Biology Reference
In-Depth Information
A Fission Yeast-Based Platform for
Phosphodiesterase Inhibitor HTSs
and Analyses of Phosphodiesterase Activity
Didem Demirbas, Ozge Ceyhan, Arlene R. Wyman, and Charles S. Hoffman
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
2 Use of Yeast to Study Mammalian Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
3 cAMP Signaling and
fbp1
Transcriptional Regulation in the Fission Yeast
S. pombe
....137
4 First Generation Screens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
5 Second Generation Screens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
6 Third Generation Screens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
7 Profiling PDE Activity in Strains Lacking Adenylyl Cyclase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
8 Additional Uses of this System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Abstract Fission yeast strains have been engineered so that their growth behavior
reflects the activity of heterologous cyclic nucleotide phosphodiesterases (PDEs).
These strains can be used in High-Throughput Screens (HTSs) for PDE inhibitors
that possess “drug-like” characteristics, displaying activity in a growth stimulation
assay over a 48-h period. Through three generations of development, a collection of
strains expressing 10 of the 11 mammalian PDE families that is appropriate for
small molecule inhibitor screening has been generated in our laboratory. Strains
unable to synthesize cyclic nucleotides allow characterization of PDE activity in
that the enzyme's potency is reflected in the amount of either cAMP or cGMP that
must be added to the growth medium to stimulate cell growth. In the future, this
system could be used to screen cDNA libraries for biological regulators of target
PDEs and for the construction of strains that co-express PDEs and associated
regulatory proteins to facilitate molecular and genetic studies of their functions
and, in particular, to identify whether different PDE-partner protein complexes
show distinct patterns of inhibitor sensitivity.
D. Demirbas, O. Ceyhan, A.R. Wyman, and C.S. Hoffman (
*
)
Biology Department, Boston College, Higgins Hall 401B, Chestnut Hill, Boston, MA 02467, USA
e-mail: hoffmacs@bc.edu
