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we identified 16 compounds that inhibited the allosteric activation of the hPDE5 via
the tandem GAF domain. Two from these inhibited hPDE5 exclusively by interac-
tion with the tandem GAF domain while the catalytic domain was not inhibited,
thus fulfilling the initial requirements. All inhibitors of the hPDE5 tandem GAF
domain were assayed in additional PDE2 assays and revealed to be inhibitory under
activating and nonactivating conditions (with and without the allosteric PDE2
activator cGMP), which indicates interference with the catalytic site, but not the
allosteric site of PDE2 (unpublished results). Efforts have been started to crystallize
the newly identified GAF inhibitors together with the hPDE5 GAF domain. Crys-
tallization data might lead to a deeper understanding of the mechanism of binding
of the hPDE5 GAF inhibitors and will help to optimize binding properties.
In addition to the GAF inhibitors, the retests with the chimeric enzyme PDE5-
GAF-CyaB1 revealed another potentially highly interesting group of chemicals
that inhibited the class III adenylyl cyclase reporter enzyme.
Acknowledgments Joachim Schultz is supported by the Deutsche Forschungsgemeinschaft,
Klaus Mann optimized the performance of the Robocon robotic system and Necdet Aslan prepared
compound dilutions for screening by using an automated system.
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