Phosphodiesterase 9: insights from protein structure and role in therapeutics.

LFS-13994; No of Pages 11 Life Sciences xxx (2014) xxx–xxx

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Phosphodiesterase 9: Insights from protein structure and role in therapeutics Nivedita Singh, Sanjukta Patra ⁎ Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India

a r t i c l e

i n f o

Article history: Received 28 October 2013 Accepted 5 April 2014 Available online xxxx Keywords: Phosphodiesterase PDE9A cGMP BAY 73-9961 PF-04447943 Brain

a b s t r a c t This review focuses on the development of drugs targeting phosphodiesterase 9A (PDE9A). PDE9A normally regulates cGMP (cyclic guanosine monophosphate) levels, which in turn regulate signal transduction. However, in pathological conditions, PDE9A inhibition is required to treat diseases that lower the level of cGMP. Hence, there is a need for specific PDE9A inhibitors. Aligning the 3D structure of PDE9A with other phosphodiesterases reveals residues crucial to inhibitor selectivity. GLU406 is unique to PDE9A and stabilizes the side chain of an invariant glutamine (GLN453). TYR424 is another relevant residue, unique only to PDE9A and PDE8A. Therefore, TYR424 could discriminate between PDE9A and all other PDEs except PDE8A. TYR424 should also be considered in the design of selective inhibitors because PDE8A has low expression levels in the brain. Hence, GLU406 and TYR424 are important target residues in the design of PDE9A-selective inhibitors. © 2014 Published by Elsevier Inc.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . Overview of cyclic nucleotide phosphodiesterase enzyme . cGMP-specific phosphodiesterase 9 . . . . . . . . . . . Catalytic mechanism of PDE9A . . . . . . . . . . . . . Distribution and localization of PDE9A in mammals . . . Cloning and characterization of PDE9A . . . . . . . . . Architecture of pde9a gene . . . . . . . . . . . . . . . Importance of PDE9A structure in drug discovery . . Therapeutic disease targets for phosphodiesterase 9A . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

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Introduction Cellular communication is important for coordinating multitudinous activities between various cells and their extracellular environment. In mammalian cells, signal transduction is carried out by various signaling molecules. Among these molecules, the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) act as mediators in transmitting a wide variety of external signals through membrane-bound receptors, which in turn regulate many ⁎ Corresponding author at: Department of Biotechnology. Tel: +91 361 2582213; fax: +91 361 2582249. E-mail address: [email protected] (S. Patra).

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intracellular metabolic processes (Kim and Park, 2003; Wang et al., 2010). Fig. 1 illustrates signal transduction through the second messengers cAMP and cGMP along with their associated metabolic activities. cGMP is involved in a myriad of cellular functions, including neurotransmission, smooth muscle relaxation, platelet aggregation inhibition, blunting cardiac hypertrophy, protection against ischemia/reperfusion injury of the heart, and improvement in cognitive function (Francis et al., 2011). The cGMP signaling pathway mainly involves three enzymes: guanylyl cyclases (GC), phosphodiesterases (PDEs) and protein kinase G (PKG). cGMP synthesis is initiated by the activation of soluble membrane-bound guanylyl cyclase. Three major targets of cGMP are cGMP-dependent protein kinase (PKG), cGMP-dependent phosphodiesterases, and cGMP-gated ion channels (CNG) (Lucas et al.,

http://dx.doi.org/10.1016/j.lfs.2014.04.007 0024-3205/© 2014 Published by Elsevier Inc.

Please cite this article as: Singh N, Patra S, Phosphodiesterase 9: Insights from protein structure and role in therapeutics, Life Sci (2014), http:// dx.doi.org/10.1016/j.lfs.2014.04.007

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N. Singh, S. Patra / Life Sciences xxx (2014) xxx–xxx

Fig. 1. Pathway of signal transduction in the cell.

