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Fission yeast-based high-throughput screens for PKA pathway inhibitors and activators
Ana Santos de Medeiros1, Grace Kwak, Jordan Vanderhooft
1Biology Department, Boston College, 140 Commonwealth Avenue, Higgins Hall 401b, Chestnut Hill, MA, 02467, USA.
Abstract:
Features of the fission yeast Schizosaccharomyces pombe cAMP/PKA pathway make S. pombe particularly amenable for heterologous expression of cAMP pathway proteins such as GαS subunits and their cognate adenylyl cyclases, PKA catalytic and regulatory subunits, and cyclic nucleotide phosphodiesterases. We have constructed two PKA-repressed reporters for use in high-throughput screens to detect compounds that elevate or reduce PKA activity, thus facilitating the discovery of both inhibitors and activators of these target proteins. Here, we describe steps to construct screening strains and to optimize and conduct these screens.
Insights
Fission yeast enables studying the cAMP/PKA pathway. Researchers developed novel reporters for high-throughput screening to discover drugs that modulate protein kinase A (PKA) activity.
Area of Science:
- Molecular Biology
- Biochemistry
- Yeast Genetics
Background:
- The cAMP/PKA pathway regulates crucial cellular processes.
- Fission yeast (Schizosaccharomyces pombe) offers advantages for heterologous protein expression.
- Studying cAMP pathway components like GαS, adenylyl cyclases, and phosphodiesterases is vital.
Purpose of the Study:
- To develop tools for high-throughput screening of cAMP/PKA pathway modulators.
- To facilitate the discovery of novel inhibitors and activators of PKA activity.
- To describe the construction and optimization of screening strains in S. pombe.
Main Methods:
- Utilizing fission yeast (Schizosaccharomyces pombe) for heterologous expression.
- Constructing two novel PKA-repressed reporter systems.
- Optimizing protocols for high-throughput screening assays.
Main Results:
- Established S. pombe strains suitable for cAMP/PKA pathway research.
- Developed functional reporter systems to detect changes in PKA activity.
- Demonstrated the utility of these reporters for drug discovery.
Conclusions:
- Fission yeast is a powerful model for studying the cAMP/PKA pathway.
- The developed reporter systems enable efficient screening for PKA modulators.
- This work facilitates the discovery of new therapeutic agents targeting the cAMP pathway.
