Abstract
The second messenger cAMP has been implicated in numerous cellular processes such as glycogen metabolism, muscle contraction, learning and memory, and differentiation and development. Genetic evidence suggests that the enzyme that produces cAMP, adenylyl cyclase (AC), may be involved in pathogenesis in many of these cellular processes. In addition, these data suggest that membrane-bound ACs may be valuable targets for therapeutics to treat pathogenesis of these processes. The development of a robust real-time adenylyl cyclase assay that can be scalable to highthroughput screening could help in the development of novel therapeutics. Here we report a novel fluorescence-based cyclase assay using Bodipy FL GTPγS (BGTPγS). The fluorescence of the Bodipy™ moiety of BGTPγS was dramatically enhanced by incubation with the minimal catalytic core of wild-type-AC (wt-AC) and a mutant with decreased purine selectivity (mut-AC), in an AC activation-dependent manner. No increase in fluorescence was observed using Bodipy FL ATPγS (BATPγS) as substrate for either wt-AC or mut-AC. Using BGTPγS, forskolin, Gsα.GTPγS and the divalent cation Mn2+ potently enhanced the rate of fluorescence increase in a concentration-dependent manner. The fluorescence enhancement of the Bodipy moiety was inhibited by known inhibitors of AC such as 2deoxy,3AMP and 2,5-dideoxy- 3ATP. Furthermore, the fluorescence assay is adaptable to 96-well and 384-well multiplate format and is thus applicable to high throughput screening methodologies.
Keywords: Adenylyl cyclase, fluorescence, high-throughput screening, Bodipy-FL-GTPγS
Combinatorial Chemistry & High Throughput Screening
Title: Fluorescence-Based Adenylyl Cyclase Assay Adaptable to High Throughput Screening
Volume: 10 Issue: 4
Author(s): Kannan Vadakkadathmeethal, Jennifer M. Cunliffe, Jennifer Swift, Robert T. Kennedy, Richard R. Neubig and Roger K. Sunahara
Affiliation:
Keywords: Adenylyl cyclase, fluorescence, high-throughput screening, Bodipy-FL-GTPγS
Abstract: The second messenger cAMP has been implicated in numerous cellular processes such as glycogen metabolism, muscle contraction, learning and memory, and differentiation and development. Genetic evidence suggests that the enzyme that produces cAMP, adenylyl cyclase (AC), may be involved in pathogenesis in many of these cellular processes. In addition, these data suggest that membrane-bound ACs may be valuable targets for therapeutics to treat pathogenesis of these processes. The development of a robust real-time adenylyl cyclase assay that can be scalable to highthroughput screening could help in the development of novel therapeutics. Here we report a novel fluorescence-based cyclase assay using Bodipy FL GTPγS (BGTPγS). The fluorescence of the Bodipy™ moiety of BGTPγS was dramatically enhanced by incubation with the minimal catalytic core of wild-type-AC (wt-AC) and a mutant with decreased purine selectivity (mut-AC), in an AC activation-dependent manner. No increase in fluorescence was observed using Bodipy FL ATPγS (BATPγS) as substrate for either wt-AC or mut-AC. Using BGTPγS, forskolin, Gsα.GTPγS and the divalent cation Mn2+ potently enhanced the rate of fluorescence increase in a concentration-dependent manner. The fluorescence enhancement of the Bodipy moiety was inhibited by known inhibitors of AC such as 2deoxy,3AMP and 2,5-dideoxy- 3ATP. Furthermore, the fluorescence assay is adaptable to 96-well and 384-well multiplate format and is thus applicable to high throughput screening methodologies.
