• 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® ADPR 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
      • CD38 Assay System
      • TREX1 Assay System
    • Recombinant Enzymes
      • Human CD38 Enzyme
      • 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
    • 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 Assays – Application
    • STING Pathway Assays
      • cGAS Assay Kits
      • ENPP1 Assays – Application
      • TREX1 Assay System
      • IKK-beta Assays – Application
      • TBK1 Assays – Application
    • Nucleotidase Assays
      • CD38 Assay System
      • CD39 Assays – Application
      • CD73 Activity Assay Kits
    • Helicase / ATPase Assays
      • DDX3 Assays – Application
      • NSP13 Assays – Application
      • p97 Assays – Application
      • Ketohexokinase 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
      • Acetyl CoA Synthetase Assays – Application
    • Exonuclease Assays
      • TREX1 Assay System
      • WRN Exonuclease Assays – Application
    • 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.

Ectonucleotidases: An Outside Chance for Drug Development

by Bellbrook Labs / Friday, 28 April 2017 / Published in Emerging Targets, HTS Assays, Uncategorized

Sometimes, the most intriguing cellular processes happen outside of the cell.

Case in point: purinergic signaling pathways, in which extracellular receptors sense levels of purines in the extracellular milieu. The result? A wide array of effects on neuronal signaling, vascular tone, thrombosis, and immune function.

But what controls the level of extracellular purines? Enter ectonucleotidases: plasma membrane-bound enzymes with externally oriented active sites that metabolize nucleotides to nucleosides.

One type is ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1), also known as CD39 or NTPDase1 protein. This enzyme catalyzes the hydrolysis of γ- and β-phosphate residues of triphospho- and diphosphonucleosides to the monophosphonucleoside derivative. It’s not a terribly picky enzyme and can hydrolyze ATP, ADP, UTP, and UDP with similar efficiency. Through its actions, it affects the amount of ligands available to P2 receptors, which are ligand-gated ion channels or G-protein coupled receptors. Medically significant roles of P2 receptors include chronic pain perception and vascular complications of diabetes.

The ability of CD39 to hydrolyze ATP to ADP to AMP holds particular biological importance, because AMP can be processed to adenosine, and extracellular adenosine has a significant impact on dozens of disease states. CD39 may directly affect cancer progression by regulating T cell and natural killer cell activity and antitumor responses in general,[1] and immunosuppression of melanoma cells specifically.[2]   During tumor pathogenesis, secreted adenosine acts as an immunomodulator, affecting tumor-specific immune response.[3]

Cancer pathogenesis and inflammation aren’t the only roles for CD39, though. Expression of CD39 in the vasculature controls the activation of platelets, as well as the size and stability of thrombi.[4] There’s even evidence that CD39 is directly involved in myocardial rupture after myocardial infarction; genetic knockout of CD39 in mice results in animals whose heart tissue is nudged toward reparative processes in wake of cardiac damage.[5] Other evidence indicates that adenosine levels and CD39 activity are directly correlated with inflammation, pathogen colonization, and the ability of pathogens to evade the host inflammatory response. [6]

The potential for purinergic pathway members to serve as drug targets isn’t theoretical. Plavix (clopidogrel) is a P2Y12 receptor inhibitor used to reduce heart disease and stroke in patients at high risk. In 2011—prior to patent expiration—global annual sales revenue for Plavix was reported to be $9.7 billion. Methotrexate, an anti-inflammatory agent, results in the release of extracellular adenosine. Although it is no longer under patent protection, annual sales of generic methotrexate have topped $100 million in recent years.

But while FDA-approved drugs targeting receptors exist, drug development efforts focusing on ectonucleotidases have taken a slower path. In large part, this has been due to lack of sufficiently sensitive and specific ectonucleotidase assays. For that reason, a recent publication describing a fluorescence polarization assay for NTPDases is promising.[7]

Transcreener AMP Ectonucleotidase Assay Principle

Transcreener AMP Assay Principle for Measuring Ectonucleotidase Activity

This assay, which utilizes antibodies, fluorescent tracers, and ADP from BellBrook Labs, enables the direct detection of the enzymatic reaction product ADP when using ATP as a substrate (for NTPDase2, NTPDase3 and NTPDase8) or of AMP upon using ADP as a substrate (for NTPDase1). The method leverages selective antibodies that can distinguish between mono-, di-, and triphosphates. ADP or AMP produced during the reaction displaces the fluorescent tracer nucleotide from the antibody, resulting in a change in fluorescent properties and production of signal. Because the fluorescence polarization immunoassay (FPIA) assay is highly sensitive, low concentrations of substrate can be used (for example, the optimal concentration of antibody for the NTPDase1 / CD39 assay was 8 ug/mL, which is a level suitable for screening moderately soluble compounds and for fragment-based screening).

