ThePDE2 (phosphodiesterase 2)enzyme is one of 21 differentphosphodiesterases (PDE) found inmammals. These different PDEs can be subdivided to 11 families (PDE1 – PDE11). The different PDEs of the same family are functionally related despite the fact that theiramino acid sequences show considerable divergence.[1] The PDEs have differentsubstrate specificities. Some arecAMP selectivehydrolases (PDE 4, -7 and -8), others arecGMP selective hydrolases (PDE 5, -6 and -9) and the rest can hydrolyse bothcAMP andcGMP (PDE1, -2, -3, -10 and -11).[2]
There is only onegene family coding for the PDE2, which is the PDE2A. Threesplice variants have been found, the PDE2A1, PDE2A2 and PDE2A3 (PDE2A2 has only been found in rats). PDE2A1 iscytosolic whereas -A2 and -A3 are membrane bound. It has been suggested that different localization of PDE2A2 and -A3 is due to a unique N-terminal sequence, which is absent in PDE2A1. Despite the PDE2Asplice variants being different, there is no known differences in their kinetic behavior.[3]
Thecrystal structure of theactive site of the PDE2 enzyme has been reported.[4] Even thoughamino acid sequences, for members of the PDE family show considerable difference (25-35% identity), the overall folding, functional and structural elements of the active sites are very similar. Theactive site is formed by residues that are highly conserved among all PDEs. Thebinding pocket contains metal ion (zinc and magnesium) binding sites. The twohistidine and twoaspartic acid residues, which bind zinc are conserved among all studied PDEs.[3] The structure of several other PDE iso-enzymes has been elucidated and among them fewco-crystal structures, with inhibitors residing in the active site.[5] The co-crystal structures for PDE4B, PDE4D andPDE5A have revealed two common features of inhibitor binding to PDEs. One is aplanar ring structure of the inhibitors, which align in theactive site of the enzymes and the other is a conservedglutamine residue (the "glutamine switch" mentioned below), which is essential fornucleotide recognition and selectivity.[1][5][6]
As mentioned above, PDE2 is able to hydrolyze bothcAMP and cGMP, whereas some other members of the PDE family are selective for either of the two cyclic nucleotides. The variability in selectivity towards either cAMP or cGMP is thought to be determined by a so-called "glutamine switch". The "glutamine switch" is an invariantglutamine found in all PDEs, for which thecrystal structure has been solved. In PDE2, this residue is the Gln859. It has potential to formhydrogen bonds with the exocyclicamino group of cAMP and the exocycliccarbonyl oxygen of cGMP. In PDEs, which canhydrolyze both cAMP andcGMP, this glutamine is able to rotate freely. In PDEs that are selective for either cAMP or cGMP, this glutamine is constrained by neighboring residues to a position favoring selectivity for either cyclicnucleotide.[1][3]
When cGMP binds to theallosteric GAF-B domain of the PDE, it causesconformational change in theprotein structure leading to higher enzyme activity. Increased hydrolysis ofcAMP due to binding ofcGMP to the GAF-B domain is well documented, however there are no known examples for the reverse.[3] It has been shown that the GAF-B domain has 30-100 fold loweraffinity forcAMP than forcGMP.[7] This information combined with what is currently known aboutintracellular cAMP concentrations, renders it unlikely that activation ofcGMP hydrolysis bycAMP can take placein vivo.[3]
PDE2 is expressed in various tissues, for example:adrenal medulla, brain, heart,platelet,macrophages andendothelial cells. The enzyme is thought to be involved in regulating many differentintracellular processes, such as:[3][8]
Several enzyme functions have been reported for the PDE2. It has been shown that PDE2 lowers cAMP through increased cGMP caused byatrial natriuretic peptide (ANP) resulting in decreasedaldosteronesecretion.[3] It has also been suggested that PDE2 might play an important role in the regulation of elevatedintracellular concentrations of cAMP and cGMP in platelets. PDE3 is an important player in platelet aggregation. It has been reported that higher concentration of cGMP causes inhibition ofPDE3, whereas it stimulates PDE2. Interplay between those two functions seems to mediate an opposing regulation of cAMP in platelets.[3] PDE2 regulates cardiac L-type Ca2+ current incardiacmyocytes, where activation of PDE2 by cGMP lowers cAMP and thereby affecting cardiac function.[3] However, it has been recently found that different cAMP pools, located within the cardiac myocyte, mediate different (moreover, sometimes opposing) effects. As different PDE types may affect different cAMP pools, the different PDEs may regulate different processes in the cell.[9] PDE2 is expressed in several regions of the brain and rat experiments have indicated that inhibition of PDE2 enhances functions such as memory.[3] PDE2 is up regulated whenmonocytes differentiate intomacrophages, but the role of PDE2 in matured macrophages is yet to be characterized. Furthermore, PDE2 has been indicated to play a role ininflammatory responses as it has been detected in microvessels, but not in larger vessels. It has been speculated thattumor necrosis factor-alpha (TNFα) might regulate the function of PDE2 inendothelial cells and thereby affecting flow of fluid and cells through the endothelial barrier asin vitro experiments on endothelial cells show up regulation of both PDE2mRNA and activity.[3]
Until now PDE2 inhibitors have mostly been used as research tools, but are presently being investigated for improving memory, for decreasing endothelialpermeability underinflammatory conditions,[3] and for preventing/improving heart failure and cardiac hypertrophy.[10]
The first specific inhibitor developed for PDE2 wasEHNA (erythro-9-(2-hydroxy-3-nonyl)adenine). It has been demonstrated to specifically act on PDE2 by inhibiting cGMP-activation of PDE2 with an IC50 value of ~1μM and an at least 50-fold selectivity over other PDEs.[11] The core structure of EHNA resembles cAMP but differentiates in the fact that EHNA has a bulkyhydrophobic carbon side chain replacing the phospho-ribosemoiety in cAMP.[1]
