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A High Throughput CD39 Assay for the Ectonucleotidase Based on the Transcreener Platform

CD39 Assay - A Transcreener AMP2 Assay Application

Transcreener AMP² Assay directly measures AMP produced by CD39 (Cluster of Differentiation 39, Ectonucleoside triphosphate diphosphohydrolase-1, or NTPDase1). These AMP measurements allow researchers to effectively determine the CD39 enzyme activity. The assay provides a powerful tool to screen compound libraries for CD39 modulators to help find new therapies for disease. 

The kit comes complete with the detection reagents required to measure activity. CD39 enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.

This assay is designed to be used with purified enzyme preparations. Transcreener assays are not validated with cell lysates, blood, serum, or other biological samples.

Important Messenger for Purinergic Signalling

Ectonucleotidases are plasma membrane-bound enzymes with externally oriented active sites that metabolize nucleotides to nucleosides and are crucial for maintaining immune homeostasis. CD39 hydrolyzes ATP and ADP to AMP.  AMP is further processed to adenosine leading to a significant impact on many disease states. 

Purinergic Signaling

How Does This CD39 Assay Work?

The Transcreener CD39 Assay determines CD39 enzyme activity by directly measuring the AMP formed by the enzyme using the Transcreener AMP Assay. By detecting AMP output, the assay provides a universal method to assess the activity of any AMP-producing enzyme in real-time.

Transcreener CD39 Assay technology uses a simple but highly effective method that consists of an antibody selective to AMP and a far-red fluorescent tracer. AMP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.

The CD39 assay is available with FP and TR-FRET. It is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS.

Direct Detection of AMP to Measure CD39 Enzymatic Activity

Fluorescent Polarization (FP)

CD39 Assay Schematic FP

The workhorse. Used in many large screens. Best deck and signal stability. 

Time-Resolved Förster Resonance Energy Transfer (TR-FRET)

CD39 Assay Schematic TR-FRET

For customers who prefer TR-FRET detection. Uses the same filter set as HTRF®

Applications

  • Measure Enzymatic Activity of CD39
  • Screen Compound Libraries for CD39 Inhibitors
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling

Features

  • Direct detection of unlabeled AMP
  • Easy to use, homogenous, one step format
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method
  • Available in FP or TR-FRET readouts

Easy-to-Use, Mix-and-Read, HTS-Ready Assay

Run your enzyme reaction, add Transcreener reagents, and read your plates. The CD39 assay is compatible with 96, 384, and 1536-well formats.

Transcreener Mix and Read Assay

Robust Assay Yields Quality Data

Z’ measurements using optimized CD39 reaction conditions indicate a robust assay.  Robust data like this is vital for sizeable high throughput screens that are difficult to complete due to massive sample quantity. Both ATP and ADP produce excellent results.

Robust Assay with Excellent Z prime

Detection of AMP Under CD39 Initial Velocity

The assay demonstrates linearity when raw data is converted to AMP using a standard curve. CD39 can use a variety of substrates. Here both ATP and ADP are shown under initial velocity conditions.

Human CD39 Enzyme Titration

Linear Response

cd39 Under Initial Velocity Conditions using ATP and ADP as substrates

Screen for Inhibitors & Perform SAR

Transcreener Assays are designed for screening compound libraries in a high throughput format. Follow up SAR can also be performed using the assay to determine inhibitor potency with ease.

1600 Compound Pilot Screen

CD39 Pilot Screen

A 1600 compound subset from Chembridge Diversity Library was screened using CD39 and ATP in the Transcreener TR-FRET format resulting in 8 hits shown in green.

Dose Response Curve

CD39 Assay Services

Interested in moving your program forward, but don't want to bring an assay in-house? Our scientists can help! BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your CD39 program.

Assay Development Services

Lead Discovery Services Include:

  • Inhibitor Screening – To identify or confirm activity with the target.
  • Inhibitor Potency Profiling – Dose-response with target and/or related proteins. Fast IC50 results.
  • Residence Time Measurements – Determination of koff using ‘jump dilution’ enzymatic assay method.
  • Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.

Contact Us to Learn More

Please fill out the form below. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.

