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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.

The GAF domain is a small-molecule-binding-domain (SMBD) identified in >7400 proteins. However, mostly the ligands are unknown. Here we mainly deal with regulatory N-terminal tandem GAF domains, GAF-A and GAF-B, of four mammalian phosphodiesterases (PDEs) and of two cyanobacterial adenylyl cyclases (ACs) which bind cyclic nucleotides. These tandem GAFs are preceded by N-terminal sequences of variable lengths and a function of their own. In mammals, GAF domains are found only in cyclic nucleotide PDEs 2, 5, 6, 10, and 11. cAMP is the ligand for phosphodiesterase 10, cGMP for the others. Two cyanobacterial ACs, CyaB1 and 2, carry regulatory cAMP-binding tandem GAF domains which are similar in sequence to the mammalian ones. These tandem GAF domains have a prominent NKFDE motif which contributes to ligand binding in an as yet unknown manner. Contradicting structures (parallel vs. antiparallel) are available for the tandem GAF domains of PDE 2 and AC CyaB2. In addition, the structures of phosphodiesterase 5 and 10 GAF monomers with bound ligands have been solved. In all instances, cyclic nucleotide binding involves specific protein-ligand interactions within a tightly closed binding pocket and minimal solvent exposure of the ligand. The PDE tandem GAF domains can functionally substitute for the tandem of the cyanobacterial AC CyaB1; e.g. cGMP-regulation is grafted onto the AC using tandem GAFs from PDEs 2, 5 and 11. Studies of GAF domain-regulated PDEs are hampered by the identities of regulator and substrate molecules. Using AC CyaB1 as a reporter which uses ATP as a substrate solves this issue and makes the tandem GAF domains of mammalian PDEs available for detailed kinetic and mechanistic studies. In addition, drugs which potentially act on PDE regulatory domains may be assayed with such a novel test system.