PDE2
Encyclopedia
The PDE2 enzyme
is one of 21 different phosphodiesterase
s (PDE) found in mammal
s. 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 their amino acid
sequences show considerable divergence. The PDEs have different substrate
specificities. Some are cAMP
(figure 1) selective hydrolase
s (PDE 4, -7 and -8), others are cGMP
(figure 1) selective hydrolases (PDE 5, -6 and -9) and the rest can hydrolyse both cAMP
and cGMP
(PDE1, -2, -3, -10 and -11).
There is only one gene
family coding for the PDE2, which is the PDE2A. Three splice variants have been found, the PDE2A1, PDE2A2 and PDE2A3 (PDE2A2 has only been found in rats). PDE2A1 is cytosol
ic 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 PDE2A splice variants being different, there is no known differences in their kinetic behavior. (See review article ).
of the active site
of the PDE2 enzyme has been reported. (Picture online: ).
Even though amino 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. The active site
is formed by residues that are highly conserved among all PDEs. The binding pocket contains metal ion (zinc and magnesium) binding sites. The two histidine
and two aspartic acid
residues, which bind zinc are conserved among all studied PDEs (See review article ).
The structure of several other PDE iso-enzymes has been elucidated and among them few co-crystal structures, with inhibitors residing in the active site. The co-crystal structures for PDE4B, PDE4D and PDE5A have revealed two common features of inhibitor binding to PDEs. One is a planar
ring structure of the inhibitors, which align in the active site
of the enzymes and the other is a conserved glutamine
residue (the “glutamine switch” mentioned below), which is essential for nucleotide
recognition and selectivity.
has been solved. In PDE2 this residue is the Gln859. It has potential to form hydrogen bonds with the exocyclic amino group of cAMP and the exocyclic carbonyl
oxygen of cGMP. In PDEs, which can hydrolyze both cAMP
and cGMP
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 cyclic nucleotide
(See review article ).
in the protein
structure leading to higher enzyme activity. Increased hydrolyzis of cAMP
due to binding of cGMP
to the GAF-B domain is well documented, however there are no known examples for the reverse (See review article ). It has been shown that the GAF-B domain has 30-100 fold lower affinity for cAMP
than for cGMP
. This information combined with what is currently known about intracellular
cAMP concentrations, renders it unlikely that activation of cGMP
hydrolysis by cAMP
can take place in vivo
. (See review article.)
, brain, heart, platelet
, macrophage
s and endothelial cells. The enzyme is thought to be involved in regulating many different intracellular
processes, such as:
(See review article )
Several enzyme functions have been reported for the PDE2. It has been shown that PDE2 lowers cAMP through increased cGMP caused by atrial natriuretic peptide
(ANP) resulting in decreased aldosterone
secretion
(See review article ).
It has also been suggested that PDE2 might play an important role in the regulation of elevated intracellular
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 of PDE3
, whereas it stimulates PDE2. Interplay between those two functions seems to mediate an opposing regulation of cAMP in platelets (See review article ).
PDE2 regulates cardiac L-type Ca2+ current in cardiac myocyte
s, where activation of PDE2 by cGMP lowers cAMP and thereby affecting cardiac function. (See review article ).
PDE2 is expressed in several regions of the brain and rat experiments have indicated that inhibition of PDE2 enhances functions such as memory(See review article ).
PDE2 is up regulated when monocyte
s differentiate into macrophages, but the role of PDE2 in matured macrophages is yet to be characterized. Furthermore, PDE2 has been indicated to play a role in inflammatory
responses as it has been detected in microvessels, but not in larger vessels. It has been speculated that tumor necrosis factor-alpha (TNFα) might regulate the function of PDE2 in endothelial cells and thereby affecting flow of fluid and cells through the endothelial barrier as in vitro
experiments on endothelial cells show up regulation of both PDE2 mRNA and activity. (See review article )
Until now PDE2 inhibitors have mostly been used as research tools, but are presently being investigated for improving memory and decreasing endothelial permeability
under inflammatory
conditions (See review article ).
