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.

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

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

ENPP1 Titration With ATP Substrate

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

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

Supplier Instrument FP Assays
TR-FRET Assays
bioteklogo TriStar²S LB 942 in review validated
Mithras² LB 943 in review validated
bioteklogo 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
BMGLABTECH Logo 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
MDS AT logo 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
perkinElmerLogo EnVision®/EnVision® Xcite validated validated
tecanLogo Infinite® M1000/M1000Pro/Safire2™ validated validated
Infinite® M200 not capable not capable
Infinite® F500 validated validated
Infinite® F200/Ultraevolution contact us contact us
Spark™ 10M validated validated

What's Included

What You Will Need

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.

Assay Plates for TR-FRET

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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