Targeting the cGAS-STING Pathway Using a Homogenous, HTS Compatible Transcreener cGAS Assay

Transcreener cGAMP cGAS Assay

Transcreener cGAMP cGAS Assay directly measures cyclic GMP-AMP (cGAMP) produced by cyclic GMP-AMP synthetase (cGAS, C6orf150, 2'3'-cGAMP Synthetase). These cGAMP measurements allow researchers to effectively determine the enzyme's activity. The assay provides a powerful tool to screen compound libraries for cGAS modulators to help find new therapies for disease.

The kit comes complete with the detection reagents required to measure activity. cGAS enzyme is not included in the inhibitor screening assay kit. Recombinant cGAS enzyme is available for separate purchase through BellBrook labs. 

Important Messenger in the STING Pathway

DNA in the cytosol of eukaryotic cells indicates infection or cellular damage, and it elicits a robust immune response, driven by type I interferon (IFN) induction. 

cGAS is an exquisitely sensitive sensor for double-stranded DNA in the cytoplasm.  Binding of DNA activates the enzyme to produce a unique cyclic dinucleotide second messenger, cGAMP, which acts as an agonist for the STING (stimulator of interferon genes) receptor, leading to induction of a type I interferon response.

cGAS STING Pathway

How Does This cGAS Assay Work?

The Transcreener cGAMP cGAS Assay determines cGAS enzyme activity by directly measuring the cGAMP formed by the enzyme using the Transcreener cGAMP Assay. By detecting cGAMP output, the assay provides a universal method to assess the activity of any cGAMP-producing enzyme in real-time.

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

The cGAS 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 cGAMP to Measure cGAS Enzymatic Activity

Fluorescent Polarization (FP)

cGAS Assay Schematic FP

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

cGAS Assay Schematic TR-FRET

Applications

  • Measure Enzymatic Activity of cGAS
  • Screen Compound Libraries for cGAS inhibitors
  • Quantify Inhibitor Potency
  • Determine Inhibitor-cGAS Residence Time

Features

  • Direct detection of unlabeled cGAMP
  • 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 cGAS assay is compatible with 96, 384, and 1536-well formats.

Transcreener Mix and Read Assay

Outstanding Signal Stability

The assay exhibits 24 hours+ signal stability. Plate stability like this is vital for sizeable high throughput screens that take significant time to complete due to massive sample quantity.

cGAS-Assay-Stability-curve

Standard curves include ATP, GTP, dsDNA, and titration of cGAMP. The same plate was read at each time point.

Detection of cGAMP Under cGAS Initial Velocity

The assay demonstrates linearity when raw data is converted to cGAMP using a standard curve. cGAS is activated by double-stranded DNA and requires ATP and GTP substrates for the production of cGAMP.

Full-Length Human cGAS Titration

Full-length human enzyme titration

Linear Response

Screen for Inhibitors & Perform SAR

The Transcreener cGAS Assay is 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.

1600 Compound Pilot Screen

Acquire Accurate Dose Response Measurements

Illustrates DNA & Substrate Dependance of the cGAS Enzyme

The assay accurately demonstrates the characteristics of the cGAS enzyme. Double-stranded DNA activates the enzyme which generates cGAMP in the presence of both GTP and ATP. A 45 base pair ISD (interferon stimulatory DNA) and a 60 base pair HSV (herpes simplex virus) DNA  both activate the enzyme. The assay has a high affinity to cGAMP and can detect even in the presence of other nucleotides.

cGAS Enzyme Activation by dsDNA

cGAS DNA Titration

Dependence on ATP and GTP

BellBrook Lab's Recombinant cGAS Enzyme

Active, full-length Human Cyclic GMP-AMP synthase enzyme, His-tagged protein expressed and purified from E.coli. The enzyme has been thoroughly validated with Transcreener cGAS Activity Assays. For specific activity, please refer to the Certificate of Analysis for each individual enzyme lot. Purity is >90% in a 20mM Tris 7.5, 200mM NaCl, 1mM DTT, 10% glycerol buffer composition. The enzyme is currently used in the Transcreener cGAS Activity Assay at 30 nM, which is 1.75 μg/mL (17.5 ng in 10 µL enzyme reaction volume in a 384-well format). Please adjust the enzyme concentration based on your requirements.

Recombinant cGAS Enzyme

cGAS 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 cGAS 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
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 cGAMP cGAS Assay Kits

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

cGAS Enzyme Ordering Information

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

The Role of cGAS As a Therapeutic Target

cGAS recognizes foreign double-stranded DNA and initiates an innate immune response, called the cGAS-STING pathway. cGAS can cause tissue or autoimmune diseases if overactivated by the body's abnormal DNA. Examples of diseases caused by this include Aicardi-Goutières syndrome, systemic lupus erythematosus, and primary biliary liver disease (Li et al, 2021).

The cGAS-STING pathway is an attractive target for autoimmune diseases and cancers. Transcreener's cGAMP cGAS Assay is an excellent tool for researchers examining the therapeutic effects of cGAS. 

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