A Fission Yeast-Based Platform for Phosphodiesterase Inhibitor HTSs and Analyses of Phosphodiesterase Activity - Phosphodiesterases as Drug Targets - page 142

Fig. 4 Effect of rolipram, BC35, and BC54 (structures shown in Fig. 3 ) on 5FOA R growth of a
PDE4B2 expressing strain. OD 600 values were measured in the presence of various concentrations
of compounds in 384 well microtiter dishes after 48-h growth

the budding yeast S. cerevisiae , S. pombe strains that lack either adenylyl cyclase or
PKA are viable. Deletion of the git2 adenylyl cyclase gene abolishes cAMP
synthesis, so that PKA can be regulated by addition of cAMP to the growth
medium. The expressed PDE activity reduces cell growth in response to exogenous
cAMP (Fig. 2 ), allowing one to determine the optimal concentration of cAMP to
add to the medium for inhibitor detection. For PDE8A-expressing cells, 40 m M
cAMP is optimal for achieving differential growth in 5FOA medium, as this level of
cAMP stimulates growth to OD 600 > 1.0 of a strain lacking PDE activity, while

having little effect on a PDE8A-expressing strain (Fig. 5 ). These conditions were
used to measure PDE8A inhibition by 56 PDE4 or PDE7 inhibitors identified from
the first generation screens and revealed that the PDE4 inhibitor BC69 (Fig. 3 )isan
effective inhibitor of PDE8A (Fig. 5 ). Further work with BC69 allowed the
optimization of a PDE8A inhibitor HTS.

6 Third Generation Screens

The screens described above exploit the fact that the expressed PDE can
hydrolyze endogenously produced or exogenously added cAMP to reduce
PKA activity. As such, these screens detect inhibitors of cAMP hydrolysis.
We have found that S. pombe PKA can also be activated by exogenously-added