(erythro-9-(2-hydroxy-3-nonyl)adenine, figure 2). 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. The core structure of EHNA resembles cAMP but differentiates in the fact that EHNA has a bulky hydrophobic carbon side chain replacing the phospho-ribose moiety in cAMP.
neurons, the inhibition of PDE2A by EHNA potentiates NMDA
(N-metyl-D-aspartate) receptor activated increase in cGMP, but has no effect on cAMP concentrations.
EHNA is also a very potent
adenosine deaminase
inhibitor with an IC50 ~2 nM. This dual inhibition would lead to the accumulation of the two inhibitory metabolite
s, adenosine and cGMP, which may act in synergy
to mediate diverse pharmacological responses including anti-viral, anti-tumour and anti-arrhythmic effects.
Although EHNA potently inhibits adenosine 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 and has shown to be effective to reverse hypoxic pulmonary vasoconstricion in perfused lung models.
EHNA has been therefore been used for two purposes:
However, the use of EHNA as a chemical tool in determing the pharmacological role of PDE2 is limited due to low PDE2 potency and high potency in inhibition of adenosine deaminase.
of EHNA
, which is more than 100-fold more potent and is highly selective for PDE2A. Other newly discovered selective PDE2 inhibitors are PDP (9-(6-Phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one, figure 5) and Oxindole (figure 3).
Table 1 shows the potency
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.
s of the enzyme, cAMP and cGMP (see figure 1 through 5) 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.
s 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 the purine
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.
of PDE2. However, a computerized docking model of the inhibitor EHNA and the substrates cAMP and cGMP bound in the catalytic site have been made
.
The docking model
of EHNA showed that the mutation
s of the amino acids Asp811 to Ala (Asp811Ala) and Ile826 to Val (Ile826Val) at the active 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.
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.
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.
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
is one of 21 different phosphodiesterase
Phosphodiesterase
A phosphodiesterase is any enzyme that breaks a phosphodiester bond. Usually, people speaking of phosphodiesterase are referring to cyclic nucleotide phosphodiesterases, which have great clinical significance and are described below...
s (PDE) found in mammal
Mammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
s. 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 their amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
sequences show considerable divergence. The PDEs have different substrate
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
specificities. Some are cAMP
Cyclic adenosine monophosphate
Cyclic adenosine monophosphate is a second messenger important in many biological processes...
(figure 1) selective hydrolase
Hydrolase
In biochemistry, a hydrolase is an enzyme that catalyzes the hydrolysis of a chemical bond. For example, an enzyme that catalyzed the following reaction is a hydrolase:-Nomenclature:...
s (PDE 4, -7 and -8), others are cGMP
Cyclic guanosine monophosphate
Cyclic guanosine monophosphate is a cyclic nucleotide derived from guanosine triphosphate . cGMP acts as a second messenger much like cyclic AMP...
(figure 1) selective hydrolases (PDE 5, -6 and -9) and the rest can hydrolyse both cAMP
Cyclic adenosine monophosphate
Cyclic adenosine monophosphate is a second messenger important in many biological processes...
and cGMP
Cyclic guanosine monophosphate
Cyclic guanosine monophosphate is a cyclic nucleotide derived from guanosine triphosphate . cGMP acts as a second messenger much like cyclic AMP...
(PDE1, -2, -3, -10 and -11).
There is only one gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
family coding for the PDE2, which is the PDE2A. Three splice variants have been found, the PDE2A1, PDE2A2 and PDE2A3 (PDE2A2 has only been found in rats). PDE2A1 is cytosol
Cytosol
The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments....
ic 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 PDE2A splice variants being different, there is no known differences in their kinetic behavior. (See review article ).
Crystal structure
The crystal structureCrystal structure
In mineralogy and crystallography, crystal structure is a unique arrangement of atoms or molecules in a crystalline liquid or solid. A crystal structure is composed of a pattern, a set of atoms arranged in a particular way, and a lattice exhibiting long-range order and symmetry...
of the active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
of the PDE2 enzyme has been reported. (Picture online: ).
