The GAF-tandem domain of phosphodiesterase 5 as a potential drug target.

Classic PDE5 inhibitors interact with and block the catalytic site of PDE5. They have been clinically validated for treatment of erectile dysfunction as well as reduction of pulmonary arterial pressure, improvement of exercise capacity, quality of life, and arterial oxygenation in patients with secondary pulmonary hypertension. Minor side effects are visual disturbances, headache, migraine, back pain, and interaction with nitrates (hypotension). Some of those side effects presumably can be ameliorated by improving selectivity and pharmacokinetics; other side effects probably are target related due to inhibition of basic physiological processes. Target related side effects may be bypassed by using PDE5 inhibitors with a different mode of action: PDE5, like PDE2, PDE6, PDE10, and PDE11, is a multidomain protein with an N-terminal tandem GAF domain, which in case of PDE5, is allosterically activated by cGMP. Potential inhibitors acting at the PDE5 GAF domain would be expected to inhibit only pathophysiologically upregulated PDE5 activity, whereas basal activity of PDE5 would remain unaffected.Here, we summarize a high-throughput screening campaign to identify inhibitors of the regulatory GAF domain of human PDE5. To target the regulatory domain independently from the catalytic site, we used a chimeric reporter enzyme: The hPDE5 GAF-tandem domain functionally replaced the GAF domain in the cyanobacterial adenylyl cyclase CyaB1. We identified inhibitors that target the GAF domain and also inhibitors that target the bacterial cyclase.Compounds binding to the PDE5 GAF domain were reanalysed with native human PDE5 to demonstrate inhibition using capillary electrophoresis. This identified 16 compounds that act on the GAF domain of PDE5. Two compounds fulfilled the initial requirement to inhibit, exclusively, activated PDE5, but not basal PDE5 activity.