Biology Reference
In-Depth Information
Keywords cAMP
Cell-based assay
cGMP
High throughput
Inhibitors
Phosphodiesterase
PKA
Schizosaccharomyces pombe
1
Introduction
Mammals express 11 families of cyclic nucleotide phosphodiesterases (PDEs),
encoded by 21 genes that generate more than 100 distinct PDE isoforms by
variations in transcriptional start sites and splicing. Each PDE family includes
proteins encoded by as many as four genes (designated A through D), and are
grouped, in part, according to substrate specificity: PDE4, PDE7, and PDE8 are
cAMP-specific; PDE5, PDE6, and PDE9 are cGMP-specific; and PDE1, PDE2,
PDE3, PDE10, and PDE11 display dual-specificity. PDE families are also grouped
according to conserved domains outside of the catalytic domain and their relative
sensitivity to various chemical inhibitors and other molecules such as calcium/
calmodulin or cGMP (Bender and Beavo
2006
; Conti and Beavo
2007
; Lerner and
Epstein
2006
; Soderling and Beavo
2000
). In addition, tissue-specific expression
and subcellular localization of PDEs permit members of this superfamily, though
acting on only two substrates, to influence tissue-specific biological processes or
even distinct processes in a single cell through compartmentalization of cAMP
signaling (Houslay
2010
). Thus, inhibition of a specific PDE subpopulation in a cell
may have a therapeutic benefit without altering other cAMP- or cGMP-controlled
processes in the affected cell.
Most, though not all, approaches to PDE inhibitor development focus on com-
pounds that act by binding to the catalytic site. This is true of medicinal chemistry
approaches that produce and characterize analogs of nonselective PDE inhibitors,
as well as rational drug design approaches that are guided by crystal structures of
target PDEs (Card et al.
2004
,
2005
). Although these methods have led to the
development of many selective PDE inhibitors, there remains a need to develop
specific inhibitors for certain PDE families (PDE1, PDE6, PDE8, PDE11), as well
as subtype-selective inhibitors for families encoded by multiple genes (PDE1,
PDE3, PDE4, PDE6, PDE7, and PDE8).
This chapter describes a fission yeast-based platform for PDE inhibitor screens
that can also be used to characterize PDEs in live cells. Differing from traditional
inhibitor screens, this approach allows inexpensive screening of full-length
enzymes expressed in a eukaryotic cell, following the yeast genetic philosophy of
discovery: “first find something that does what you are looking for and then figure
out the mechanism”. As such, this screening platform is open to the discovery of
both active-site and allosteric inhibitors that are structurally unrelated to current
PDE inhibitors. In addition, it can be used to characterize the PDEs under condi-
tions that closely resemble their natural cellular environment in contrast to in vitro
enzyme assays that are frequently carried out on biochemically stable fragments of
PDEs expressed in and purified from
E. coli
. Finally, this system is amenable to
conducting cDNA library screens for genes that encode biological regulators of a
target PDE and genetic screens for mutations that affect PDEs or their regulators.
