• Assay Selection Tool

BellBrook Labs

  • Products
    • Transcreener® HTS Assay Kits
      • Transcreener® ADP² Kinase Assay Kits
        • Transcreener® ADP2 Assay Kit – FP Readout
        • Transcreener® ADP2 Assay Kit – FI Readout
        • Transcreener® ADP2 Assay Kit – TR-FRET Readout
      • Transcreener® ADO CD73 Assay Kit
      • Transcreener® AMP²/GMP² Phosphodiesterase Assay Kits
        • Transcreener® AMP2/GMP2 FP Assay
        • Transcreener® AMP2/GMP2 Assay Kit – TR-FRET Readout
      • Transcreener® cGAMP cGAS Assay Kits
        • Transcreener® cGAMP Assay Kit – FP Readout
        • Transcreener® cGAMP Assay Kit – TR-FRET Readout
      • Transcreener dAMP Exonuclease Assay Kit
      • Transcreener® EPIGEN SAH Methyltransferase Assay Kit
      • Transcreener® GDP GTPase Assay Kits
        • Transcreener® GDP Assay Kit – FP Readout
        • Transcreener® GDP Assay Kit – FI Readout
        • Transcreener® GDP Assay Kit – TR-FRET Readout
      • Transcreener® UDP² Glycosyltransferase Assay Kits
        • Transcreener® UDP2 Assay Kit – FP Readout
        • Transcreener® UDP2 Assay Kit – FI Readout
        • Transcreener® UDP2 Assay Kit – TR-FRET Readout
      • Transcreener® 2-5A OAS Assay Kit
    • AptaFluor® HTS Assay Kits
      • AptaFluor® SAH Methyltransferase Assay Kit
    • Enzyme Assay Systems
      • TREX1 Assay System
    • Recombinant Enzymes
      • Human cGAS Enzyme
      • Mouse cGAS Enzyme
      • Human DDX3 Enzyme
      • Human OAS1 Enzyme
      • Human TREX1 Enzyme
    • Assay Plates
    • Ordering Information
  • Services
    • Assay Development Services
    • Lead Discovery Services
    • CD38 Assay Services
    • GTPase Profiling Services
    • ATPase Profiling Services
  • Assays by Target
    • Kinase Assays
      • ADK Assays – Application
      • AMPK Assays – Application
      • IKK-beta Assays – Application
      • IRAK4 Assays – Application
      • JAK1 Assays – Application
      • JAK3 Assays – Application
      • MAPK8 Assays – Application
      • PKR Assays – Application
      • RIPK1 Assays – Application
      • RIPK2 Assays – Application
      • TBK1 Assays – Application
    • GTPase Assays
      • GAP Assays – Application
      • GEF Assays – Application
      • KRAS Assays – Application
      • HRAS Assays – Application
      • NRAS Assays – Application
      • RRAS Assays – Application
      • Rac1 Assays – Application
      • RhoA Assays – Application
      • RhoC Assays – Application
      • Cdc42 Assays – Application
      • Ran Assays – Application
    • Methyltransferase Assays
      • EZH2 Assays – Application
      • G9a Assays – Application
      • SET7/9 Assays – Application
      • SET8 Assays – Application
      • PRMT1 Assays – Application
      • PRMT3 Assays – Application
      • PRMT4 Assay – Application
    • STING Pathway Assays
      • cGAS Assay Kits
      • ENPP1 Assays – Application
      • TREX1 Assay System
      • IKK-beta Assays – Application
      • TBK1 Assays – Application
    • Nucleotidase Assays
      • CD38 Assay Services
      • CD39 Assays – Application
      • CD73 Activity Assay Kits
    • Helicase / ATPase Assays
      • DDX3 Assays – Application
      • NSP13 Assays – Application
      • P97 Assays – Application
    • Glycosyltransferase Assays
      • Toxin B Assays – Application
      • GALNT2 Assays – Application
      • GALNT3 Assays – Application
      • BGalT1 Assays – Application
    • Phosphodiesterase Assays
      • PDE3 Assays – Application
      • PDE4 Assays – Application
      • PDE5 Assays – Application
      • PDE7 Assays – Application
    • Ligase and Synthetase Assays
      • SUMO E1 Assays – Application
      • Acyl CoA Synthetase Assays – Application
      • S-Acetyl CoA Synthetase Assays – Application
    • Exonuclease Assays
      • TREX1 Assay System
    • OAS Assays
      • OAS1 Assay Kits
    • Other Enzyme Assays
      • NUDT5 Assays – Application
  • Resources
    • Technical Manuals
    • Transcreener® Assays – Instrument Compatibility
    • Application Notes
    • Posters and Presentations
    • Publications
    • Transcreener® FAQ’s
    • Guides
      • Residence Time Guide
      • Hit Prioritization Guide
      • Kinases in Innate Immunity
  • Company
    • President’s Message
    • International Distributors
    • Careers
    • Downloads
    • Contact Us
  • Blog
  • MY CART
    No products in cart.