Inprimary cultures of ratcortical neurons, the inhibition of PDE2A by EHNA potentiatesNMDA (N-methyl-D-aspartate) receptor activated increase in cGMP, but has no effect on cAMP concentrations.[12]EHNA is also a verypotentadenosine deaminase inhibitor with an IC50 ~2 nM.[13] This dual inhibition would lead to the accumulation of the two inhibitorymetabolites, adenosine[13][14] and cGMP,[12][15] which may act insynergy to mediate diverse pharmacological responses including anti-viral, anti-tumour and anti-arrhythmic effects.[11]Although EHNA potently inhibitsadenosine deaminase, it has been successfully used with the proper controls as a tool to probe PDE2 functions. EHNA has been used to study implication of PDE2 in calcium control in cardiac myocytes[16] and has shown to be effective to reverse hypoxic pulmonary vasoconstriction in perfused lung models.[17]EHNA has been therefore been used for two purposes:
However, the use of EHNA as a chemical tool in determining the pharmacological role of PDE2 is limited due to its low potency to inhibit PDE2 and high potency in inhibition of adenosine deaminase.[18] Theoretically, this problem can be resolved if the effect of adenosine accumulated by EHNA, a result of adenosine deaminase inhibition, can be accounted and corrected for. This way, a positive inotropic effect elicited by EHNA, as a result of PDE2 inhibition, was observed.[19][20]
BAY 60-7550 is ananalog ofEHNA, which is more than 100-fold more potent and is highly selective for PDE2A.[13] Other newly discovered selective PDE2 inhibitors are PDP (9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one)[21] and oxindole.[18]
| Inhibitor | IC50 | Reference |
| EHNA | 1 μM | [11] |
| Oxindole | 40 nM | [16] |
| Bay 60-7550 | 4,7 nM | [13] |
| PDP | 0,6 nM | [21] |
The table above shows thepotency of PDE2 inhibitors including EHNA. There is a large increase in potency between EHNA, Bay 60-7550 and PDP. The large dimethoxybenzyl group in position 2 of the purine moiety of Bay-60 7550 and PDP might be contributing to the added potency.
Comparison of these inhibitors with the naturalsubstrates of the enzyme, cAMP and cGMP reveal some common characteristics of the molecules. The main characteristic of all the molecules is the flat moiety comprising at least two fused ring structures, a six atom ring and a five atom ring. This ring system in cGMP and cAMP is a purine ring system, and the same is true for EHNA and PDP. Bay 60-7550 and oxindole lack the purine core but do possess a related ring system. Hydrogen bond acceptors, mostly nitrogen but also oxygen, reside in the ring system of the inhibitors. These atoms might interact with hydrogen bond donators, which are part of amino acids in the active site of the enzyme and thereby contribute to the inhibition of the enzyme from hydrolyzing cAMP and cGMP similar to how the natural substrates bind to the active site.[1]
The structures of Bay 60-7550 and PDP are very similar. The difference between these molecules is the exocyclicmethyl group on Bay 60-7550, which replaces the nitrogen atom in PDP decreasing the possibility to formhydrogen bonds with the enzyme in an important site for substrate and inhibitor binding.The oxindole structure differs from the other inhibitors since it is more divergent from thepurine ring system and has less hydrogen binding possibilities. The molecule also lacks the large side group, analogous to the dimethoxybenzyl group of Bay 60-7550 and PDP. It is difficult to predict possible interactions to the enzyme without a co-crystal structure of the phenomenon.
There is a lack of co-crystal structures of inhibitors bound in theactive site of PDE2. However, a computerizeddocking model of the inhibitor EHNA and the substrates cAMP and cGMP bound in the catalytic site have been made.[1] Thedocking model of EHNA showed that themutations of the amino acids Asp811 to Ala (Asp811Ala) and Ile826 to Val (Ile826Val) at theactive site, where the only amino acid substitutions that significantly affected the inhibition by EHNA. The Asp811 mutation to alanine increased IC50 value for EHNA 6-fold and the Ile826 mutation to valine leads to a 7-fold increased IC50 value for EHNA compared to wild type PDE2A.[1]
EHNA is in close proximity to Gln859 at the active site, which could donate two hydrogen bonds to N1 and N6 of the nitrogen atoms in the adenine ring of EHNA. On the other site of the binding pocket the Asp811 could donate another hydrogen bond to N7 in the adenine ring in order to stabilize the bond inhibitor. This hypothesis is supported by the fact that the Asp811Ala mutant has decreased activity toward cAMP, whereas activity toward cGMP is unchanged.[1]
The residues in the lower binding pocket may lie too far away for interaction with the inhibitor and therefore might be irrelevant for EHNA selectivity.However the residues may play an indirect role of EHNA selectivity. Ile826 is positioned below the purine ring of EHNA and thereby limits the space for EHNA. Substitution with smaller valine (Ile826Val mutation) could increase the space for EHNA and cause the loss of hydrogen binding with residues in the upper binding pocket, while improving hydrogen binding within the lower binding pocket. This shift of interactions could destabilize binding of the adenine ring of EHNA, which could be the reason for higher IC50 value.[1]
No models are available for the other inhibitors than EHNA, which align in the active site. Therefore, it is more difficult to interpret the molecular binding. When looking at the inhibitors and their overall similarity, it is likely that they bind with a similar mechanism to the active site and that the different side groups determine potency of the inhibitor.The determinants of inhibitor specificity within the PDE2 active site are not very well known and with better understanding of these determinants it would facilitate the development of inhibitors with increased potency.[1]