Transcreener® AMP²/GMP² Phosphodiesterase Assays

Download this application note to find out how the Transcreener dAMP exonuclease assay is a powerful tool for the discovery of TREX1 antagonists that will accelerate efforts to validate the target pharmacologically, possibly leading to a new class of immune checkpoint inhibitors.

The Transzyme Methyltransferase kit offers a complete HTS assay solution, with a validated purified enzyme, an optimal acceptor substrate and enzyme buffer and predetermined reaction conditions that yield a Z’ of at least 0.6 in BellBrook’s laboratories. This Application Note describes how to generate a dose response curve for an inhibitor using a Transzyme Methyltransferase Assay kit.

The Transcreener® EPIGEN Methyltransferase Assay is a universal biochemical HTS assay for enzymes that produce S-adenosylhomocysteine (SAH), including all enzymes within the histone (HMTs) and DNA (DNMTs) methyltransferase families. It combines the extensively validated Transcreener AMP2 which relies on fluorescent immunodetection of AMP, with coupling enzymes that convert SAH to AMP. This application note demonstrates a streamlined approach to develop a Transcreener EPIGEN Methyl transferase Assay for PRMT1. These steps can be followed to easily adapt the Transcreener EPIGEN Assay for doing high throughput screening with PRMT1.

The Transcreener® assays rely on antibodies that are able to differentiate between nucleotides on the basis of subtle structural differences. This makes detection of the product nucleotide possible even in the presence of excess substrate nucleotide. This case study focuses on the biochemical characterization of a broad range of targets, using Jump Dilution protocol to make residence time determinations for Transcreener® assays.

This application note will serve as a guide for using the Transcreener® EPIGEN MethyltransferaseAssay to detect the initial velocity enzyme activity of HMT G9a with an assay window suitable for inhibitor screening and dose response measurements. It should be used as an adjunct to the Transcreener EPIGEN MT Assay.

Identifying glycosyltransferase inhibitors remains a challenging task in drug discovery, because for many enzymes in this diverse class, screening methods have been limited to expensive and cumbersome radiolabeled assays or HPLC/MS analyses. As shown here, over the last few years Transcreener® glyscosyltransferase assays have been developed based on the simple and direct immunodetection of nucleotides, which are easily adaptable to automation and systemized high throughput screening protocols.

This poster demonstrates the sensitivity and signal stability of the MT and AT assay. The assay signal is stable for almost 48 hours, making the method very flexible for automated HTS protocols. By combining a novel enzymatic coupling step with the well characterized Transcreener® AMP2/GMP2 assay, the study has developed and validated a robust HTS assay that is sensitive and generic and can be used for a variety of HMTs, DNMTs and ATs.

Glycosyltransferase enzymes participate in extremely diverse metabolic and regulatory roles by catalyzing the transfer of sugar molecules to protein, lipid and carbohydrate acceptors as well as endogenous and xenobiotic small molecules. From a drug discovery point of view, they are gaining increasing interest as targets for “substrate reduction therapy” in lysosomal storage diseases, and as anti-microbial targets for disrupting bacterial cell wall biosynthesis. This study indicates that a suite of Transcreener assays may be a powerful approach for screening and profiling diverse glycosyltransferases in humans.

The exploration of more extensive drug target space by pharmaceutical and academic labs is creating a need for HTS compatible methods to detect diverse enzymes. The Transcreener AMP2/GMP2 Assay is simple, single step, homogenous, amenable to HTS and enables the detection of any AMP/GMP producing enzyme while using any precursor substrate. Importantly, the assay relies on direct immunodetection of AMP and GMP without using coupling enzymes, which eliminates the need for counter-screening. The readout options have been extended to include far-red, time-resolved Förster-resonance-energy-transfer (TR-FRET) in addition to the original fluorescence polarization based assay. This poster describes the development, optimization and validation of the Transcreener AMP2/GMP2 TR-FRET Assay, including detection of different classes of AMP/GMP-producing enzymes.

Watch this webinar from Meera Kumar, Senior Scientist at BellBrook Labs, to learn more about CD73 as a target for immunotherapy, including information on how the transcreener CD73 assay was developed and how this assay can be used to help measure adenosine.

The grant will be used to accelerate the discovery of new treatments for autoimmune diseases by targeting the cGAS-STING pathway