Using a Glycosyltransferase Activity Assay to Study Glycosylation and the Function of GTs

by Bellbrook Labs / Tuesday, 09 July 2019 / Published in Emerging Targets, HTS Assays
Glycosyltransferase Activity Assay and Lysosomal Storage Disorders

Glycosyltransferase (GT) enzymes are incredibly diverse in their abilities to catalyze the transfer of sugar molecules to protein, carbohydrate, and lipid substrates. GTs have various metabolic and regulatory roles in biology. Glycosyltransferase targets provide different areas of therapeutic potential in a wide array of disease, including cancer, metabolic disorders, and infectious disease. A glycosyltransferase activity assay provides a means of which to thoroughly interrogate GT targets for novel treatments.

From a drug discovery point of view, GTs are gaining increasing interest as targets for “substrate reduction therapy” in lysosomal storage disorders (LSDs) such as Gaucher and Fabry diseases, and as anti-microbial targets for disrupting bacterial cell wall biosynthesis. From an HTS assay perspective, GTs are a challenging target class because of the diversity of both donor and acceptor substrates.

Although GTs use a diverse mix of acceptor substrates from proteins to small molecules, the majority of human GTs use only three different nucleotide-sugars as donors: UDP, GDP, or CMP. Because there is only one “donor product” for each type of group transfer reaction, a single set of detection reagents can cover an entire family.

Studying Lysosomal Storage Disorders with a Glycosyltransferase Activity Assay

Lysosomal storage disorders (LSDs), a group of over 40 inheritable diseases present in one out of every 7700 births, arise from enzyme deficiencies within the glycosphingolipid pathway that result in toxic accumulations of non-degraded substrates. LSDs include Gaucher, Tay-Sachs, Sandhoff, Fabry, Neimann-Pick type C, and GM1 gangliosidosis diseases.

Enzyme replacement therapy (ERT) is a clinically validated treatment for some LSDs, including Gaucher and Pompe diseases, but it requires an injection of the recombinant enzyme and it is not effective for neuropathic symptoms due to the inability of the replacement enzyme to cross the blood-brain barrier.

Substrate reduction therapy (SRT) is an alternative therapeutic approach that relies on the development of small molecule inhibitors that reduce biosynthesis of the toxic metabolite. In most cases, the target enzyme is a glycosyltransferase.

Glycosyltransferase Activity Assay and LSDs

LSDs are caused by deficiencies in glycosphingolipid degradative enzymes. Inhibiting β-glucocerebrosidase (GCS) decreases flux into the biosynthetic pathway, resulting in lower levels of toxic intermediates.

Issues remain with glycosyltransferase activity assay methods. For example, measurement of activity with the traditional use of radioactive sugar-nucleotide donors, requires management of radioactive materials and waste and relies on a cumbersome extraction process to recover and measure glucosylated products. Other non-radioactive assays for glycosylated products tend to be specific for just one enzyme, can require modified sugar-nucleotide donors, and may require a separation step.

A viable alternative is UDP detection through a universal assay for any UDP-sugar glycosyltransferase. Invariably, this method has involved enzyme coupling to convert the UDP to a detectable product, such as phosphate, or coupling with UDP-glucose dehydrogenase to produce NADH before conversion to a fluorescent resazurin by diaphorase. However, these coupled assays are inherently prone to false positives from compound interference with the coupling enzymes, and thus require many additional wells for counter-screening.

