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The Transcreener CD73 Assay: A High Throughput, Sensitive Assay to Measure Adenosine

Transcreener ADO CD73 Assay

The Transcreener CD73 Assay is a specialized enzymatic assay designed to measure CD73 (ecto-5'-nucleotidase) activity. The assay is known for high sensitivity, robustness, and ease of use for detecting CD73 activity. Leveraging the trusted Transcreener technology, this assay provides an innovative platform for screening and profiling inhibitors.

The kit comes complete with the detection reagents required to measure activity. CD73 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. On the cell surface, CD39 hydrolyzes ATP and ADP to AMP.  AMP is further processed to Adenosine by the nucleotidase CD73 leading to a significant impact on many disease states. Learn about targeting CD39 with the Transcreener AMP² Assay.

Purinergic Signaling

How Do You Measure CD73 Activity?

The Transcreener ADO Assay determines CD73 enzyme activity by using a coupling enzyme that converts ADO produced by CD73 to the products AMP and ADP. ADP can then be measured using the trusted Transcreener ADP2 Assay components. The assay provides a universal method to assess activity of any ADP-producing enzyme.

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

The assay is available with FP readout. 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.

Detection of ADO to Measure CD73 Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener CD73 Assay Schematic

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

Applications

  • Measure Enzymatic Activity of CD73
  • Screen Compound Libraries for CD73 Inhibitors
  • Quantify Inhibitor Potency (IC50)
  • Inhibitor Selectivity Profiling

Features

  • Detection of unlabeled Adenosine
  • Easy to use, homogenous, one-step format
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readout minimize compound interference
  • A safe, non-radioactive method

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

The CD73 assay is in a simple two-step format: Run your enzyme reaction, add Transcreener reagents, and then your plate is ready to read. It is compatible with  96, 384, and 1536-well formats. 

Transcreener Mix and Read Assay

Excellent Z' Data Indicates Robust Assay

Z’ measurements using optimized CD73 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. 

Z Prime of CD73 Assay

Detection of Adenosine Under CD73 Initial Velocity

The assay demonstrates linearity when raw data is converted to Adenosine using a standard curve. 

Human CD73 Enzyme Titration

CD73 Enzyme Titration

Linear Response

CD73 Under Initial Velocity

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. Assay conditions to produce dose response curve used 5 pM CD73.

1600 Compound Pilot Screen

CD73 Pilot Screen

A subset from Chembridge Diversity Library was screened using the CD73 assay. This subset resulted in a hit rate of 0.85%, while a robust Z' of 0.81 was achieved.

Dose-Response - PSB 12379

Dose Response Curve CD73 Assay

IC50 = 19 nM

CD73 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 CD73 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 Readout Validated on Major Multimode Readers

SupplierInstrumentFP Assays
TriStar2S LB 942In Review
Tristar 5In Review
Mithras2 LB 943In Review
CytationTM 5Validated
CytationTM 3Validated
CytationTM 1Validated
SynergyTM H1Validated
SynergyTM 2/H4/4Click Me ArrowValidated
SynergyTM HTXNot Capable
SynergyTM Neo 2Validated
POLARstar® OmegaClick Me ArrowValidated
FLUOstar® OmegaNot Capable
PHERAstar® FSXClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidated
SenseIn Review
Analyst® GT/HTClick Me ArrowValidated
Gemini® XPS/EMNot Capable
SpectraMax® M2/M2eNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not Capable
SpectraMax® ParadigmClick Me ArrowValidated
SpectraMax® iD3/iD5Click Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidated
Infinite® M200Not Capable
Infinite® F500Click Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidated

What's Included

What You Will Need

Transcreener CD73 Assay Kits

* For custom or bulk orders (over 10,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

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

Why is CD73 a Promising Target for Drug Discovery?

CD73 is important in the Purinergic Signalling Pathway, processing AMP produced by CD39 into adenosine. This makes CD73 a main contributor in producing extracellular adenosine, aiding tumor growth and metastasis. Targeting CD73 inhibitors is a promising target in drug discovery to reduce extracellular adenosine produced by CD73 (Battastini et al, 2021).

Studies show a key role for Adenosine in immunosuppression in the tumor microenvironment with ectonucleotidases emerging as promising immuno-oncology targets. 

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