The GAF-Tandem Domain of Phosphodiesterase 5 as a Potential Drug Target - Phosphodiesterases as Drug Targets - page 154

domains, and long isoforms in the PDE4 family, encoded by four separate genes,
have a tandem of two UCR domains N-terminal to the catalytic domain (the
acronym UCR stands for upstream-conserved region), and phosphorylation in this
region augments PDE4 activity (Houslay and Adams 2003 ; Sette and Conti 1996 ).
However, the biochemical characterization of the relationship between regulatory
and catalytic domains remains cumbersome. For PDEs 2, 5, 6, 10, and 11, the
allosteric activator cGMP (cAMP for PDE10) concomitantly serves as a substrate.
This seriously limits the kinetic characterization and renders the design of high-
throughput assays for modulators of these allosteric sites difficult. In 2002, we
demonstrated that the GAF-tandem domain of PDE2 is functionally interchange-
able with the GAF-tandem domain from the cyanobacterial adenylyl cyclase
CyaB1, i.e., the adenylyl cyclase, which intrinsically is regulated by its enzymatic
product cAMP, is turned into a cGMP-regulated adenylyl cyclase by appending the
region of PDE2 that contains the tandem GAF domains (Kanacher et al. 2002 ).
Activation is robust and the biochemical properties of the tandem GAF domain in
the chimera reflect those reported in studies of recombinant isolated GAF domains
or in conjunction with its catalytic PDE domain (Corbin et al. 2000 , 2003 ). Several
subsequent studies involving the tandem GAF domains from PDE5, PDE10, and
PDE11 in chimeras with the cyanobacterial cyclase have generally confirmed that
the cyclase can be used as an excellent reporter enzyme to characterize biochemical
properties of mammalian PDE GAF-tandem domains (Bruder et al. 2006 ; Gross-
Langenhoff et al. 2006 ; Hofbauer et al. 2008 ). In fact, this has been extended to the
tandem calmodulin-binding domains from PDE1 and the tandem UCR domains
from PDE4 isoforms (Banjac and Schultz, unpublished). The constructs with the
tandem GAF domains offer the advantage that the allosteric activator/regulator and
enzyme substrate are separate, noninterfering chemicals.
Here, we describe the use of a chimeric protein comprising the tandem GAF
domain of human PDE5 (hPDE5) fused to the adenylyl cyclase CyaB1. We have
exploited this novel construct for use in a high-throughput screen of a diverse
chemical library with several 100,000 compounds to identify substances that
might inhibit adenylyl cyclase activation via interaction with the regulatory tandem
GAF domains from hPDE5. Here, we critically review the assay parameters and the
feasibility of such a large-scale assay.
2 Requirements to Run a High-Throughput Screen
The PDE5 tandem GAF-CyaB1 adenylyl cyclase chimera (Fig. 1 ; PDE5-GAF-
CyaB1) converts ATP to equimolar amounts of cAMP and pyrophosphate. A key
question in developing a high-throughput screen involves determining which prod-
uct of the enzymatic reaction can be measured in a simple, reliable, quick, and cost-
effective way cAMP determination via ELISA, although well established, is costly
and due to several washing steps unsuitable for automation. Sensitive colorimetric
methods to determine inorganic phosphate using ammonium molybdate and