PDE3

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phosphodiesterase 3A, cGMP-inhibited
Identifiers
Symbol	PDE3A
HUGO	8778
Entrez	5139
OMIM	123805
RefSeq	NM_000921
UniProt	Q14432
Other data
Locus	Chr. 12 p12

phosphodiesterase 3B, cGMP-inhibited
Identifiers
Symbol	PDE3B
HUGO	8779
Entrez	5140
OMIM	602047
RefSeq	NM_000922
UniProt	Q13370
Other data
Locus	Chr. 11 p15.2

PDE3 is a phosphodiesterase. The PDE's belong to at least eleven related gene families which are different in their primary structure, substrate affinity, responses to effectors and regulation mechanism. Most of the PDE families are comprised of more than one gene. PDE3 is clinically significant because of its role in regulating heart muscle, vascular smooth muscle, and platelet aggregation, and PDE3 inhibitors have been developed as pharmaceuticals; but their use is limited by arrhythmic effects and they can increase mortality in some applications.

Contents 1 Structure 2 Substrate affinity 3 PDE3 isoforms 4 Localization of PDE3 5 The gene profile 6 Regulation 7 Function of PDE3 8 SAR (structure-activity relationships) 8.1 First generation PDE inhibitors 8.2 Second generation PDE inhibitors 8.3 Examples of a selective PDE3 inhibitors: 9 References

[edit] Structure

The mammalian PDE's share a common structural organization and contain three functional domains which include the conserved catalytic core, a regulatory N-terminus and the C-terminus. The conserved catalytic core is much more similar within PDE families, with about 80% amino acid identity, than between different families. It is believed that the core contains common structural elements that are important for the hydrolysis of cAMP and cGMP phosphodiester bonds. It is also believed that it contains family specific determinants for differences in affinity for substrates and sensitivity for inhibitors. ^[1]
The catalytic domain of PDE3 is characterized by a 44 amino acid insert, but this insert is unique to the PDE3 family and is a factor when determining a structure for a potent and selective PDE3 inhibitor. ^[1]
The crystal structure of the catalytic domains of several PDE's including PDE3B, have shown that they contain three helical subdomains :

1. N-terminal cyclin fold region
2. Linker region
3. C-terminal helical bundle ^[2][3]

At the interface of these domains a deep hydrophobic pocket is formed by residues that are highly conserved among all PDE's. This pocket is the active site and is composed of four subsites :

1. Metal binding site (M site)
2. Core pocket (Q pocket)
3. Hydrophobic pocket (H pocket)
4. Lid region (L region) ^[2][3]

The M site is at the bottom of the hydrophobic binding pocket and contains two divalent metal binding sites. The metal ions that can bind to these sites are either zinc or magnesium. The zinc binding site has two histidine and two aspartic acid residues that are absoulutely conserved among those PDE's studied to date. ^[2][3]
The N-terminal portions of PDE's are widely divergent and contain determinants which are associated with regulatory properties that are specific to different gene families. For PDE3 those determinants are the hydrophobic membrane associaton domains and cAMP-dependent protein kinase phosphorylation sites. ^[1]

[edit] Substrate affinity

At first the PDE3's were purified and described as enzymes that hydrolyse both cGMP and cAMP with K_m values between 0.1 – 0.8 µM. However the V_max for cAMP hydrolysis is 4 - 10 times higher than V_max for cGMP hydrolysis. ^[1]
When different PDE's were first identified, two types of PDE's (PDE3 and PDE4) that exhibited high affinities for cAMP were isolated. PDE3 exhibited high affinity for both cGMP and cAMP but PDE4 only had high affinity for cAMP. For that reason the PDE3 was called the “cGMP inhibited PDE” to distinguish it from PDE4. ^[1]
The 44 amino acid insertion in the catalytic domain of PDE3's is believed to be involved in PDE3's interaction with its substrate and inhibitors but that remains to be estabilished. ^[1]
The proposed molecular mechanism of cyclic nucleotide specificity of PDE's is the so called glutamine switch mechanism.
In the PDE's which have had their structure solved there seems to be an invariant glutamine residue that stabilizes the binding of the purine ring in the active site (binding pocket). The g-amino group of the glutamine residue can alternatively adopt two different orientations:

1. The hydrogen bond network supports guanine binding – cGMP selectivity
2. The hydrogen bond network supports adenine binding – cAMP selectivity

In PDE's that can hydrolyse both cGMP and cAMP (PDE3's) the glutamine can rotate freely and therefore switch between orientations. ^[2][3]

[edit] PDE3 isoforms

The PDE3 family in mammals consists of two members, PDE3A and PDE3B. The PDE3 isoforms are structurally similar, containing an N-terminal domain important for the localization and a C-terminus end. ^[4] The 44 amino acid insertion in the catalytic domain differs in the PDE3 isoforms and the N-terminal portions of the isoforms are quite divergent. PDE3A and PDE3B have strikingly similar pharmacological and kinetic properties but the distinction is in expression profiles and affinity for cGMP. ^[2]

[edit] Localization of PDE3

PDE3A is mainly implicated in cardiovascular function and fertility but PDE3B is mainly implicated in lipolysis. ^[2] Table 1 is an overview of localization of the PDE3 isoforms.