2000; Friebe and Koesling, 2003; Mullershausen et al., 2004; de Vente, 2004). Among these targets, cGMP-specific phosphodiesterases play important roles in regulating cellular signals by controlling the intracellular cGMP level. However, in various pathophysiological conditions, cGMP-specific phosphodiesterases act as a potential biomarker. These conditions include male erectile dysfunction, neurodegenerative disease (including Alzheimer's disease, schizophrenia, Parkinson's disease (PD), Creutzfeldt–Jakob disease (CJD), diabetes, obesity, chronic obstructive pulmonary disease (COPD), and certain cardiovascular diseases (Oeckl et al., 2012)). Thus, cGMP-dependent phosphodiesterase inhibition has become the routine choice in drug development for such diseases. Its inhibition leads to persistent signaling by increasing the level of cGMP (Reneerkens et al., 2009). Several inhibitors are available against various PDEs. Sildenafil (synthesized by a group of pharmaceutical chemists at Pfizer's research facility in Sandwich, Kent, England), is a phosphodiesterase 5 (PDE5) inhibitor widely used to treat male erectile dysfunction (Goldstein et al., 1998). However, the search for specific inhibitors against a particular PDE member still remains a challenge due to the scarcity of available knowledge on this subject (Wang et al., 2007; Liu et al., 2008; Hou et al., 2011). Therefore, very few drugs that specifically inhibit this particular member of the phosphodiesterase superfamily have successfully reached the market. A plethora of reviews have been published on the phosphodiesterase superfamily (Card et al., 2004; Zhang et al., 2004; Lugnier, 2006; Bender and Beavo, 2006; Halpin, 2008; Francis et al., 2011; Keravis and Lugnier, 2012). However, very few reviews exist on one particular PDE family. This review emphasizes the details of PDE9, including its structure, mechanism of action, inhibition pattern and drug development. Furthermore, this review summarizes research progress on PDE9 with the intent to develop PDE9-specific inhibitors.

Overview of cyclic nucleotide phosphodiesterase enzyme Phosphodiesterases are the most prominent family of enzymes that degrade cAMP and cGMP by hydrolysis to 5′-AMP and 5′-GMP, respectively (Braumann et al., 1986; Trong et al., 1990). Figs. 1 and 2

depict phosphodiester bond cleavage by phosphodiesterase enzymes, resulting in cell signal disruption. PDEs can be divided into three classes: class I, class II and class III. Mammalian PDEs belong to class III (Omori and Kotera, 2007). The human genome contains genes that encode 21 PDEs from 11 different families (1–11). This classification is based on amino acid sequence, a conserved C-terminal catalytic domain of ~270–300 amino acids, and a regulatory domain present between the N-terminal splicing region and the C-terminal catalytic domain. Each family contains several isoforms generated by using various transcriptional start sites and alternate mRNA splicing (Bender and Beavo, 2006; Lugnier, 2006; Ke and Wang, 2007). The regulatory domain is the most diverse region of the phosphodiesterase enzyme structure and is therefore the basis for classification within the superfamilies (Fig. 3). PDE members also differ in terms of substrate affinity, specificity, and subcellular localization. These differences can be exploited in the development of specific inhibitors (Russell et al., 1973). Table 1 provides complete details of the PDE families, including the various subtypes, Km values for cGMP/cAMP, site of localization, available inhibitors, and the cellular activities influenced by the inhibition of PDE. On the basis of substrate specificity, phosphodiesterases are divided into three groups: (1) cAMP specific-PDE 4, 7 and 8; (2) cGMP specificPDE 5, 6 and 9; and (3) both cAMP and cGMP specific-PDE 1, 2, 3, 10 and 11 (Mehats et al., 2002; Conti and Beavo, 2007). While these specificities are determined by the highly conserved catalytic domain, the specific mechanism of substrate recognition remains a question (Ke et al., 2011; Hou et al., 2011). According to Zhang et al. (2004), a “glutamine switch mechanism” may be important for selectivity. This is because the γ-amino group of a conserved or invariant glutamine in the PDE active site can adopt two different orientations. In one orientation, the hydrogen bond network supports guanine binding, resulting in cGMP selectivity. In the second orientation, the network supports adenine binding, resulting in cAMP selectivity. In contrast, in the dualspecificity PDEs, the side chain of glutamine can switch between the two orientations, resulting in specificity towards both the cyclic nucleotides (Zhang et al., 2004; Jeon et al., 2005). Binding patterns of the invariant glutamine of different phosphodiesterases are shown in Fig. 4.