This approach allows screening at levels near the Km value of the enzyme, facilitating high-throughput methods. This is exceptionally helpful, because screens conducted at much higher concentrations require the competitive inhibitor to be present at similarly high concentration—which is problematic for inhibitors that are poorly soluble. The conventional malachite green assay for ectonucleotidase activity detects phosphate and not the nucleoside reaction product; because many biological reactions release phosphates, this often results in high background and lack of signal specificity. The FPIA assay does not suffer from this limitation.

The authors validated the FPIA assays statistically, and used the assay to calculate the Ki of a known NTPDase3 inhibitor (an anthraquinone derivative named PSB-06126). The determined Ki value agreed with the published value. The researchers performed a small library high-throughput screen to identify inhibitors of NTPDase3, yielding two potential novel inhibitors.

Could ectonucleotidase inhibitors be viable drug candidates? If lessons from other purinergic pathway members serve as a guide, the answer is yes. But success will require highly specific and sensitive HTS assays—tools that are now within reach, enabling a world of possibilities.

– Robyn M. Perrin, PhD


On Demand Webinar: Development of Ectonucleotidase Assay Methods Using the Transcreener® HTS Assay Platform

In this webinar we discussed:

  • Assay development for CD39 using the Transcreener® AMP2/GMP2 Assay with fluorescence polarization and TR-FRET readouts
  • Demonstrated assay robustness for use in high-throughput screening
  • Inhibitor potency profiling for CD39
  • Recent external publications using the Transcreener® platform for studying ectonucleotidase activity

Watch the Video


[1] Bastid J, Cottalorda-Regairaz A, Alberici G, Bonnefoy N, Eliaou JF, Bensussan A. 2013. ENTPD1/CD39 is a promising therapeutic target in oncology. Oncogene. 32(14):1743-51.

[2] Umansky V, Shevchenko I, Bazhin AV, Utikal J. 2014. Extracellular adenosine metabolism in immune cells in melanoma. Cancer Immunol Immunother. 63(10):1073-80.

[3] Kumar V. 2013. Adenosine as an endogenous immunoregulator in cancer pathogenesis: where to go? Purinergic Signal. 9(2):145-65.

[4] Deaglio S, Robson SC. 2011. Ectonucleotidases as regulators of purinergic signaling in thrombosis, inflammation, and immunity. Adv Pharmacol. 61:301-32.

[5] Sutton NR, Hayasaki T, et al. 2017. Ectonucleotidase CD39-driven control of postinfarction myocardial repair and rupture. JCI Insight. 2(1):e89504.

[6] Alam MS, Costales MG, Cavanaugh C, Williams K, 2015. Extracellular adenosine generation in the regulation of pro-inflammatory responses and pathogen colonization.  Biomolecules.5(2):775-92.

[7] Fiene A, Baqi Y, Lecka J, Sévigny J, Müller CE. 2015. Fluorescence polarization immunoassays for monitoring nucleoside triphosphate diphosphohydrolase (NTPDase) activity. Analyst. 140(1):140-8.

Tagged under: Assay Development Services, drug discovery services, Lead Discovery Services, Transcreener ADP Kinase Assay, Transcreener AMP/GMP Assay

What you can read next

JNK1 And Cancer Pathogenesis
The Double-Edged Sword in Cancer Pathogenesis – JNK1
CDK12 Activity Assays Find Inhibitors Cancer Treatment
Finding Inhibitors for a Cancer Target: CDK12 Activity Assays Accelerate the Search
Scientist Using a GTPase Assay
Researchers Employ GTPase Assay to Investigate Rab Proteins

Categories

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

Recent Posts

  • Preview of DNA Damage Repair Webinar

    [Webinar] Targeting DNA Damage Repair for Drug Discovery Using the Transcreener ADPR® Assay

    Interested in DNA damage repair pathways for dr...
  • Calculating MAPK14 Residence Time

    Transcreener® ADP2 Assay Delivers Precise Measurement of Functional MAPK14 Residence Time

    Researchers at the University of Tubingen use T...
  • DDX1 as a versatile RNA Helicase

    DDX1: A Versatile RNA Helicase

    DDX1 (Dead-Box Helicase 1) belongs to the DEAD-...
  • RAB2 Transports Membrane Bound Vesicles

    Investigating RAB2 as a Vesicle Transporter & Autophagy Initiator

    RAB2 is part of the RAB family of small GTPases...
  • PARP1 as a Hero vs Villain

    Is PARP1 a Hero or Villain?

    Not counting histones, PARP1 [Poly(ADP-ribose) ...

Archives

BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400

info@bellbrooklabs.com

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

TOP