Export Options
About this article
Cite this article as:
Vadakkadathmeethal Kannan, Cunliffe M. Jennifer, Swift Jennifer, Kennedy T. Robert, Neubig R. Richard and Sunahara K. Roger, Fluorescence-Based Adenylyl Cyclase Assay Adaptable to High Throughput Screening, Combinatorial Chemistry & High Throughput Screening 2007; 10 (4) . https://dx.doi.org/10.2174/138620707780636600
DOI https://dx.doi.org/10.2174/138620707780636600 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
Call for Papers in Thematic Issues
Artificial Intelligence Methods for Biomedical, Biochemical and Bioinformatics Problems
Recently, a large number of technologies based on artificial intelligence have been developed and applied to solve a diverse range of problems in the areas of biomedical, biochemical and bioinformatics problems. By utilizing powerful computing resources and massive amounts of data, methods based on artificial intelligence can significantly improve the ...read more
Eco-friendly Agents for Biological Control of Pathogenic Diseases
The discovery of an alternative biological approach to disease management includes work on medicinal products derived from natural sources as a starting point for the development of eco-friendly agents for these diseases and the injuries they cause, as well as reducing human contact with hazardous chemicals and their residues. We ...read more
Emerging trends in diseases mechanisms, noble drug targets and therapeutic strategies: focus on immunological and inflammatory disorders
Recently infectious and inflammatory diseases have been a key concern worldwide due to tremendous morbidity and mortality world Wide. Recent, nCOVID-9 pandemic is a good example for the emerging infectious disease outbreak. The world is facing many emerging and re-emerging diseases out breaks at present however, there is huge lack ...read more
Exploring Spectral Graph Theory in Combinatorial Chemistry
Scope of the Thematic Issue: Combinatorial chemistry involves the synthesis and analysis of a large number of diverse compounds simultaneously. Traditional methods rely on brute force experimentation, which can be time-consuming and resource-intensive. Spectral Graph Theory, a branch of mathematics dealing with the properties of graphs in relation to the ...read more
- Author Guidelines
- Graphical Abstracts
- Fabricating and Stating False Information
- Research Misconduct
- Post Publication Discussions and Corrections
- Publishing Ethics and Rectitude
- Increase Visibility of Your Article
- Archiving Policies
- Peer Review Workflow
- Order Your Article Before Print
- Promote Your Article
- Manuscript Transfer Facility
- Editorial Policies
- Allegations from Whistleblowers
Related Articles
-
Potential Targets and Their Relevant Inhibitors in Anti-influenza Fields
Current Medicinal Chemistry Quorum-Sensing Inhibitors and Biofilms
Anti-Infective Agents in Medicinal Chemistry An Aggressive Medical Approach for Inflammatory Bowel Disease: Clinical Challenges and Therapeutic Profiles in a Retrospective Hospitalbased Series
Current Clinical Pharmacology Evolution of Novel Non-Steroidal Anti-Inflammatory Drugs with Reduced Gastrointestinal Adverse Effects
Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry Population Based Helicobacter pylori Screening and Eradication: Advances Versus Side Effects
Current Pharmaceutical Design Melatonin in Bacterial and Viral Infections with Focus on Sepsis: A Review
Recent Patents on Endocrine, Metabolic & Immune Drug Discovery Catalytic Activity of Certain Antibodies as a Potential Tool for Drug Synthesis and for Directed Prodrug Therapies
Current Medicinal Chemistry A Purified Fraction of Ocimum tenuiflorum (L) Inhibits LTC4, LTA4 and COX-2 Activities, Down-Regulates mRNA Expression in HL-60 Cells and Reduces Lung Inflammation in an OVA Induced Asthma Model in BALB/c Mice
Immunology, Endocrine & Metabolic Agents in Medicinal Chemistry (Discontinued) Involvement of Leukotriene Pathway in the Pathogenesis of Ischemia- Reperfusion Injury and Septic and Non-Septic Shock
Current Vascular Pharmacology Non-Injection Routes for Allergen Immunotherapy: Focus on Sublingual Immunotherapy
Inflammation & Allergy - Drug Targets (Discontinued) Anti-Inflammatory Mechanisms of Glucocorticoids Targeting Granulocytes
Current Drug Targets - Inflammation & Allergy Mast Cells in Lung Homeostasis: Beyond Type I Hypersensitivity
Current Respiratory Medicine Reviews Editorial [ Hot Topic:Immunomodulation in Helminth Infections
Endocrine, Metabolic & Immune Disorders - Drug Targets Drug Repurposing for the Treatment of Staphylococcal Infections
Current Pharmaceutical Design Role of Nitric Oxide in the Modulation of Angiogenesis
Current Pharmaceutical Design Microbiota Regulation of Inflammatory Bowel Disease
Inflammation & Allergy - Drug Targets (Discontinued) Role of FoxP3<sup>+</sup> Treg Cells Mediating Immune Suppression in Leprosy
Current Immunology Reviews (Discontinued) Recombinant Secretory Immunoglobulin A in Passive Immunotherapy: Linking Immunology and Biotechnology
Current Pharmaceutical Biotechnology Angiotensin-TGF-β1 Crosstalk in Human Idiopathic Pulmonary Fibrosis:Autocrine Mechanisms in Myofibroblasts and Macrophages
Current Pharmaceutical Design Hypersensitivity Reactions to Last Generation Chimeric, Umanized and Human Recombinant Monoclonal Antibodies for Therapeutic Use
Current Pharmaceutical Design