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

Discover ENPP1 Inhibitors with the Transcreener AMP²/GMP² Assay

The Transcreener AMP²/GMP² ENPP1 Assay directly measures the GMP and (or) AMP produced by ectonucleotide pyrophosphatase/phosphodiesterase 1 (also known as ENPP1). By measuring the production of AMP and GMP researchers can effectively determine the activity of the ENPP1 enzyme. The assay provides a powerful tool to screen entire compound libraries for ENPP1 modulators to help find new therapeutics for diseases such as cancer.

ENPP1 is Critical for Purinergic Signalling

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.

Direct Detection of AMP and GMP to Measure ENPP1 Enzymatic Activity

AMP²/GMP² FP Assay - LEARN MORE

Transcreener AMP²/GMP² FP Assay Tech Manual (PDF)

AMP²/GMP² TR-FRET Assay - LEARN MORE

Transcreener AMP²/GMP² TR-FRET Assay Tech Manual (PDF)

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

The Transcreener ENPP1 Assay is in a simple, HTS-ready, mix-and-read format. Run your enzyme reaction, add Transcreener reagents, and read your plates. The assay is compatible with 96, 384, and 1536-well formats.

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

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

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

Transcreener AMP²/GMP² ENPP1 Assay Kits

* 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

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

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Transcreener® AMP²/GMP² ENPP1 Assay

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

Discover ENPP1 Inhibitors with the Transcreener AMP²/GMP² Assay

ENPP1 is Critical for Purinergic Signalling

Direct Detection of AMP and GMP to Measure ENPP1 Enzymatic Activity

Transcreener AMP²/GMP² FP Assay Tech Manual (PDF)

Transcreener AMP²/GMP² TR-FRET Assay Tech Manual (PDF)

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