Potency and mechanism of action of E4021, a type 5 phosphodiesterase isozyme-selective inhibitor, on the photoreceptor phosphodiesterase depend on the state of activation of the enzyme

The ability of inhibitors selective for the type 5 phosphodiesterase isozym e (PDE5) to act on the photoreceptor PDE isozyme (PDE6, the central effecto r enzyme for visual transduction) is poorly understood. Because PDES inhibi tors are currently used as therapeutic agents, it is important to assess th e potency and mechanism of action of this class of PDE inhibitor on PDE6. W e show that E4021 (sodium 1-[6-chloro-4-(3,4-methylenedioxybenzyl)-aminoqui nazolin-2-yl]piperidine-4-carboxylate carboxylate sesquihydrate) inhibits a ctivated PDE6 (K-1 = 1.7 nM) as potently as PDE5. This makes E4021 the most potent inhibitor of PDE6 discovered to date. The effectiveness of E4021 to inhibit nonactivated PDE6 (with bound inhibitory gamma subunits) is reduce d 40-fold compared with the activated enzyme. Furthermore, at intermediate E4021 concentrations and high cGMP concentrations, nonactivated PDE undergo es activation of cGMP hydrolysis rather than inhibition. We demonstrate dir ect competition of E4021 and the gamma subunits for binding to the catalyti c site. Measurements of cGMP binding to noncatalytic regulatory sites on th e catalytic subunits of PDE6 rule out an allosteric effect of E4021 by dire ct binding to these noncatalytic sites. We conclude that E4021 is a competi tive inhibitor of cGMP hydrolysis and that the gamma subunit also competes with both E4021 and substrate for catalytic site binding. An understanding of the effects of PDE5-targeted drugs on retinal PDE6 requires a knowledge of the complex interactions among substrate, drug, and inhibitory gamma sub unit at the catalytic site of both nonactivated and activated forms of PDE6 .