Learn More About Transcreener Method Used for This AMPK Assay

Discover AMPK Modulators with the Transcreener ADP2 Assay

The Transcreener ADP² Kinase Assay directly measures the ADP produced by  AMPK (also known as AMP-Activated Protein Kinase or 5' Adenosine Monophosphate-Activated Protein Kinase). By measuring the production of ADP researchers can effectively determine the activity of the AMPK enzyme. The assay provides a powerful tool to screen compound libraries for AMPK modulators to help find new therapeutics for a variety of different diseases including coronavirus, autoimmune diseases, and cancer.

The Role of AMPK As Therapeutic Target

AMPK is a drug target in many areas, including metabolism, inflammatory diseases, tumor suppression, and more recently, the coronavirus. SARS-CoV-2 uses the ACE2 receptor to infect the human body. Research has shown that AMPK can increase the expression and stability of ACE2.¹ Once the coronavirus is in the body, it leads to a downregulation of ACE2. This causes a chain of events leading to deadly cardio-pulmonary issues.¹ One drug currently on the market Metformin, activates AMPK via phosphorylation. Currently used as a Diabetes drug, Metformin has positive effects on both lipid and glucose metabolism via AMPK.² Another AMPK activator AICAR has shown to improve alveolar and capillary barriers preventing damage from an infection like SARS-CoV-2 in mice.³ Further work needs to be done but AMPK is a target of interest for coronavirus treatment.

Direct Detection of ADP to Measure AMPK Enzymatic Activity

ADP FP Kinase Assay

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

ADP FI Kinase Assay

Positive FI signal. Compatible with simple fluorescence plate readers. Faster read time than FP or TR-FRET.

ADP TR-FRET ATPase Assay

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

Applications

  • Measure Enzymatic Activity of AMPK
  • Screen Compound Libraries for AMPK Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling
  • Measure Drug-Target Residence Time

Features

  • Direct detection of unlabeled ADP
  • 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, FI, or TR-FRET readouts

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

The Transcreener ADP² AMPK 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 ADP Under AMPK Initial Velocity - FP Readout

The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use both 25 µM ATP with 20 µM SAMS peptide substrates. Linearity is shown here under initial velocity conditions when raw data is converted to ADP formed. The AMPK enzyme reaction took place for 60 minutes at room temperature. The enzymatic reaction was then halted using Stop & Detect Buffer B included in the kit and assay plates were read.

Human AMPK Enzyme Titration

Linear Response

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. Assay conditions include 0.5 µg/mL AMPK, 25 µM ATP, 20 µM SAMS for an enzyme reaction of 60 minutes at room temperature.

Dose-Response - Staurosporine

Staurosporine Dose Response with AMPK

IC50 = 2.18 nM

Robust Assay Yields Quality Data

Z’ measurements using optimized AMPK 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. Both reactions have 25 μM ATP and 20 μM SAMS. Z' shown here = 0.85.

Three Fluorescent Readouts Available

Choose the readout that is the best fit for your lab based on preference and plate reader compatibility. 

Fluorescent Intensity

Assay conditions for the AMPK titration shown include 25 µM ATP, 20 µM SAMS, and 24 µg/mL ADP² antibody.

TR-FRET

Assay conditions for the AMPK titration shown include 25 µM ATP, 20 µM AMPK,  55 nM tracer, and 8 nM ADP² Tb Antibody.

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

What's Included

What You Will Need

AMPK 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 AMPK 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

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References:

  1. Liu, J., Li, X., Lu, Q., Ren, D., Sun, X., Rousselle, T., ... & Leng, J. (2019). AMPK: a balancer of the renin–angiotensin system.Bioscience Reports39(9), BSR20181994.
  2. Sharma, S., Ray, A., & Sadasivam, B. (2020). Metformin in COVID-19: A possible role beyond diabetesdiabetes research and clinical practice164, 108183.
  3. Ayres, J. S. (2020). A metabolic handbook for the COVID-19 pandemic. Nature Metabolism, 1-14.
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