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Fig. 2. Phosphodiesterase hydrolysis reaction.

cGMP-specific phosphodiesterase 9 Phosphodiesterase 9A (PDE9A) is cGMP-specific and has the highest cGMP affinity of any PDE. To date, only one gene of PDE9 has been reported; hence, it is commonly known as “PDE9A”. It has 20 known variants based on N-terminal alternative splicing. In PDE9A, the conserved catalytic domain contains three subdomains: an N-terminal cyclin-fold region, a linker region, and a C-terminal helical bundle. A deep hydrophobic pocket exists at the interface of the three subdomains. This pocket is composed of four subsites: a metal-binding site (M site), a core pocket (Q pocket), a hydrophobic pocket (H pocket) and the lid region (L region) (Jeon et al., 2005). Zinc (Zn2+) and magnesium (Mg2+) ions are present at the metal binding site and participate in interactions with inhibitors and with a histidine-rich region in the active site. The hydrophobic pocket is mainly composed of TRP416, LEU420, LEU421, PHE251 and VAL417. The Q pocket of PDE9A contains the conserved catalytic residue GLN453, which is involved in substrate/ inhibitor selectivity by hydrogen bonding. Fig. 5 illustrates the configuration of amino acid residues in the PDE9A active site, which contains the ligand IBMX (1-methyl 3-isobutyl xanthine).

inhibitor), these metal ions form coordination bonds with protein side chains and water molecules. M1 forms four coordination bonds with the PDE9A active site residues ASP293, ASP402, HIS292 and HIS256 and two with the water molecules W1 and W0. W1 has partial occupancy in the active site. M2 forms one coordination bond each with ASP293 and the five water molecules W0, W2, W3, W4 and W5. cGMP binds in the catalytic center by displacing W1 and W2 and then interacts with M1 and M2, which form coordination bonds with the axial and equatorial phosphate groups of cGMP, respectively. This process results in the formation of an enzyme-substrate (ES) complex. W0 acts as a nucleophile in the hydrolytic cleavage of cGMP, leading to the formation of 5′-GMP. Thus, this is the rate-limiting phenomenon in which metal ions act as Lewis acids in catalyzing hydrolysis. Alternatively, HIS252 acts as general acid. This study reveals that the specificity of PDE9 towards cGMP could be due to hydrogen bond formation between the guanine base and the side chain of GLN453, whose orientation is fixed by the interaction between the Nε atom of GLN453 and the Oε atom of GLU406. Here, GLU406 assists in polarizing the amide side chain of GLN453, which is a feature unique to PDE9 (Liu et al., 2008). Fig. 6 presents the catalytic mechanism of PDE9, which breaks cyclic phosphodiester bonds in the presence of metal ions and cGMP.

Catalytic mechanism of PDE9A Distribution and localization of PDE9A in mammals Liu et al. (2008) captured the enzymatic reaction of PDE9A2mediated hydrolysis by using a crystallographic freeze-trapping method (Huai et al., 2004; Liu et al., 2008). The hydrolytic center of PDE9A contains two metal cations, M1 and M2, and six water molecules, W0, W1, W2, W3, W4 and W5. In the absence of ligand (substrate or

In mammals, PDE9A is expressed in all tissues except blood (Guipponi et al., 1998; Rentero et al., 2003). The highest expression levels of PDE9A are in the brain, spleen, small intestine and kidneys (Fisher et al., 1998; Rentero et al., 2003). In the brain, PDE9A is the

Fig. 3. Structure-based classification of PDEs.


4

PDE family

Subtypes Properties

Km Values

Tissue expression

PDE1

3

Ca++/calmodulin stimulated

PDE1A = 112.7⁄5.0 μM [cAMP⁄cGMP] PDE1B = 24.3⁄2.7 μM [cAMP⁄cGMP] PDE1C = 1.7⁄1.3 μM [cAMP⁄cGMP] PDE2 = 10/30 μM [cAMP⁄cGMP]

PDE2

1

cGMP-stimulated

PDE3

2

cGMP-inhibited, cAMP-selective

PDE4

4

PDE5

Inhibitors

Cellular function associated with PDE inhibition

References

Brain, heart, lung, smooth Neurodegenerative diseases muscle such as Neuronal ischemia, epilepsy, Parkinson's & Alzheimer's disease

Calimidazolium, Dioclein, Phenethiazines, SCH51866, Vinpocetine, Zaprinast

Apoptosis, phosphorylation of AMPA receptors, enhancement of pulmonary vasodilation

Jiang et al. (1996); Bender et al. (2004); Evgenov et al. (2006); Dunkern and Hatzelmann (2007); Goncalves et al. (2009); Medina (2011).