	PDE3A	PDE3B
Localization in tissues	- Heart * - Vascular smooth muscle* - Platelets - Oocyte - Kidney	- Vascular smooth muscle - Adipocytes - Hepatocytes - Kidney - β cells - Developing sperm - T-lymphocytes - Macrophages
Intracellular localization	- Membrane-associated or cytosolic	- Membrane-associated (predominantly)
Table 1: Overview of PDE3 isoform localization. *Variants of PDE3A have differential expression in cardiovascular tissues [3]

In general PDE3 can be either cytosolic or membrane bound and has been associated to plasma membrane, sarcoplasmic reticulum, golgi and nucleus envelope. ^[4]
PDE3B is predominantly membrane-associated and is localized to endoplasmic reticulum and microsomal fractions. ^[3]
PDE3A can be either membrane-associated or cytosolic depending on the variant and the cell type it is expressed in. ^[3]

[edit] The gene profile

The PDE3 family is composed of two genes, PDE3A and PDE3B. In cells expressing both genes PDE3A is usually dominant. Three different variants of PDE3A (PDE3A1-3) are products of alternate startcodon usage of the PDE3A gene. The PDE3B encodes a single isoform only. ^[3][5]

In their full-length both PDE3A and PDE3B contain two N-terminal hydrophobic membrane association regions, NHR1 and NHR2 (figure 2). The difference of the PDE3A1-3 variants lies in whether they include:

• both NHR1 and NHR2
• only NHR2
• neither NHR1 nor the NHR2

The last can be predicted to be exclusively on soluble/cytosolic form. ^[5][6]

[edit] Regulation

PDE3A and PDE3B activity is regulated by several phosphorylation pathways. Protein kinase A and Protein kinase B both activate PDE3A and PDE3B via phosphorylation at two different phosphorylation sites (P1 and P2) between NHR1 and NHR2 (figure 2). Hydrolysis of cAMP by PDE3 isoforms is also directly inhibited by cGMP, although PDE3B is only ≈10% as sensitive to cGMP inhibition as PDE3A. ^[5]
The PDE3B has been extensively studied for its importance in mediating the antilipolytic and antiglycogenlytic effect of insulin in adipose and liver tissues. The activation of PDE3B in adipocytes is associated with phosphorylation of serine residue by an insulin-stimulated protein serine kinase (PDE3IK). By blocking insulin activation of PDE3IK and in turn phosphorylation/activation of PDE3B the antilipolytic effect of insulin can be antagonized. Activation of PDE3B decreases concentrations of cAMP which in turn reduces Protein kinase A activity. Protein kinase A is responsible for activation of lipase which induces lipolysis as well as other physilogical pathways. ^[1][5]
Whether phosphorylation pathways, which regulate activity of PDE3A or PDE3B, could serve as a potential drug targets rather than the catalytic domain of the PDE3 enzyme itself is unclear and beyond the scope of this text.

[edit] Function of PDE3

PDE3 enzymes are involved in regulation of cardiac and vascular smooth muscle contractility. Molecules that inhibit PDE3 were originally investigated for the treatment of heart failure but because of unwanted arrhythmic side effects they are not studied for that indication any longer. Nonetheless, the PDE3 inhibitor milrinone is approved for use in heart failure in intravenous form. ^[3]
Both PDE3A and PDE3B are expressed in vascular smooth muscle cells and are likely to modulate contraction. Their expression in vascular smooth muscle is altered under specific conditions such as elevated cAMP and hypoxia. ^[3]

Inhibitors of the PDE3 enzyme:

• antagonize platelet aggregation
• block oocyte maturation
• increase contractility of the heart
• enhance vascular smooth muscle relaxation
• enhance airway smooth muscle relaxation

It has been demonstrated that PDE3A inhibition prevents oocyte maturation in vitro and in vivo. ^[3] For example when mice are made completely deficient of PDE3A they become infertile. ^[4]
Aggregation of platelets is highly regulated by cyclic nucleotides. PDE3A is a regulator of this process and PDE3 inhibitors effectively prevent aggregation of platelets. Cilastazol is approved for treatment of intermittent claudication and is thought to involve inhibition of platelet aggregation and also inhibition of smooth muscle proliferation and vasodilation.
The most studied roles of PDE3B have been in the areas of insulin, IGF1 and leptin signaling. ^[3] When PDE3B is overexpressed in β-cells in mice, it causes impaired insulin secretion and glucose intolerance. ^[4]
The involvement of PDE3B in regulation of these important pathways has inspired researchers to begin studying the possible roles of this enzyme in disorders such as obesity, diabetes and cellulite. ^[7]