Even though amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
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. The active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
is formed by residues that are highly conserved among all PDEs. The binding pocket contains metal ion (zinc and magnesium) binding sites. The two histidine
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
and two aspartic acid
Aspartic acid
Aspartic acid is an α-amino acid with the chemical formula HOOCCHCH2COOH. The carboxylate anion, salt, or ester of aspartic acid is known as aspartate. The L-isomer of aspartate is one of the 20 proteinogenic amino acids, i.e., the building blocks of proteins...
residues, which bind zinc are conserved among all studied PDEs (See review article ).
The structure of several other PDE iso-enzymes has been elucidated and among them few co-crystal structures, with inhibitors residing in the active site. The co-crystal structures for PDE4B, PDE4D and PDE5A have revealed two common features of inhibitor binding to PDEs. One is a planar
Planar
In computer graphics, planar is the method of representing pixel colours with several bitplanes of RAM. Each bit in a bitplane is related to one pixel on the screen...
ring structure of the inhibitors, which align in the active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
of the enzymes and the other is a conserved glutamine
Glutamine
Glutamine is one of the 20 amino acids encoded by the standard genetic code. It is not recognized as an essential amino acid but may become conditionally essential in certain situations, including intensive athletic training or certain gastrointestinal disorders...
residue (the “glutamine switch” mentioned below), which is essential for nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
recognition and selectivity.
Substrate selectivity
As mentioned above PDE2 is able to hydrolyze both cAMP and cGMP (figure 1), 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 invariant glutamine found in all PDEs, for which the crystal structureCrystal structure
In mineralogy and crystallography, crystal structure is a unique arrangement of atoms or molecules in a crystalline liquid or solid. A crystal structure is composed of a pattern, a set of atoms arranged in a particular way, and a lattice exhibiting long-range order and symmetry...
has been solved. In PDE2 this residue is the Gln859. It has potential to form hydrogen bonds with the exocyclic amino group of cAMP and the exocyclic carbonyl
Carbonyl
In organic chemistry, a carbonyl group is a functional group composed of a carbon atom double-bonded to an oxygen atom: C=O. It is common to several classes of organic compounds, as part of many larger functional groups....
oxygen of cGMP. In PDEs, which can hydrolyze both cAMP
Cyclic adenosine monophosphate
Cyclic adenosine monophosphate is a second messenger important in many biological processes...
and cGMP
Cyclic guanosine monophosphate
Cyclic guanosine monophosphate is a cyclic nucleotide derived from guanosine triphosphate . cGMP acts as a second messenger much like cyclic AMP...
this glutamine
Glutamine
Glutamine is one of the 20 amino acids encoded by the standard genetic code. It is not recognized as an essential amino acid but may become conditionally essential in certain situations, including intensive athletic training or certain gastrointestinal disorders...
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 cyclic nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
(See review article ).
Regulation
When cGMP binds to the allosteric GAF-B domain of the PDE, it causes conformational changeConformational change
A macromolecule is usually flexible and dynamic. It can change its shape in response to changes in its environment or other factors; each possible shape is called a conformation, and a transition between them is called a conformational change...
in the protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
structure leading to higher enzyme activity. Increased hydrolyzis of cAMP
Cyclic adenosine monophosphate
Cyclic adenosine monophosphate is a second messenger important in many biological processes...
due to binding of cGMP
Cyclic guanosine monophosphate
Cyclic guanosine monophosphate is a cyclic nucleotide derived from guanosine triphosphate . cGMP acts as a second messenger much like cyclic AMP...
to the GAF-B domain is well documented, however there are no known examples for the reverse (See review article ). It has been shown that the GAF-B domain has 30-100 fold lower affinity for cAMP
Cyclic adenosine monophosphate
Cyclic adenosine monophosphate is a second messenger important in many biological processes...
than for cGMP
Cyclic guanosine monophosphate
Cyclic guanosine monophosphate is a cyclic nucleotide derived from guanosine triphosphate . cGMP acts as a second messenger much like cyclic AMP...