Adrenal gland, heart, lung, liver, platelets

Acute respiratory Distress Syndrome (ARDS), sepsis

EHNA, Bay 60-7550, MPB– forskolin, PDP

Endothelial cell permeability, presynaptic inhibition

PDE3 = 0.2/0.1 μM [cAMP⁄cGMP]

Adipose tissue, heart, lung, liver, inflammatory cells, platelets

2–4 μM for cAMP

Sertoli cells, kidney, brain, liver, lung, inflammatory cells

Amrinone, Bucladesine, Cilostazol, Cilostamide Enoximone, Milrinone, Org 9935, Olprinone, SK&F 95654, Siguazodan, Saterinone Apremilast, NCS 613, Rolipram, Roflumilast

Induction of insulin secretion

cGMP-insensitive, cAMP-specific

1

cGMP-specific, PKA/PKG phosphorylated

2.9 ± 0.8 μM for cGMP (full length PDE5A) 5.1 μM for cGMP (catalytic domain PDE5A)

Cardiomyocytes lung, platelets, vascular, smooth muscle,

Asthma, chronic heart disease, cardiovascular disease, intermittent caudation, vascular smooth muscle relaxation Autoimmune disease, inflammatory disease, neurodegenerative disease, cancer, allergic disorder. Coronary heart disease, cardiovascular disease, pulmonary hypertension, renal failure, sexual dysfunction, stroke

Michie et al. (1996); Boess et al. (2004); Surapisitchat et al. (2007); Wunder et al. (2009); Witzenrath et al. (2009); Hu et al. (2012) Kieback and Baumann (1999); Ahmad et al. (2000); Surapisitchat et al. (2007)

PDE6

3

2.5 μM for cGMP

PDE7

2

Transducin-activated, cGMP-specific cAMP-specific, Rolipram-insensitive

Photoreceptors, pineal gland Lung, hematopoietic cells, placenta, pancreas, brain, heart, thyroid, and skeletal muscle, immune cells

PDE8

2

cAMP-specific, Rolipram-insensitive IBMX-insensitive

PDE9

1

cGMP-specific, IBMX-insensitive

Testes, eye, liver, skeletal muscle, heart, kidney, ovary, brain, T lymphocytes 0.070–0.25 μM for cGMP Brain, kidney, spleen, 230 μM for cAMP small intestine.

PDE 10

1

Dual specific

0.05–0.26 μM for cAMP 3–7.2 μM for cGMP

PDE 11

1

Dual specific

0.52 μM for cAMP 1.04 μM for cGMP

0.03–0.2 μM for cAMP

0.04–0.15 μM for cAMP

Conditions treated by inhibition of PDE

Long-term potentiation

Mackenzie and Houslay (2000); Schett et al. (2010); Rabe (2011); Yougbare et al. (2011)

Dipyridamole, SK&F 96231, Tadalafil, Vardenafil, Sildenafil, Zaprinast

Antihypertrophic effects, inflammatory immune response, apoptosis etc

Sometimes for adverse effect on vision Immune and inflammatory disorders, neurological disease such as spinal cord injury

Dipyridamole, Sildenafil, Vardenafil, Zaprinast ASB16165, BAY 73-6691, PF04447943, S14, VP1.15

N/A

Das et al. (2005); Wang et al. (2006); Kass et al. (2007); Zoraghi et al. (2007); Tedford et al. (2008); Rao and Xi (2009); Westermann et al. (2012) Lugnier (2006); Cahill et al. (2012) Bender and Beavo (2006); Castaño et al. (2009); Paterniti et al. (2011)

Polycystic ovary syndrome (PCOS)

Dipyridamole

Cardiovascular diseases, BAY73-6691, PF-04447943 Insulin-resistance syndrome and diabetes, obesity, neurodegenerative disorders Testes, striatum (brain) Anxiety, cognition MP10 deficiency disorder, neurodegenerative disease Skeletal muscle, prostate, Asthma, adrenal, testicular, Tadalafil testis and salivary glands. bipolar disorder, depression and prostatic cancers

Lessen inflammatory response, regulation of pro-inflammatory and immune T-cell functions, reduction in tissue injury, reduction in TNF-α, IL-6, COX-2 and iNOS expression Suppression of Teff cell functions, lipid accumulation

Synaptic plasticity, LTP (longterm potentiation)

Lugnier (2006); Vang et al. (2010); Shimizu-Albergine et al. (2008)

Long-term potentiation

Fisher et al. (1998); Soderling et al. (1998); Wang et al. (2003); Huai et al. (2004); Wang et al. (2010) Abdel-Magid (2013)

Spermatogenesis

Makhlouf et al. (2006)



Table 1 Overview of cyclic nucleotide phosphodiesterase superfamily.


