Truly Generic Assays
Group transfer reactions, such as phosphorylation, glycosylation, and methylation, are important on/off switches for signaling proteins in a variety of disease pathways. The activity of drugs and hormones is also controlled by group transfer reactions. The enzymes that catalyze these reactions are generally assayed by detecting the amount of product formed or substrate depleted. This requires individual assays and reagents, however, since most enzymes within a group transfer family have different substrates and products.
BellBrook Labs’ Transcreener platform takes a different approach. The generic assays detect nucleotide products of a donor molecule cleavage, such as ADP for kinases and ATPases; UDP (uridine diphosphate) for glycosyltransferases; coenzyme A for acetyltransferases; and AMP (adenosine monophosphate) or GMP (guanine monophosphate) for phosphodiesterases.
Each type of group transfer reaction generates just one donor product, so a single set of Transcreener reagents can be used for all family members, regardless of the starting substrate or end product. “The assays are truly generic,” says Dr. Lowery.
The Transcreener HTS assays are based on competitive fluorescence polarization immunoassays to monitor the binding of a donor product to a specific antibody. This detection method has less interference and a lower false positive rate than assays that measure substrate depletion.
Moreover, the same assays can help pharmaceutical companies to predict adverse side effects, which are responsible for nearly half of all clinical failures. Glycotransferase enzymes, for instance, play an important role in the metabolism of drugs and the production of toxic metabolites. The development of new assays for targeted classes of proteins will enable the screening of many more enzymes in emerging target families, such as ubiquitin ligases, which produce AMP.
“We want to give people access to targets that they otherwise would not be able to screen, to make a real difference,” comments Dr. Lowery.
The main advantage of the Transcreener assays over existing HTS methods is the wide diversity of targets that can be addressed with just a few assays, says Dr. Lowery.
For instance, there are about 2,000 human proteins that utilize ATP. Since most of them generate AMP or ADP as a product, just two Transcreener assays are needed to detect them. The assays come in a single-addition, mix-and-read format, making them ideal for automated HTS. The reagents and consumables work with existing HTS equipment, and the results are read on standard multiwell plate readers. The nucleotide product of a group transfer reaction is usually generated in stoichiometric amounts, giving a direct measure of enzyme turnover.