. This information combined with what is currently known about intracellular
Intracellular
Not to be confused with intercellular, meaning "between cells".In cell biology, molecular biology and related fields, the word intracellular means "inside the cell".It is used in contrast to extracellular...
cAMP concentrations, renders it unlikely that activation of cGMP
Cyclic guanosine monophosphate
Cyclic guanosine monophosphate is a cyclic nucleotide derived from guanosine triphosphate . cGMP acts as a second messenger much like cyclic AMP...
hydrolysis by cAMP
Cyclic adenosine monophosphate
Cyclic adenosine monophosphate is a second messenger important in many biological processes...
can take place in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
. (See review article.)
Clinical value of PDE2
PDE2 is expressed in various tissues, for example: adrenal medullaAdrenal medulla
The adrenal medulla is part of the adrenal gland. It is located at the center of the gland, being surrounded by the adrenal cortex. It is the innermost part of the adrenal gland, consisting of cells that secrete epinephrine , norepinephrine , and a small amount of dopamine in response to...
, brain, heart, platelet
Platelet
Platelets, or thrombocytes , are small,irregularly shaped clear cell fragments , 2–3 µm in diameter, which are derived from fragmentation of precursor megakaryocytes. The average lifespan of a platelet is normally just 5 to 9 days...
, macrophage
Macrophage
Macrophages are cells produced by the differentiation of monocytes in tissues. Human macrophages are about in diameter. Monocytes and macrophages are phagocytes. Macrophages function in both non-specific defense as well as help initiate specific defense mechanisms of vertebrate animals...
s and endothelial cells. The enzyme is thought to be involved in regulating many different intracellular
Intracellular
Not to be confused with intercellular, meaning "between cells".In cell biology, molecular biology and related fields, the word intracellular means "inside the cell".It is used in contrast to extracellular...
processes, such as:
- aldosteroneAldosteroneAldosterone is a hormone that increases the reabsorption of sodium ions and water and the release of potassium in the collecting ducts and distal convoluted tubule of the kidneys' functional unit, the nephron. This increases blood volume and, therefore, increases blood pressure. Drugs that...
secretionSecretionSecretion is the process of elaborating, releasing, and oozing chemicals, or a secreted chemical substance from a cell or gland. In contrast to excretion, the substance may have a certain function, rather than being a waste product...
from the adrenal glandAdrenal glandIn mammals, the adrenal glands are endocrine glands that sit atop the kidneys; in humans, the right suprarenal gland is triangular shaped, while the left suprarenal gland is semilunar shaped... - intracellular concentrations of cAMP and cGMP in platelets
- cGMP in neurons and effect on long-term memory
- barrier function of endothelial cells under inflammatoryInflammationInflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process...
conditions
(See review article )
Several enzyme functions have been reported for the PDE2. It has been shown that PDE2 lowers cAMP through increased cGMP caused by atrial natriuretic peptide
Atrial natriuretic peptide
Atrial natriuretic peptide , atrial natriuretic factor , atrial natriuretic hormone , or atriopeptin, is a powerful vasodilator, and a protein hormone secreted by heart muscle cells. It is involved in the homeostatic control of body water, sodium, potassium and fat...
(ANP) resulting in decreased aldosterone
Aldosterone
Aldosterone is a hormone that increases the reabsorption of sodium ions and water and the release of potassium in the collecting ducts and distal convoluted tubule of the kidneys' functional unit, the nephron. This increases blood volume and, therefore, increases blood pressure. Drugs that...
secretion
Secretion
Secretion is the process of elaborating, releasing, and oozing chemicals, or a secreted chemical substance from a cell or gland. In contrast to excretion, the substance may have a certain function, rather than being a waste product...
(See review article ).