5

Fig. 4. Comparative binding patterns of invariant glutamine with AMP or GMP, or both: (A) interaction of AMP with cAMP-specific PDE4D by forming two H-bonds between adenine moiety of AMP and invariant Q369. (B) Interaction of GMP with cGMP-specific PDE5A by two H-bonds between guanine base of GMP and invariant Q817, this interaction is in the opposite orientation of glutamine side chain as shown in AMP-PDE4D interaction. (C) Interaction pattern of dual-specificity PDE10A with AMP. (D) Interaction pattern of dual-specificity PDE10A with GMP.

most highly expressed PDE (Andreeva et al., 2001) and almost all cell signaling pathways pass through cGMP. Regulation of the synthesis and degradation of cGMP fluctuates in different regions of the brain depending on physiological and pathological states. cGMP signaling is important for numerous functions in the brain, such as synaptic plasticity, phototransduction, learning, memory and stem cell differentiation.

Differentiation of stem cells to neurons is promoted by a high level of cGMP, whereas a low level of cGMP promotes differentiation to non-neuronal cells (i.e., glial cells) (Erceg et al., 2005; Kleppisch, 2009; Gomez-Pinedo et al., 2011). PDE9A is highly expressed in the basal forebrain, cerebellum and olfactory bulb (Andreeva et al., 2001). Hence, inhibiting PDE9A could be an approach to treat various psychiatric and

Fig. 5. Surface view of IBMX-bound PDE9A active site.


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Fig. 6. Structural representation of PDE9A hydrolytic center. (A) Active site with two metal ions M1 and M2 (can be Mn2+–Mn2+ or Mn2+–Mg2+ or Mg2+–Mg2+ or Mg2+–Zn2+, etc..) in absence of ligand. (B) Substrate cGMP interacting with PDE9A hydrolytic center leads to the formation of an ES complex. (C) Transition from ES to EP complex after the breaking at 3′-5′ cyclic bond, but 5′-GMP is still interacting with the PDE9A hydrolytic center (D) partial interaction of 5′-GMP in PDE9 active center as E + P complex.

neurodegenerative diseases with associated lowered cGMP (Kleiman et al., 2012). Though a few inhibitors targeting PDE9A have been developed, all of them to date lack specificity (Verhoest et al., 2009; Wunder et al., 2009; Wang et al., 2010; Claffey et al., 2012; Meng et al., 2012).

variant was introduced. Hence, more than 20 variants of PDE9A have currently been reported (Rentero and Puigdomènech, 2006).

Architecture of pde9a gene Cloning and characterization of PDE9A In 1998, Fisher et al. first recognized an EST (expressed sequence tag) clone of PDE9A from the EST database (Incyte Pharmaceuticals Inc., Palo Alto, CA) by BLAST (Basic Local Alignment Search Tool). The BLAST search was based on the sequence homology with the catalytic domain of PDE4B. However, the query clone was not identical to any known PDE family member clones. Subsequently, they isolated the identified clone from a prostate cDNA library and extended its 5′ end with nested PCR from a testes cDNA library. Finally, the cDNA of PDE9A encoding a full-length protein of 593 amino acid residues was confirmed by sequencing (Fisher et al., 1998). PDE9A was reported to have the highest of all PDE affinities for cGMP (Soderling et al., 1998). In the same year, four splice variants (PDE9A1, PDE9A2, PDE9A3 and PDE9A4) were reported by Guipponi et al. (1998). These variants were also confirmed as new sub-families of PDE9A (Guipponi et al., 1998). The full length mouse clone of PDE9A1 has also been characterized. After a few years, another variant, PDE9A5, was reported by Wang et al. (2003). In terms of the length of amino acid residues, it was found that PDE9A5 was smaller than PDE9A1 and PDE9A2 but longer than PDE9A3 and PDE9A4. PDE9A5 is similar in enzymatic properties to PDE9A1 but has different tissue distribution and subcellular localization. PDE9A1 is exclusively nuclear because of the presence of a pat7 motif, a nuclear localization signal, while other PDEs are cytosolic (Wang et al., 2003). In the same year, another group separately reported 16 other alternative splicing variants, including PDE9A5 from the same PDE9A mRNA transcript (Rentero et al., 2003). In 2006, another PDE9A

