A Fission Yeast-Based Platform for Phosphodiesterase Inhibitor HTSs and Analyses of Phosphodiesterase Activity - Phosphodiesterases as Drug Targets - page 143

1.2

1

0.8

0.6

0.4

no PDE
no PDE + BC69
PDE8A
PDE8A + BC69

0.2

0

0 40 80 120 160 200 240

[cAMP] ( µ m)

Fig. 5 5FOA R growth conferred by exogenous cAMP on strains lacking adenylyl cyclase. OD 600
values were determined for strains lacking adenylyl cyclase and either lacking PDE activity or
expressing PDE8A. The X -axis indicates the concentration of cAMP ( m M) added to the 5FOA
medium. At 40 m M cAMP ( vertical dashed line ), the OD 600 for the strain lacking PDE activity is
fivefold higher than for the strain expressing PDE8A. Addition of 20 m M BC69 (Fig. 3 ; identified
in a PDE4 inhibitor screen) shifts the cAMP response curve of the PDE8A-expressing strain
toward that of the strain lacking PDE activity

cGMP, promoting 5FOA R growth of a strain that lacks both adenylyl cyclase
and PDE activity (Fig. 6 ). Similar to the second-generation screens, cGMP
stimulated growth is reduced upon expression of a PDE that hydrolyzes
cGMP. This allows for screens to detect inhibitors of cGMP-specific PDEs,
while inhibitors of dual specificity PDEs can be screened for using exogenous
cAMP or cGMP (Fig. 2 ).
The ability to use exogenous cGMP for screening was validated using strains
expressing cGMP-specific PDE5A (McAllister-Lucas et al. 1993 ) and PDE9A
(Fisher et al. 1998b ). As expected for a strain lacking both adenylyl cyclase and
PDE activity, a higher concentration of cGMP is required to stimulate growth
relative to cAMP. 40 0.6 as compared to

12.5 m M cAMP, while 100 m M cGMP results in saturated growth as compared to
25 m M cAMP. Expression of either PDE5A or PDE9A completely eliminates the
response to cGMP at these, as well as significantly higher, concentrations. As
described above for PDE8A, a screen of inhibitors obtained in either PDE4 or
PDE7 HTSs led to the identification of compound BC76 (Fig. 3 ) as an effective
PDE5A inhibitor. In vitro enzyme assays confirm that BC76 is a PDE5A inhibitor
with an IC 50 of ~250 nM (Sharron Francis, personal communication).
These three iterations of screening strains and methods have allowed us to
construct strains that express mammalian PDEs from 10 of the 11 PDE families
(with the exception of the PDE6 family of the visual system), all of which display
sufficient activity to permit inhibitor screens based on 5FOA R growth.

m M cGMP produces an OD 600 ¼