It has also been suggested that PDE2 might play an important role in the regulation of elevated intracellular
Intracellular
Not to be confused with intercellular, meaning "between cells".In cell biology, molecular biology and related fields, the word intracellular means "inside the cell".It is used in contrast to extracellular...
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 of PDE3
PDE3
PDE3 is a phosphodiesterase. The PDEs belong to at least eleven related gene families, which are different in their primary structure, substrate affinity, responses to effectors, and regulation mechanism....
, whereas it stimulates PDE2. Interplay between those two functions seems to mediate an opposing regulation of cAMP in platelets (See review article ).
PDE2 regulates cardiac L-type Ca2+ current in cardiac myocyte
Myocyte
A myocyte is the type of cell found in muscles. They arise from myoblasts.Each myocyte contains myofibrils, which are long, long chains of sarcomeres, the contractile units of the cell....
s, where activation of PDE2 by cGMP lowers cAMP and thereby affecting cardiac function. (See review article ).
PDE2 is expressed in several regions of the brain and rat experiments have indicated that inhibition of PDE2 enhances functions such as memory(See review article ).
PDE2 is up regulated when monocyte
Monocyte
Monocytes are a type of white blood cell and are part of the innate immune system of vertebrates including all mammals , birds, reptiles, and fish. Monocytes play multiple roles in immune function...
s differentiate into macrophages, but the role of PDE2 in matured macrophages is yet to be characterized. Furthermore, PDE2 has been indicated to play a role in inflammatory
Inflammation
Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process...
responses as it has been detected in microvessels, but not in larger vessels. It has been speculated that tumor necrosis factor-alpha (TNFα) might regulate the function of PDE2 in endothelial cells and thereby affecting flow of fluid and cells through the endothelial barrier as in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
experiments on endothelial cells show up regulation of both PDE2 mRNA and activity. (See review article )
Until now PDE2 inhibitors have mostly been used as research tools, but are presently being investigated for improving memory and decreasing endothelial permeability
Vascular permeability
Vascular permeability, often in the form of capillary permeability, characterizes the capacity of a blood vessel wall to allow for the flow of small molecules or even whole cells in and out of the vessel. Blood vessel walls are lined by a single layer of endothelial cells...
under inflammatory
Inflammation
Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process...
conditions (See review article ).
PDE2A inhibitors
EHNA
The first specific inhibitor developed for PDE2 was EHNAEHNA
EHNA is a phosphodiesterase inhibitor that selectively inhibits phosphodiesterase type 2 ....
(erythro-9-(2-hydroxy-3-nonyl)adenine, figure 2). 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. The core structure of EHNA resembles cAMP but differentiates in the fact that EHNA has a bulky hydrophobic carbon side chain replacing the phospho-ribose moiety in cAMP.
Inhibitory effects of EHNA
In primary cultures of rat corticalCerebral cortex
The cerebral cortex is a sheet of neural tissue that is outermost to the cerebrum of the mammalian brain. It plays a key role in memory, attention, perceptual awareness, thought, language, and consciousness. It is constituted of up to six horizontal layers, each of which has a different...
neurons, the inhibition of PDE2A by EHNA potentiates NMDA
NMDA
N-Methyl-D-aspartic acid or N-Methyl-D-aspartate is an amino acid derivative which acts as a specific agonist at the NMDA receptor mimicking the action of glutamate, the neurotransmitter which normally acts at that receptor...
(N-metyl-D-aspartate) receptor activated increase in cGMP, but has no effect on cAMP concentrations.