Phosphodiesterase 9A is encoded by the pde9A gene. This gene is 122 kb in length, consists of 22 exons, and is located on chromosome 21q22.3 between the TFF1 and D21S360 genes in the human genome. It is located on chromosome 17 in the mouse genome (Guipponi et al., 1998; Rentero et al., 2003). On the basis of PCR amplification and EST sequence analysis, 20 different PDE9A mRNA transcripts have currently been identified. These variants are produced as a result of alternative splicing at the 5′mRNA region. However, the C-terminal region of the mRNA transcript consists of 12 exons that code for the conserved catalytic domain. The first 10 exons participate in multiple splicing. The occurrence of these splice variants can be tissue dependent. The majority of the variants are found in almost all tissues, but some PDE9 mRNAs are restricted to a few tissues (i.e., PDE9A11 in peripheral blood leukocytes, PDE9A12 in prostate, PDE9A14 in ovary, and PDE9A15 in thymus). The most abundant variant, PDE9A1, is present in the prostate, colon, rectum, fetal brain, fetal kidney, and the intestine. It is also moderately expressed in the cerebellum and forebrain (Rentero et al., 2003). According to Rentero and Puigdomènech (2006), translation of the pde9A gene is performed from more than one start codon (ATG) (Rentero and Puigdomènech, 2006). The first start codon is in exon 1, while the second is located 52 bases downstream. Two other start codons may be found in exons 7 and 8, as shown in Fig. 7. Some splice variants are involved in producing proteins targeted to the cell membrane and cellular vesicles, whereas the rest are targeted to the cytoplasm (Rentero and Puigdomènech, 2006). Fig. 7 illustrates the structural details of the pde9A gene, its splice variants, and start codon variations.



7

Fig. 7. Schematic diagram of human pde9A gene and its mRNA transcripts.

Importance of PDE9A structure in drug discovery All PDE9A transcripts have a conserved catalytic domain at the Cterminus. The C-terminus contains the active site, including a nucleotide recognition pocket with a hydrophobic clamp created by PHE456 and LEU420, along with the hydrolytic center (Zhang et al., 2004; Liu et al.,

2008). The crystal structure of IBMX in complex with the catalytic domain of PDE9A2 was reported by Huai et al. (2004) and is shown in Fig. 8 (Huai et al., 2004). It has been observed that the N-terminal regulatory domain does not alter catalytic activity. The catalytic domain contains 16 α-helices with 181–506 amino acid residues. The catalytic domain of PDE9A2 is similar to the catalytic domain of PDE4D2 but is


8


Fig. 8. Catalytic domain of phosphodiesterase 9A showing 16 α-helices.

vastly different from that of PDE5A1. The differences have been demonstrated by superimposing the catalytic domain of PDE9A2 over those of PDE5A1 and PDE4D2. A root mean square (RMS) deviation of 1.5 Å was observed between the Cα atoms of residues 207–495 in PDE9A2 and residues 115–411 in PDE4D2 showing structural similarity (Huai et al., 2004). The catalytic domain of PDE9A2 has also been shown to form a dimer similar to that formed by PDE4D2. The residues involved in dimerization are TYR315, ASN316, ASP317, ASN323, and ARG353. Dimerization involves the formation of hydrogen bonds between two subunits, which provides a major force for dimerization. Unlike PDE4D2, PDE9A does not form a tetramer. PDE9A is highly specific for cGMP, as shown by a comparative study of the Km and Vmax values of cGMP and cAMP. The Km and Vmax of PDE9A for cGMP are 139 ± 18 nM and 1.53 ± 0.24 μmol.min−1.mg−1, respectively (Huai et al., 2004). In contrast, for cAMP, the Km and Vmax values are 181 ± 17 μmol and 0.08 ± 0.015 μmol.min−1.mg−1, respectively (Huai et al., 2004). The catalytic domain of PDE9A is shown in Fig. 8. Table 2 shows various crystallographic details of PDE9A, such as resolution, length in amino acid residues, and the release date of the PDE9A submitted structure in the protein data bank. These data are useful in understanding the three-dimensional structure of the protein and further aid research. With the objective of exploring the nuances of the protein structure, we conducted comparative analyses of catalytic domains from various PDEs. We observed a unique feature of the PDE9A catalytic domain,

