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

ENPP1 Assay Method

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Assay Services

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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.

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)

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

Learn More About The FP Assay

Transcreener AMP²/GMP² FP Assay Tech Manual

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

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

Learn More About The TR-FRET Assay

Transcreener AMP²/GMP² TR-FRET Assay Tech Manual

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.

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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

Detection of AMP and GMP Under ENPP1 Initial Velocity

The assay demonstrates linearity when raw data is converted to AMP using a standard curve. ENPP1 can use a variety of substrates. Here both ATP and cGAMP have been tested. ATP is shown here under initial velocity conditions.

Human ENNP1 Enzyme Titration

Linear Response

ENPP1 Titration with ATP Initial Velocity

Hydrolysis of cGAMP by ENPP1

Titration of ENPP1 in the presence of 10 µM cGAMP was done to determine 1 pM ENPP1 to be used for assay with cGAMP. Titration of cGAMP with ENPP1 shows the EC₅₀ to be around 80 nM.

ENPP1 Titration - cGAMP Substrate

cGAMP Titration with ENPP1

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

IC₅₀ = 0.36

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.

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 k_off 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.

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

Instrument	FP Assays	TR-FRET Assays
TriStar²S LB 942	in review	validated
Mithras² LB 943	in review	validated

Cytation™ 5	validated	validated
Cytation™ 3	validated	validated
Cytation™ 1	validated	validated
Synergy™ H1	validated	validated
Synergy™ 2/H4/4	validated	validated
Synergy™ HTX	not capable	not capable
Synergy™ Neo 2	validated	validated

POLARstar^® Omega	validated	validated
FLUOstar^® Omega	not capable	validated
PHERAstar^® FSX	validated	validated
PHERAstar^® Plus/FS	validated	validated
CLARIOstar^® /Plus	validated	validated
VANTAstar^™	validated	validated

Analyst^® GT/HT	validated	validated
Gemini^® XPS/EM	not capable	not capable
SpectraMax^® M2/M2e	not capable	not capable
SpectraMax^® M5/M5e/FlexStation^® 3	not capable	validated
SpectraMax^® Paradigm	validated	contact us
SpectraMax^® iD3/iD5	in review	validated

EnVision^®/EnVision^® Xcite	validated	validated

Infinite^® M1000/M1000Pro/Safire2™	validated	validated
Infinite^® M200	not capable	not capable
Infinite^® F500	validated	validated
Infinite^® F200/Ultra^evolution	contact us	contact us
Spark™ 10M	validated	validated

What's Included


Component	Notes
AMP/GMP Antibody	Antibody specific to the nucleotides AMP and GMP.
AMP/GMP Tracer	When displaced, changes in fluorescence can be read in an FP or TR-FRET depending on the assay format.
Tris Solution	Tris solution is used to buffer the detection mixture.
AMP and GMP	AMP and GMP are used to create the ATP/AMP or cGAMP/AMP & GMP standard curve.

What You Will Need


Component	Notes
ENPP1 Enzyme	We have successfully used recombinant human ENPP1 from R&D Systems Cat. #6136.
Enzyme Buffer	Enzyme buffer components optimized for your target. See the technical manual for potential interfering components.
ATP or cGAMP	Use the nucleotide required for your experiment. It is possible to purchase nucleotides direct from BellBrook Labs.
Stop Buffer B, 5X	Buffer 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 Plates	Assay 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 Reader	A multi-detection microplate reader configured to measure the output of the AMP/GMP tracer is required.
Liquid Handling Devices	Use liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates.
Ultrapure Water	Some 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.

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Transcreener AMP²/GMP² Assay Kits - For ENPP1 Activity Assays

* For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.


Cat. No.	Product Description	Price	Add to Cart
3015-1K	Transcreener AMP²/GMP² FP Assay (1,000 Assay, 384 Well)	$659.00	Add to Cart
3015-10k	Transcreener AMP²/GMP² FP Assay (10,000 Assay, 384 Well)	$3,789.00	Add to Cart
3015-100k	Transcreener AMP²/GMP² FP Assay (10 X 10,000 Assay, 384 Well)	$18,945.00	Add to Cart
3020-1K	Transcreener AMP²/GMP² TR-FRET Assay (1,000 Assay, 384 Well)	$719.00	Add to Cart
3020-10K	Transcreener AMP²/GMP² TR-FRET Assay (10,000 Assay, 384 Well)	$4,109.00	Add to Cart
3020-100K	Transcreener AMP²/GMP² TR-FRET Assay (10 X 10,000 Assay, 384 Well)	$20,545.00	Add to Cart

Assay Plates for FP

Cat. No.	Product Description	Price
2233	Corning 96-Well Black Assay Plates (#3686), 3-Pack (200+ Assays)	$50.00	Add to Cart
2229	Corning 384-Well Black Assay Plates (#4514), 3-Pack (1,000+ Assays)	$55.00	Add to Cart
2230	Corning 384-Well Black Assay Plates (#4514), 30-Pack (10,000+ Assays)	$449.00	Add to Cart

Assay Plates for TR-FRET

Cat. No.	Product Description	Price
2234	Corning 96-Well White Assay Plates (#3642), 3-Pack (200+ Assays)	$50.00	Add to Cart
2231	Corning 384-Well White Assay Plates (#4513), 3-Pack (1,000+ Assays)	$60.00	Add to Cart
2232	Corning 384-Well White Assay Plates (#4513), 30-Pack (10,000+ Assays)	$499.00	Add to Cart

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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.

ENPP1 Assay – Transcreener Application

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

ENPP1 Assay Method

Ordering Information

Assay Services

Related Resources

ENPP1 Assay - A Transcreener AMP2/GMP2 Assay Application

How Does This ENPP1 Assay Work?

Direct Detection of AMP and GMP to Measure ENPP1 Enzymatic Activity

Fluorescent Polarization (FP)

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

Applications

Features

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

Robust Assay Yields Quality Data

Detection of AMP and GMP Under ENPP1 Initial Velocity

Human ENNP1 Enzyme Titration

Linear Response

Hydrolysis of cGAMP by ENPP1

ENPP1 Titration - cGAMP Substrate

cGAMP Titration with ENPP1

Screen for Inhibitors & Perform SAR

Dose-Response - ATP Substrate

IC50 = 0.36

Dose-Response cGAMP Substrate

IC50 = 0.20

ENPP1 Assay Services

Lead Discovery Services Include:

Contact Us to Learn More

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

What's Included

What You Will Need

Transcreener AMP2/GMP2 Assay Kits - For ENPP1 Activity Assays

Assay Plates for FP

Assay Plates for TR-FRET

The Role of ENPP1 As a Therapeutic Target

Related Resources

ENPP1 Assay - A Transcreener AMP²/GMP² Assay Application

IC₅₀ = 0.36

IC₅₀ = 0.20

Transcreener AMP²/GMP² Assay Kits - For ENPP1 Activity Assays