EHNA is also a very potent
Potent
Potent may refer to:*Vair#Potent for the heraldic fur*Warren Potent for the Australian Olympic medalist in shootingSee also:*Potency...
adenosine deaminase
Adenosine deaminase
Adenosine deaminase is an enzyme involved in purine metabolism. It is needed for the breakdown of adenosine from food and for the turnover of nucleic acids in tissues.-Reactions:...
inhibitor with an IC50 ~2 nM. This dual inhibition would lead to the accumulation of the two inhibitory metabolite
Metabolite
Metabolites are the intermediates and products of metabolism. The term metabolite is usually restricted to small molecules. A primary metabolite is directly involved in normal growth, development, and reproduction. Alcohol is an example of a primary metabolite produced in large-scale by industrial...
s, adenosine and cGMP, which may act in synergy
Synergism
In theology, synergism is the position of those who hold that salvation involves some form of cooperation between divine grace and human freedom...
to mediate diverse pharmacological responses including anti-viral, anti-tumour and anti-arrhythmic effects.
Although EHNA potently inhibits adenosine deaminase
Adenosine deaminase
Adenosine deaminase is an enzyme involved in purine metabolism. It is needed for the breakdown of adenosine from food and for the turnover of nucleic acids in tissues.-Reactions:...
, 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 and has shown to be effective to reverse hypoxic pulmonary vasoconstricion in perfused lung models.
EHNA has been therefore been used for two purposes:
- to serve as a lead structure for the rational design of more selective and potent PDE2 inhibitors, and
- to define some of PDE’s biological targets.
However, the use of EHNA as a chemical tool in determing the pharmacological role of PDE2 is limited due to low PDE2 potency and high potency in inhibition of adenosine deaminase.
BAY 60-7550, Oxindole and PDP
BAY 60-7550 (figure 4) is an analogAnalog (chemistry)
In chemistry, a structural analog , also known as chemical analog or simply analog, is a compound having a structure similar to that of another one, but differing from it in respect of a certain component. It can differ in one or more atoms, functional groups, or substructures, which are replaced...
of EHNA
EHNA
EHNA is a phosphodiesterase inhibitor that selectively inhibits phosphodiesterase type 2 ....
, which is more than 100-fold more potent and is highly selective for PDE2A. Other newly discovered selective PDE2 inhibitors are PDP (9-(6-Phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one, figure 5) and Oxindole (figure 3).
| Inhibitor | IC50 | Reference |
| EHNA | 1 µM | |
| Oxindole | 40 nM | |
| Bay-60 7550 | 4,7 nM | |
| PDP | 0,6 nM | |
Table 1 shows the potency
Potency
Potency may refer to:* Potency , a measure of the activity of a drug in a biological system* Virility* Potency is a measure of the differentiation potential of stem cells...
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.
Structure and bonding of the inhibitors
Comparison of these inhibitors with the natural substrateSubstrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
s of the enzyme, cAMP and cGMP (see figure 1 through 5) 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.
Structural similarity of the inhibitors
The structures of Bay 60-7550 and PDP are very similar (see figure 3 and 4). The difference between these molecules is the exocyclic methyl group on Bay 60-7550, which replaces the nitrogen atom in PDP decreasing the possibility to form hydrogen bondHydrogen bond
A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine, that comes from another molecule or chemical group. The hydrogen must be covalently bonded to another electronegative atom to create the bond...
s 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 the purine
Purine
A purine is a heterocyclic aromatic organic compound, consisting of a pyrimidine ring fused to an imidazole ring. Purines, including substituted purines and their tautomers, are the most widely distributed kind of nitrogen-containing heterocycle in nature....
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.
Possible structure-activity relationship for PDE2 inhibitors
There is a lack of co crystal structures of inhibitors bound in the active siteActive site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
of PDE2. However, a computerized docking model of the inhibitor EHNA and the substrates cAMP and cGMP bound in the catalytic site have been made
.
The docking model
Protein-ligand docking
Protein–ligand docking is a molecular modelling technique. The goal of protein-ligand docking is to predict the position and orientation of a ligand when it is bound to a protein receptor or enzyme...
of EHNA showed that the mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
s of the amino acids Asp811 to Ala (Asp811Ala) and Ile826 to Val (Ile826Val) at the active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
, 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.
Upper binding pocket: Gln859 and Asp811
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.The lower binding pocket: Ile 826
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.
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.