cAMP Signaling pp 181-190 | Cite as

A Yeast-Based High-Throughput Screen for Modulators of Phosphodiesterase Activity

  • Ana Santos de Medeiros
  • Charles S. HoffmanEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1294)


Cell-based high-throughput screens (HTSs) targeting heterologously expressed proteins in yeast identify compounds that often display relevant biological activity when tested in cell culture. We developed a fission yeast-based HTS to detect small-molecule inhibitors of mammalian cyclic nucleotide phosphodiesterases (PDEs). These screens are carried out in Schizosaccharomyces pombe using a PKA-repressed fbp1-ura4 reporter whose expression due to low PKA activity prevents cells from growing in medium containing the pyrimidine analog 5-fluoro orotic acid (5FOA). We describe here the steps required to construct strains for screening and to optimize conditions for successful screens.


Cyclic nucleotide phosphodiesterase Fission yeast Schizosaccharomyces pombe fbp1 High-throughput screen Inhibitors 



This work was supported by NIH grant GM079662, the Peter Rieser Lectureship Fund, and a grant from Boston College to C.S.H.


  1. 1.
    Hoffman CS (2005) Glucose sensing via the protein kinase A pathway in Schizosaccharomyces pombe. Biochem Soc Trans 33:257–260CrossRefPubMedGoogle Scholar
  2. 2.
    Hoffman CS, Winston F (1990) Isolation and characterization of mutants constitutive for expression of the fbp1 gene of Schizosaccharomyces pombe. Genetics 124:807–816PubMedCentralPubMedGoogle Scholar
  3. 3.
    Hoffman CS, Winston F (1991) Glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene occurs by a cAMP signaling pathway. Genes Dev 5:561–571CrossRefPubMedGoogle Scholar
  4. 4.
    Ivey FD, Wang L, Demirbas D et al (2008) Development of a fission yeast-based high-throughput screen to identify chemical regulators of cAMP phosphodiesterases. J Biomol Screen 13:62–71CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Demirbas D, Ceyhan O, Wyman AR et al (2011) Use of a Schizosaccharomyces pombe PKA-repressible reporter to study cGMP metabolising phosphodiesterases. Cell Signal 23:594–601CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Alaamery MA, Wyman AR, Ivey FD et al (2010) New classes of PDE7 inhibitors identified by a fission yeast-based HTS. J Biomol Screen 15:359–367CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Ceyhan O, Birsoy K, Hoffman CS (2012) Identification of biologically active PDE11-selective inhibitors using a yeast-based high-throughput screen. Chem Biol 19:155–163CrossRefPubMedGoogle Scholar
  8. 8.
    Demirbas D, Wyman AR, Shimizu-Albergine M et al (2013) A yeast-based chemical screen identifies a PDE inhibitor that elevates steroidogenesis in mouse leydig cells via PDE8 and PDE4 inhibition. PLoS One 8:e71279CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Ivey FD, Taglia FX, Yang F et al (2010) Activated alleles of the Schizosaccharomyces pombe gpa2 + Gα gene identify residues involved in GDP-GTP exchange. Eukaryot Cell 9:626–633CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272CrossRefPubMedGoogle Scholar
  11. 11.
    Wang L, Griffiths K Jr, Zhang YZ et al (2005) Schizosaccharomyces pombe adenylate cyclase suppressor mutations suggest a role for cAMP phosphodiesterase regulation in feedback control of glucose/cAMP signaling. Genetics 171:1523–1533CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Biology DepartmentBoston CollegeChestnut HillUSA

Personalised recommendations