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Targeting Cancer Immunotherapy: Development of an HTS Method Using the Transcreener® ENPP1 Assay

ENPP1 Assay - A Transcreener AMP2/GMP2 Assay Application

Transcreener AMP²/GMP² ENPP1 Assay directly measures the AMP/GMP produced by ENPP1 (ectonucleotide pyrophosphatase / phosphodiesterase 1). These AMP/GMP measurements allow researchers to effectively determine the activity of the ENPP1 enzyme. The assay provides a powerful tool to screen compound libraries for ENPP1 modulators to help find new therapies for diseases. 

The kit comes complete with the detection reagents required to measure activity. ENPP1 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.

How Does This ENPP1 Assay Work?

The Transcreener ENPP1 Assay determines ENPP1 enzyme activity by directly measuring the AMP/GMP formed by the enzyme using the Transcreener AMP2/GMP2 Assay. By detecting AMP/GMP output, the assay provides a universal method to assess the activity of any AMP/GMP producing enzyme in real-time.

Transcreener ENPP1 Assay technology uses a simple but highly effective method that consists of an antibody selective to AMP/GMP and a far-red fluorescent tracer. AMP/GMP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout. 

The ENPP1 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 and GMP to Measure ENPP1 Enzymatic Activity

Fluorescent Polarization (FP)

ENPP1 Assay with FP Readout

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

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

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

Applications

  • Measure Enzymatic Activity of ENPP1
  • Screen Compound Libraries for ENPP1 Inhibitors
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling

Features

  • Direct detection of unlabeled AMP and GMP
  • 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 ENPP1 assay is compatible with 96, 384, and 1536-well formats. 

Transcreener Mix and Read Assay

Robust Assay Yields Quality Data

Z’ measurements using optimized ENPP1 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' shown here = 0.72

ENPP1 Titration in Presence of ATP or cGAMP

ENPP1 was titrated in the presence of 10 μM ATP or cGAMP in buffer containing 50 mM Tris, 5 mM MgCl2, 1% DMSO, and 0.01% Brij (pH 7.5). The plate was mixed well and incubated for 60 minutes. The enzyme reaction was quenched with an equal volume of EDTA containing stop buffer with 4 nM tracer and 10 μg/mL antibody. The plate was mixed well and read in PHERAstar Plus. Raw data and polarization values were converted into AMP demonstrating initial velocity conditions. The EC50 using cGAMP as a substrate was 48.12 μM. The EC50 using ATP as a substrate was 46.47 μM.

ENPP1 Titration

ENPP1 Assay Titration in Presence of ATP or cGAMP substrate

Linear Response

Linear Conversion ENPP1 Assay with ATP or cGAMP Substrate

Determination of Kinetic Parameters for ATP or cGAMP Substrate

ENPP1 (500 pM) was used to determine the substrate Km values in reactions containing varying amounts of ATP or cGAMP. Staggered incubation periods of 5, 10, 20, 30, 45, and 60 min were used prior to addition of Stop and Detect Mix to allow for initial velocity measurements at all substrate concentrations. Raw data (millipolarization units for FP) were converted to AMP using separate standard curves for each ATP or cGAMP concentration. The initial velocity from each of those time points was fitted to a Michaelis–Menten curve using GraphPad Prism to calculate the apparent Km values.

Kinetic Parameters for ATP Substrate

ENPP1 Kinetic Parameters for ATP Substrate

Km = 7.670 μM

Kinetic Parameters for cGAMP Substrate

ENPP1 Kinetic Parameters for cGAMP Substrate

Km = 4.729 μM

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.

Dose-Response - ATP Substrate

ENPP1 Suramin Dose Response ATP Substrate

IC50 = 0.36

Dose-Response cGAMP Substrate

ENPP1 Suramin Dose Response cGAMP Substrate

IC50 = 0.20

ENPP1 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 ENPP1 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
AMP2/GMP2 AntibodyAntibody specific to the nucleotides AMP and GMP.
AMP2/GMP2 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.
AMP and GMPAMP and GMP are used to create the ATP/AMP or cGAMP/AMP & GMP standard curve.

What You Will Need

ComponentNotes
ENPP1 EnzymeWe have successfully used recombinant ENPP1 from R&D Systems Cat. #6136.
Enzyme BufferEnzyme buffer components optimized for your target. See the technical manual for potential interfering components.
ATP or cGAMPUse the nucleotide required for your experiment. It is possible to purchase nucleotides direct from BellBrook Labs.
Stop Buffer B, 5XBuffer containing EDTA capable of stopping the ENPP1 reaction. If you'd like to use this please mention it in your order and it will be included from BellBrook Labs Cat. #2139 - 1 mL, Cat. #2140 - 10 mL.
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/GMP 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 ENPP1 As a Therapeutic Target

ENPP1 is a nucleotide pyrophosphatase and phosphodiesterase that regulates purinergic signaling and can degrade different nucleotides including ATP and cGAMP. ENPP1 has been identified as the primary enzyme responsible for degrading cGAMP and therefore is under intense investigation as a therapeutic target for cancer immunotherapy.

The Transcreener ENPP1 Assay is an excellent tool for researchers examining the therapeutic effects of ENPP1.

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