Far-Red FP & TR-FRET Readouts Validated on Major Multimode Readers

SupplierInstrumentFP AssaysTR-FRET Assays
TriStar2S LB 942In ReviewClick Me ArrowValidated
Tristar 5In ReviewIn Review
Mithras2 LB 943In ReviewClick Me ArrowValidated
CytationTM 5ValidatedValidated
CytationTM 3ValidatedValidated
CytationTM 1ValidatedValidated
SynergyTM H1ValidatedValidated
SynergyTM 2/H4/4Click Me ArrowValidatedClick Me ArrowValidated
SynergyTM HTXNot CapableNot Capable
SynergyTM Neo 2ValidatedValidated
POLARstar® OmegaClick Me ArrowValidatedClick Me ArrowValidated
FLUOstar® OmegaNot CapableClick Me ArrowValidated
PHERAstar® FSXClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidatedClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidatedClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidatedClick Me ArrowValidated
SenseIn ReviewIn Review
Analyst® GT/HTClick Me ArrowValidatedClick Me ArrowValidated
Gemini® XPS/EMNot CapableNot Capable
SpectraMax® M2/M2eNot CapableNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not CapableClick Me ArrowValidated
SpectraMax® ParadigmClick Me ArrowValidatedClick Me ArrowContact Us
SpectraMax® iD3/iD5Click Me ArrowValidatedClick Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidatedClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M200Not CapableNot Capable
Infinite® F500Click Me ArrowValidatedClick Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact UsClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidatedClick Me ArrowValidated

What's Included

ComponentNotes
AMP AntibodyAntibody specific to the nucleotide AMP.
AMP TracerWhen displaced, changes in fluorescence can be read in an FP or TR-FRET readout, depending on the assay format.
Tris SolutionUsed to buffer the detection mixture.
AMPAMP is used to create the ATP/AMP or ADP/AMP standard curve.

What You Will Need

ComponentNotes
CD39 EnzymeWe have successfully used recombinant human CD39 from R&D Systems Cat. #4397-EN
Enzyme BufferWe have had success with a 50 mM Tris, 10 mM CaCl2, and 0.01% Brij-35 buffer at pH 7.5.
ATP or ADPUse the nucleotide required for your experiment. It is possible to purchase nucleotides direct from BellBrook Labs.
Assay PlatesAssay Plates Can Be Purchased Directly Through BellBrook Labs

FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514

TR-FRET - An entirely white plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4513
Plate ReaderA multi-detection microplate reader configured to measure the output of the AMP tracer is required.
Liquid Handling DevicesUse liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates.
Ultrapure WaterSome deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem.

Interconnections Between the DNA Damage Response and Innate Immunity

There is extensive crosstalk between the DNA damage response (DDR) pathways and innate immune pathways. Both the individual pathways and the interconnections between them are a focus for exciting new small molecule drug therapeutics that target cancers and debilitating autoimmune disorders. The Transcreener HTS Assay platform accelerates these efforts by providing a robust and easy-to-use biochemical assay to measure activity of key enzymes in the innate immune and DDR pathway.

In this guide, we provide an overview of the DDR and innate immune pathway, and describe Transcreener Assays and Assay Systems for key therapeutic targets. The enzyme targets discussed include:

DDR

  • POLQ
  • WRN
  • PARP1
  • PARG
  • DNA PK

Innate Immunity

  • cGAS
  • TREX1
  • TBK1
  • IKKβ
  • IRAK4
  • ENPP1
  • CD38
  • OAS-1
  • RIG-1/MDA5
  • DDX3
  • CD39
  • CD73
DNA Damage Response and Innate Immunity Preview

The Role of CD39 As a Therapeutic Target

CD39 is important in Purinergic Signalling, hydrolyzing ATP & ADP to AMP. CD73 completes this pathway by processing AMP into extracellular adenosine. Extracellular adenosine aids tumor growth and metastasis. Inhibiting this pathway shows potential for cancer treatment. CD39 inhibitors also show potential for use in combination with chemotherapy (Guo et al, 2022).

Studies show a key role for adenosine in immunosuppression in the tumor microenvironment, and ectonucleotidases are emerging as promising immuno-oncology targets. The Transcreener AMP2 CD39 Assay is an excellent tool for researchers examining the therapeutic effects of CD39. 

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