Jackie D. Corbin

3 Cyclic Nucleotide-Dependent Protein Kinases

Publisher Summary This chapter describes the characteristics and functions of cyclic nucleotide-dependent protein kinases and methods for purifying the enzyme. The characteristics of the enzymes are described as they relate to functional aspects of the isozymes, to evolutionary relationships and homologies among protein kinases and other cyclic nucleotide binding proteins, and to the mechanisms of catalytic and regulatory subunit action. The regulation of enzyme activity by cyclic nucleotides and other effectors is reviewed and the role the enzymes play in cellular function is assessed in the chapter. The role of these kinases in the short-term control of cellular function through the regulation of enzyme activities and the long-term control of processes—such as transcription, protein induction and cell growth, and differentiation—is highlighted. In addition, several methods used for deducing the biological role of the kinases are compared and critiqued. It remains to be clearly demonstrated whether the regulatory subunit plays a role in the regulation of gene transcription.

High biochemical selectivity of tadalafil, sildenafil and vardenafil for human phosphodiesterase 5A1 (PDE5) over PDE11A4 suggests the absence of PDE11A4 cross-reaction in patients

The physiological role of phosphodiesterase (PDE)11 is unknown and its biochemical characteristics are poorly understood. We have expressed human His-tagged PDE11A4 and purified the enzyme to apparent homogeneity. PDE11A4 displays Km values of 0.97 μM for cGMP and 2.4 μM for cAMP, and maximal velocities were 4- to 10-fold higher for cAMP than for cGMP. Given the homology between PDE11 and PDE5, we have compared the biochemical potencies of tadalafil (Cialis™, Lilly-ICOS), vardenafil (Levitra™, Bayer-GSK), and sildenafil (Viagra™, Pfizer Inc.) for PDE11A4 and PDE5A1. PDE5A1/PDE11A4 selectivities are 40-, 9300-, and 1000-fold for tadalafil, vardenafil, and sildenafil, respectively. This suggests that none of these three compounds is likely to crossreact with PDE11A4 in patients.

Regulatory subunit of cAMP-dependent protein kinase inhibits phosphoprotein phosphatase.

The activity of a purified high molecular weight phosphoprotein phosphatase was inhibited by purified type II cAMP-dependent protein kinase. This effect required cAMP and was obtained in the absence of ATP. The isolated type II regulatory subunits (R-subunits) from several species also inhibited the phosphatase activity in both crude extracts and purified preparations. Half maximal inhibition was observed at 0.06-0.25 microM, well within the physiological range of R-subunit concentrations. The inhibitory potency of R-subunit was greater using the thiophosphorylated form. Limited trypsinization of the R-subunit abolished the inhibitory activity. The C-subunit released the bound cAMP when combined with R-subunit, but the phosphatase did not, implying that the inhibited species is a R.cAMP-phosphatase complex. The results suggest that the R-subunit might have at least one physiological role in addition to inhibition of the C-subunit, i.e., inhibition of phosphatase. The latter would occur only when cAMP is elevated.

Single step isolation of sildenafil from commercially available Viagra™ tablets

Sildenafil, the active ingredient in Viagra™, ahs been purified from commercially available tablets. The purification, using Sephadex G25 chromatography under conditions of low ionic strength, is simple and inexpensive. Sildenafil purified according to this protocol has been characterized with respect to its IC50 for PDE5, its ultraviolet absorption profile, and by collision-induced dissociation fingerprinting, positive ion nanospray, and MALDI mass spectrometry. Tritated sildenafil (6 Ci/mmol) was prepared commercially using the sildenafil purified by this protocol and was verified to retain the potency of unlabeled sildenafil. This protocol and similar procedures will allow investigators to easily isolate sufficient amounts of sildenafil or other PDE5 inhibitors for conducting biochemical and in vitro studies of drug action.

Tyrosine-612 in PDE5 contributes to higher affinity for vardenafil over sildenafil

Despite close structural similarity, vardenafil (Levitra®) is 32-fold more potent than sildenafil (Viagra®) to inhibit cGMP-binding cGMP-specific PDE (PDE5); this is due to differences between their heterocyclic rings. In co-crystals with PDE5, one of the rings of vardenafil or sildenafil interacts with Tyr612, a catalytic site AA, via (1) a hydrogen bond with a water molecule and (2) hydrophobic bonds. For mutant PDE5Y612F, which ablates hydrogen-bonding potential, vardenafil or sildenafil inhibition was strengthened (2.2- or 3.0-fold, respectively), implying that the Tyr612 hydroxyl is a negative determinant for these inhibitors. For mutant PDE5Y612A, which ablates both hydrogen bonding and hydrophobic-bonding potential, vardenafil inhibition was weakened much more than sildenafil inhibition (122- and 26-fold, respectively), suggesting that hydrophobic bonds involving Tyr612 are stronger for vardenafil than for sildenafil.

Phosphodiesterase-5 Inhibition

Mammalian cyclic nucleotide phosphodiesterases (PDE) are a super-family of enzymes that currently consists of 11 families named PDE1–PDEll (Fig. 1; ref. 1). Some of these families contain multiple genes so that now there are known to be more than 20 PDE genes, and splicing variation of most of the genes yields even more PDEs with a final total of more than 50 isoforms (2). Some of the PDEs degrade cyclic guanosine monophosphate (cGMP), some degrade cyclic adenine monophosphate (cAMP), and some degrade both cyclic nucleotides. These enzymes vary in tissue distributions and are believed to have different physiological roles. PDE1, PDE2, PDE3, PDE4, and PDE7 are present in cardiac tissue, and PDE1, PDE2, PDE3, PDE4, PDE5, and PDEll are present in vascular smooth muscle.

Signal Transduction through cAMP and cGMP

Cells respond to hormones, neurotransmitters and other agents in two opposing ways. These are referred to as amplification (enhancement) and adaptation (diminution) (1). Amplification enables an organism or cell to respond to a very faint signal such as a low blood hormone concentration. Adaptation prevents constant background stimulation, or excessive stimulation, of a pathway. The cAMP cascade system illustrates two different kinds of amplification: the first is magnitude amplification, which is an increase in output molecules in greater numbers than input molecules; and the second is sensitivity amplification, which is a greater percentage increase in ouput than the percentage increase in input. An example of magnitude amplification would be the production of 100 cAMP molecules from 1 active molecule of adenylate cyclase, and sensitivity amplification could be an increase in cAMP-dependent protein kinase activity of 200% by 100% increase in cAMP. Both kinds of amplification can occur at each step of a cascade, although the overall magnification in a cascade can also be calculated. For example, the overall magnitude amplification for glucagon stimulation of glycogen breakdown in the liver is represented by the number of glucose molecules produced divided by the number of glucagon molecules added, and can be greater than 10,000.