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Biological Chemistry

Assay For Key Ghrelin Enzyme

Click Chemistry: Approach could help researchers find antiobesity drug leads

by Stu Borman
September 27, 2010 | A version of this story appeared in Volume 88, Issue 39

Credit: Adapted from Angew. Chem. Int. Ed. © 2010
Credit: Adapted from Angew. Chem. Int. Ed. © 2010

A new type of assay based on click chemistry will make it possible to carry out high-throughput screens to find inhibitors of the gut hormone ghrelin, a target for obesity and diabetes medications.

Ghrelin promotes hunger, helps regulate energy use, and impairs glucose control, and researchers have been actively seeking agents that lower ghrelin levels as obesity or diabetes treatments. Some have focused on finding inhibitors for ghrelin-O-acyltransferase (GOAT), which activates ghrelin by esterifying an inactive form with a fatty acid group.

But measuring GOAT activity has been hard, impeding the search for GOAT inhibitors. Current procedures, such as radiolabeling tests, are difficult and time-consuming, and they can’t be used in high-throughput studies to screen for enzyme inhibitors.

Now, research associate Amanda L. Garner and chemistry professor Kim D. Janda of Scripps Research Institute, in La Jolla, Calif., have fixed that problem by devising a simple assay called cat-ELCCA (catalytic enzyme-linked click-chemistry assay) that’s highly sensitive for GOAT and amenable to high-throughput screening (Angew. Chem. Int. Ed., DOI: 10.1002/anie. 201003387). They substitute an alkyne-containing substrate for the fatty acid that GOAT attaches when it activates ghrelin. They then use click chemistry to attach an azide-tagged version of horseradish peroxidase (HRP) to the alkyne-derivatized ghrelin.

When GOAT is active, HRP, in the presence of a reagent called Amplex Red, generates an amplified signal denoting the enzyme’s activity by catalyzing production of a fluorescent product, resorufin. When GOAT is inactive because it has been turned off by an inhibitor, no resorufin is produced.

The technique could be useful for assaying not only GOAT activity but also that of other enzymes that make posttranslational modifications to peptides and proteins, such as palmitoylation enzymes and histone acetylases and deacetylases. Such enzymes have been widely studied for their role in cancer and other diseases.

If a functional group “involved in posttranslational modification can carry an alkyne, and the enzyme doing the posttranslational modification accepts that group as a substrate,” then the new technique could be used to measure that enzyme’s activity, says bioassay specialist Eric V. Anslyn of the University of Texas, Austin. “What’s unique here,” compared with existing assays, “is that click chemistry brings along an enzyme that catalytically amplifies a signal.”

Obesity researcher Paul T. Pfluger of the University of Cincinnati College of Medicine, whose group helped demonstrate GOAT's role in energy balance, says: "People are interested in seeing if there are inhibitors of GOAT and if GOAT has a real physiological function" that can be exploited for drug discovery. The Scripps assay "gives us the first high-throughput method of measuring GOAT activity, and that’s something the pharmaceutical industry will be very interested in."



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