[edit] SAR (structure-activity relationships)

From early studies an initial model of PDE active site topography was derived. This early model can be summarized into the following steps concerning cAMP active site topography:

1. cAMP substrate with its adenine and ribose moieties in an “anti” relationship
2. The phosphate atom in cAMP binds to PDE active site, using an arginine residue and a water molecule, which was initially associated with Mg²⁺. A second arginine residue and the Mg²⁺ may also play roles during binding and/or play roles in the next step
3. S_N2 attack of phosphorus by H₂O with formation of a trigonal bipyramid transition state
4. 5´-AMP is formed as an “inverted” product. Electronic charges conserve the net charge overall and across the transition state ^[8]
   

[edit] First generation PDE inhibitors

Recognition that the knowledge about PDE could be used to develop drugs that were PDE inhibitors led to extensive research. Most studies used analogues of the nucleotide substrates or derivatives of natural product inhibitors such as xanthine (e.g. theophylline) and papaverine. ^[8][9]

The cAMP PDE active site can be considered as a summary of ideas about receptor topography resulting from the first generation inhibitors. The model of the Wells et al. version as cited in Erhardt and Chou (1991) includes the following:

1. A phosphate binding area
2. A lipophilic area that accommodates the non-polar side of the ribose moiety
3. A pyrimidine binding site
4. An imidazole binding site portion of the pyrimidine binding site
5. A sterically hindered site
6. An area with bulk tolerance ^[8]

[edit] Second generation PDE inhibitors

Since selective PDE3 inhibitors were recognised to be cardiotonic drugs there has been great interest in developing new drugs in this category. A large number of heterocyclic compounds have been synthesized during related research. These compounds constitute a second generation of PDE inhibitors. Although they have been directed mostly at PDE3, they present significant SAR for the PDE's in general. ^[8]
A “heterocycle-phenyl-imidazole” (H-P-I) pattern (figure 3) has been considered to be necessary for positive inotropic activity in cardiac muscle and many second generation inhibitors fit this pattern. ^[8]

The heterocycle region: Within each heterocycle there is the presence of a dipole and an adjacent acid proton (an amide function). These atoms are believed to mimic the electrophilic center in the phosphate group in cAMP and are confirmed as the primary site of binding. The heterocycle is a transition state analogue inhibitor of PDE. Alkyl groups, limited to either methyl or ethyl, on the heterocyclic ring usually enhance potency, with occasional exceptions. ^[8][9]

The phenyl region: It seems that an electron rich, centre such as phenyl, needs to be present. The beneficial effects of small alkyl groups on the heterocycle could be to twist the central ring away from exact coplanarity with the heterocyclic ring. There is a similar twist in cAMP and there is general agreement that high affinity PDE3 inhibitors should adopt an energetically favoured planar confomation that mimics the anti conformation of cAMP. ^[8][9]

The imidazole region: Various substituents have been placed at the para-position of the central phenyl ring. They are electron rich moieties and apparently a positively charged moiety cannot be tolerated in this region of the PDE receptor. There is general agreement about this inhibitor potency: Lactam ≥ Alkyl-CONH- ≥ Imidazoyl = Pyridine in place of the central phenyl with its nitrogen in the analogous 4 position ≥ Alkyl-S- > Simple [ether|ethers] > Halide = Amines > Imidazolium (which is totally inactive). ^[8]

Identification of features common to the most selective inhibitors has led to a “five-point model” with:

1. Presence of a strong dipole (carbonyl moiety) at one end of the molecule.
2. An adjacent acid proton.
3. A small sized alkyl substituent on the heterocyclic ring.
4. A relatively flat overall topography.
5. An electron rich centre and/or a hydrogen bond acceptor site opposite to the dipole. ^[9]

[edit] Examples of a selective PDE3 inhibitors:

Theophylline is a non-selective agent. In contrast, meribendan is a very selective inhibitor (figure 4). ^[9]

Also, meribendan, has a higher level of selectivity in comparison with the parent compound CI-930 because, beside the basic nitrogen adjacent to the lactam moiety it possesses another basic nitrogen (benzimidazole ring), opposite to the primary binding site (figure 5). ^[9]

Many PDE3 inhibitor drugs have been developed, e.g. cilostazol, milrinone, vesnarionone, enoximone and pimobendan. PDE3 inhibitors have been studied for their effectiveness as inotropic agents in severe heart failure but unfortunately have shown increased mortality in well controlled studies. These drugs are therefore only used if the benefit exceeds the risk. ^[10]
To date no isoform selective inhibitors have been discovered but research in that field is of great interest. ^[6]

[edit] References

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