Therefore, a robust, in vitro high-throughput assay targeting glycosyltransferases that enables screening of chemical libraries or optimizing leads could enhance the ability to develop inhibitors with improved therapeutic properties.

The Advantages of a Direct Detection Platform

BellBrook Labs pioneered the development of HTS-compatible assays for glycosyltransferases with the introduction of the Transcreener® UDP Glycosyltransferase Activity Assay in 2005. The Transcreener platform offers the only HTS-compatible GT assay method that relies on direct detection of reaction products. Unlike other methods, it has fewer reagents and does not depend on coupling enzymes, which can interfere with the compound screening process and increase the risk of false positives or missing a hit.

Two Reagents Do the Work

The direct detection method used by the Transcreener platform relies on the interaction between an antibody and a tracer. When the donor nucleotide displaces the tracer, a change in fluorescence enables detection. The antibody is covalently labeled with terbium (Tb) for the time-resolved Förster-resonance-energy-transfer (TR-FRET) format and unmodified for the fluorescence polarization (FP) format.

Differentiate Product from Substrate

The ability to differentiate between closely related nucleotides is a central key to the Transcreener technology—enabling detection of nucleotide products in the presence of an excess of substrate (e.g, UDP detection in the presence of excess UDP-glucose), which is a requirement for measuring enzyme initial velocity. The antibodies in Transcreener assays can resolve subtle structural differences between nucleotides, such as a single phosphate or methyl group, using a simple mix-and-read protocol. This antibody selectivity for the product nucleotide versus the substrate ranges from 150-fold to greater than 1000-fold.

Reagent Stability Compatible with HTS

Transcreener reagents are extremely robust, allowing outstanding deck and signal stability. Signal readouts from some coupled assays are limited in read times, while Transcreener fluorescent readouts can be read overnight or longer. Plates can be read long after the addition of detection reagents. This makes Transcreener a great fit for automated high throughput screening.

Learn More About the Transcreener Glycosyltransferase Assay

Tagged under: Glycosyltransferase Activity Assay, Lysosomal Storage Disorders, Transcreener UDP Assay

What you can read next

View Our Kinase Inhibitor Residence Time Webinar Anytime!
JNK1 And Cancer Pathogenesis
The Double-Edged Sword in Cancer Pathogenesis – JNK1
Pink Stethascope DOT1L Breast Cancer Target 420 X 280
How a Methyltransferase Activity Assay Could Help Identify Novel Breast Cancer Treatments

Categories

  • Company
  • Emerging Targets
  • Epigenetics
  • HTS Assays
  • Innate Immunity
  • Neurodegenerative Diseases
  • News
  • Products
  • Resources
  • Success Stories
  • Uncategorized

Recent Posts

  • PARP1 as a Hero vs Villain

    Is PARP1 a Hero or Villain?

    Not counting histones, PARP1 [Poly(ADP-ribose) ...
  • Ongoing Puzzle of c-SRC in Cancer Treatment

    Advancements in The Ongoing Puzzle to Understand c-SRC

    Nearly a half-century ago, sequences from the R...
  • SLAS 2023 Conference Exhibitor Announcement

    SLAS 2023 – HTS Assays and Discovery Services

    BellBrook Labs will exhibit and present posters...
  • BTK's Involved in Systemic lupus erythematosus

    The Challenging Search for BTK Inhibitors

    Bruton’s Tyrosine Kinase (BTK) is a 76kDa...
  • SARM1 Causes Axonal Death

    SARM1 Forefronts Research into Major Neurological Diseases

    SARM1 [Sterile alpha & toll/interleukin rec...

Archives

BellBrook Labs
5500 Nobel Drive, Suite 230
Madison, Wisconsin 53711 USA
(608) 443-2400

info@bellbrooklabs.com

 Copyright © 2023 BellBrook Labs | All Rights Reserved | Privacy Policy | Terms of Use | FCOI | Sitemap

TOP