High-throughput assay for long chain fatty acyl-CoA elongase using homogeneous scintillation proximity format.
Elongase of very-long-chain fatty acid (Elovl) 6 is a rate-limiting enzyme that is responsible for the elongation of long-chain fatty acids such as palmitoic acid (C16). Elovl6 is abundantly expressed in liver and adipose tissue, and the expression levels in these tissues are up-regulated in obese animals. Furthermore, Elovl6-deficient mice display improved glucose homeostasis and insulin sensitivity, suggesting that Elovl6 might be a potential therapeutic target for metabolic disorders. From the drug discovery point of view, it is critical to establish a high-throughput screening (HTS) assay for the identification of therapeutic agents. Conventional assay methods for fatty acid elongases include an extraction step for respective radioactive products from the reaction mixtures, which is labor-intensive and not feasible for HTS. In this study, we utilized the acyl-coenzyme A (CoA) binding protein (ACBP) as a molecular probe to detect radioactive long-chain acyl-CoA, a direct product of Elovl6. Recombinant ACBP binds stearoyl-CoA but not malonyl-CoA, enabling specific detection of the radioactive product in the homogenous reaction mixture without the liquid extraction step. Finally, combination of ACBP and scintillation proximity assay beads led to specific detection of Elovl6 activity with appropriate window and reproducibility amenable to HTS (signal-to-background noise ratio of approximately 13.0-fold, Z' = 0.85). The assay system described here has the potential to enable identification of small compounds that modify fatty acid elongase activity and assessment of the therapeutic potential of acyl-CoA elongases.
Shimamura
Ken
K
Tsukuba Research Institute, Banyu Pharmaceutical Company, Ltd., Ibaraki, Japan.
Miyamoto
Yasuhisa
Y
Kitazawa
Hidefumi
H
Kobayashi
Tsutomu
T
Kotani
Hidehito
H
Tokita
Shigeru
S
D000123:Acetyltransferases / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000378:metabolism; D026261:Diazepam Binding Inhibitor / Q000378:metabolism; D055808:Drug Discovery; D000081031:Fatty Acid Elongases; D006801:Humans; D012588:Scintillation Counting
D000214:Acyl Coenzyme A; D026261:Diazepam Binding Inhibitor; C000630955:ELOVL6 protein, human; C000630968:Elovl6 protein, mouse; C017725:stearoyl-coenzyme A; D000123:Acetyltransferases; D000081031:Fatty Acid Elongases