether a compound is useful for modulating DGAT biological activity. The method is capable of being utilized for mass screening of compounds as modulators of the biological activity of DGAT.
REFERENCES:
patent: 2002/0127627 (2002-09-01), Ramharack et al.
Gaziano J., Hennekens C., O'Donnell C., Breslow J., Buring J., Fasting triglycerides, high-density lipoprotein, and risk of myocardial infarction. Circulation 1997; 96: 2520-2525.
Coleman R., Bell R., Triacylglycerol synthesis in isolated fat cells, J. Biol. Chem. 1976: 251: 4537-4543.
Bostrom K., Boren J., Wettesten M., et al., Studies on the assembly of apo B-100-containing lipoproteins in HepG2 cells, J. Bio. Chem., 1988; 263: 4434-4442.
Pullinger C., North J., Teng B., Rifici V., Ronhild de Brito A; Scott J., The apoliopoprotein B gene is constituitively expressed in HepG2 cells: regulation of secretion by oleic aid, albumin, and insulin, and measurement of the mRNA half-life, J. Lipid Res., 1989; 30: 1065-1077.
Coleman R., Diacylglycerol acyltransferase and monoacylglycerolacyltransferase from liver and intestine, Methods in Enzymology, 1992; 209: 98-204.
Lehner R, Kuksis A., Biosynthesis of triacylglycerols, Prog. Lipid Res., 1996; 35 (No. 2): 169-201.
Bell R., Enzymes of glycerolipid synthesis in eukaryotes, Ann Rev. Biochem, 1980; 49: 459-487.
Ramharack Randy Ranjee
Spahr Mark Allan
Kurlandsky David R.
Leary Louise N.
Warner-Lambert & Company
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