Intracellular cyclic nucleotides are eliminated by phosphodiesterases (PDEs) and by ATP Binding cassette transporters such as ABCC4 and ABCC5. PDE5 and ABCC5 have similar affinity for cGMP whereas ABCC5 has much higher affinity for cGMP compared with cAMP. Since the substrate (cGMP) is identical for these two eliminatory processes it is conceivable that various PDE inhibitors also modulate ABCC5-transport. Cyclic GMP is also transported by ABBC4 but the affinity is much lower with a Km 50-100 times higher than for that of ABBCC5. The present study aimed to determine Ki-values for specific or relative specific PDE5 inhibitors (vardenafil, tadalafil, zaprinast and dipyridamole) and the non-specific PDE inhibitors (IBMX, caffeine and theophylline) for ABCC5 and ABCC4 transport. The transport of [(3)H]-cGMP (2 µM) was concentration-dependently inhibited with the following Ki-values: vardenafil (0.62 µM), tadalafil (14.1 µM), zaprinast (0.68 µM) and dipyridamole (1.2 µM), IBMX (10 µM), caffeine (48 µM) and theophylline (69 µM). The Ki-values for the inhibition of the [(3)H]-cAMP (2 µM) transport were: vardenafil (3.4 µM), tadalafil (194 µM), zaprinast (2.8 µM), dipyridamole (5.5 µM), IBMX (16 µM), caffeine (41 µM) and theophylline (85 µM). The specificity for ABCC5 we defined as ratio between Ki-values for inhibition of [(3)H]-cGMP and [(3)H]-cAMP transport. Tadalafil showed the highest specificity (Ki-ratio: 0.073) and caffeine the lowest (Ki-ratio: 1.2).

Elevated intracellular levels of cyclic guanosine monophosphate (cGMP) may induce apoptosis, and at least some cancer cells seem to escape this effect by increased efflux of cGMP, as clinical studies have shown that extracellular cGMP levels are elevated in various types of cancer. The human ATP binding cassette (ABC) transporter ABCC5 transports cGMP out of cells, and inhibition of ABCC5 may have cytotoxic effects. Sildenafil inhibits cGMP efflux by binding to ABCC5, and in order to search for potential novel ABCC5 inhibitors, we have identified sildenafil derivates using structural and computational guidance and tested them for the cGMP efflux effect. Eleven compounds from virtual ligand screening (VLS) were tested in vitro, using inside-out vesicles (IOV), for inhibition of cGMP efflux. 7 of 11 compounds predicted by VLS to bind to ABCC5 were more potent than sildenafil, and the two most potent showed Ki-values of 50 #x02013;100 nM.

In the present study we have characterized ATP-dependent transport of cAMP and cGMP in physiological, but also supraphysiological concentrations. The uptake into inside-out vesicles from human erythrocytes could be dissected into two components with high and low affinity. The respective K_m-values were 30.8 ± 5.2 and 352 ± 26 μM for cAMP and 2.6 ± 0.4 and 260 ± 15 μM for cGMP. The two cyclic nucleotides were unable to mutually inhibit cellular efflux for concentrations up to about 100 μM. At higher concentrations the inhibition curve showed a steep fall. The IC₅₀-value for cAMP reduction of high affinity [³H]-cGMP transport was 695 ± 9 μM. The respective value for cGMP inhibition of [³H]-cAMP efflux was 284 ± 20 μM. These observations are compatible with two selective high affinity transport systems. Other endogenous substances such as prostaglandins did not discriminate between cyclic nucleotide transport. The IC₅₀ values for inhibition of [³H]-cAMP and [³H]-cGMP were 4.1 and 4.2 μM for PGE₁, 2.7 and 4.4 μM for PGE₂, respectively. However, the prostaglandin analog misoprostol discriminated distinctly between cAMP and cGMP transport with respective IC₅₀-values of 4.5 and 24 μM. The assumption that the specific PDE5-inhibitor sildenafil could distinguish between the two cyclic nucleotides was disproved with respective IC₅₀ values of 3.8 and 2.9 μM for inhibition of [³H]-cAMP and [³H]-cGMP, respectively. However, at least one sildenafil analog (PHAR0099048) showed a clear difference with respective IC₅₀ values of 2.0 and 0.52 μM. The other tested sildenafil analogs showed no or minor ability to discriminate with IC₅₀ values of 0.16 and 0.17 μM for IS-39213, and 0.35 and 0.16 μM for IS-60049, respectively. In agreement with previous reports, the present study shows that proteins responsible for cyclic nucleotide transport are multiorganic anion pumps. However, the observation that drug analogs may discriminate between these two efflux systems makes them potential drug targets.