Novel High-Throughput Screen Focuses on Appetite Regulating Enzyme

A fluorescence-based assay method has been used as a high-throughput screen for drugs to inhibit the enzyme ghrelin O-acyltransferase (GOAT), which is critically linked to molecular pathways regulating appetite.

Ghrelin is a 28 amino acid, appetite-stimulating peptide hormone secreted by the food-deprived stomach. Serine-3 of ghrelin is acylated with an eight-carbon fatty acid, octanoate, which is required for its endocrine actions.

GOAT is a membrane-bound enzyme that attaches octanoate to serine-3 of ghrelin. Analysis of the mouse genome has revealed that GOAT belongs to a family of 16 hydrophobic membrane-bound acyltransferases. GOAT is the only member of this family that octanoylates ghrelin when coexpressed in cultured endocrine cell lines with prepro-ghrelin. GOAT activity requires catalytic asparagine and histidine residues that are conserved in this family. Consistent with its function, GOAT mRNA is largely restricted to stomach and intestine, the major ghrelin-secreting tissues.

Investigators at the Scripps Research Institute (La Jolla, CA, USA) applied a novel fluorescence-based assay to the problem of assessing GOAT activity. The assay, which they called cat-ELCCA (catalytic assay using enzyme-linked click chemistry assay), associates "click chemistry” with an ELISA-style approach. Click chemistry is a proprietary development of the Scripps Research Institute.

"There has not been a simple screen until now,” said senior author Dr. Kim D. Janda, professor of chemistry and immunology at the Scripps Research Institute. Dr. Janda and his research associate Dr. Amanda Garner reported in the September 15, 2010, online edition of the journal Angewandte Chemie that "This new assay technology is both highly sensitive and reproducible, making it an excellent assay for high-throughput screening.”

Related Links:
Scripps Research Institute