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Fluorescence-based adenylyl cyclase assay adaptable to high throughput screening
Kannan Vadakkadathmeethal1, Jennifer M Cunliffe, Jennifer Swift
1Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109-0632, USA.
Combinatorial Chemistry & High Throughput Screening
|May 18, 2007
Summary
A novel fluorescence assay using Bodipy FL GTPgammaS (BGTPgammaS) enables real-time measurement of adenylyl cyclase (AC) activity. This high-throughput screening method can accelerate the development of new therapeutics targeting AC-related diseases.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Cyclic adenosine monophosphate (cAMP) is a crucial second messenger regulating diverse cellular functions.
- Adenylyl cyclase (AC) enzymes synthesize cAMP, and their dysregulation is linked to various pathologies.
- Targeting membrane-bound ACs offers therapeutic potential for treating diseases associated with cAMP signaling.
Purpose of the Study:
- To develop a robust, real-time, and scalable assay for adenylyl cyclase (AC) activity.
- To facilitate high-throughput screening (HTS) for novel AC-modulating therapeutics.
- To characterize a new fluorescence-based assay utilizing Bodipy FL GTPgammaS (BGTPgammaS).
Main Methods:
- A novel fluorescence assay employing Bodipy FL GTPgammaS (BGTPgammaS) as a substrate.
- Monitoring fluorescence enhancement upon incubation with wild-type AC (wt-AC) and mutant AC (mut-AC).
- Evaluating the effects of known AC activators (forskolin, Gsalpha.GTPgammaS, Mn2+) and inhibitors (2'deoxy,3'AMP, 2',5'-dideoxy-3'ATP).
- Assessing assay adaptability to 96-well and 384-well formats for HTS.
Main Results:
- BGTPgammaS exhibited significantly enhanced fluorescence upon incubation with wt-AC and mut-AC in an activation-dependent manner.
- No fluorescence enhancement was observed with Bodipy FL ATPgammaS (BATPgammaS) as a substrate.
- Forskolin, Gsalpha.GTPgammaS, and Mn2+ dose-dependently increased BGTPgammaS fluorescence.
- Known AC inhibitors effectively reduced the fluorescence enhancement, validating the assay's specificity.
- The assay demonstrated scalability for 96-well and 384-well plate formats.
Conclusions:
- The developed BGTPgammaS fluorescence assay provides a sensitive and specific method for measuring AC activity.
- This assay is suitable for high-throughput screening, potentially accelerating the discovery of novel AC-targeted therapeutics.
- The assay's adaptability to multi-well plate formats supports its utility in drug discovery pipelines for AC-related diseases.