namely, glutamate residue (GLU406), which forms a 2.8 Å hydrogen bond between the side chain Nε of GLN453 and the Oε of Glu406. This interaction fixes the orientation of the invariant glutamine (GLN453), resulting in its selectivity towards the substrate by protonation of GLU406. This has been confirmed by mutation studies on crucial catalytic residues, GLN453GLU and GLU406ALA (Hou et al., 2011). Based on these mutational studies, Hou et al. (2011) suggested that substrate recognition is not determined by the orientation of the invariant glutamine side chain (Hou et al., 2011). This study questioned the previously proposed “glutamine switch mechanism”. TYR424 is the other unique residue present at the entrance of the active site pocket of both PDE9A and PDE8A, while phenylalanine is present in the corresponding position of other PDEs. TYR424 further increases the polarity of the active site and might be a useful residue for increasing inhibitor selectivity (Huai et al., 2004). Table 3 lists active site residues present in a 5 Å radius from the cGMP binding site in PDEs. These data were obtained by superimposing the PDE9A–cGMP complex over other PDE–cGMP complexes based on PyMol's sequence alignment method. From Table 3, we conclude that PHE251 is a crucial residue that is present only in PDE9A, which may confer substrate selectivity. As described above, GLU406 has a specific role in the substrate selectivity of PDE9A. Hence, by thorough analysis of all PDE active site pockets, we suggest targeting these residues in further study of the PDE9A active site network. Therapeutic disease targets for phosphodiesterase 9A Diminished cGMP signaling results in various disorders. These disorders might be cured by inhibiting cGMP-specific PDE9A, which might in turn lead to signal enhancement. The presence of the pde9A gene at mapping position 21q22.3 might be a reason for a number of human chromosome 21 mapped genetic diseases, such as Down syndrome and bipolar affective disorder. In addition to its mapping position, it also contributes to the regulation of cyclic nucleotide levels by biochemical and genetic mechanisms (Guipponi et al., 1998). Some diseases reported to be targeted by inhibiting PDE9A are hyperglycemia, dyslipidemia, type 1 and type 2 diabetes, insulin resistance syndrome, obesity and several neurodegenerative diseases such as Alzheimer's disease, schizophrenia, and age-based cognitive decline (Bell et al., 2004; Fryburg and Gibbs, 2005; Black et al., 2008; Vardigan et al., 2011). In the brain, the inhibition of PDE9A improves synaptic transmission and alleviates vulnerable synapses, reducing cognitive deficit in Alzheimer's disease (Verhoest et al., 2009). In 2005, the first potent and selective inhibitor of PDE9A, BAY 73-6691 (1-(2-chlorophenyl)-6-((2R)-3,3,3trifluoro-2-methylpropyl)-1,5-dihydro-4H-pyrazolo(3,4-d)pyrimidine-

Table 2 Entries of PDE9A in protein data bank [http://www.rcsb.org]. PDB ID Resolution (Å) R-value Length Structure description (residues)

Date of release References

2HD1 2YY2 3DYS 3DYQ 3DYN 3DYL 3DY8 3N3Z 4GH6 3QI4 3JSI 3JSW 3K3E 3K3H 3QI3 4E90 4G2J 4G2L

27-06-2006 30-10-2007 16-09-2008 16-09-2008 16-09-2008 16-09-2008 16-09-2008 13-04-2011 03-10-2012 27-04-2011 01-12-2009 01-12-2009 16-02-2010 16-02-2010 27-04-2011 27-02-2013 29-05-2013 29-05-2013

2.23 2.80 2.30 2.50 2.10 2.70 2.15 2.75 2.70 2.50 2.72 2.30 2.70 2.50 2.30 2.50 2.40 3.00

0.215 0.206 0.188 0.199 0.183 0.191 0.177 0.202 0.212 0.213 0.205 0.183 0.230 0.223 0.222 0.193 0.202 0.186

326 333 329 329 329 329 329 326 326 533 329 329 326 326 533 329 329 329

Crystal structure with IBMX Crystal structure with IBMX PDE9A-5′ GMP complex PDE9 inhibited by omitting divalent cation in complex with cGMP PDE9 in complex with cGMP (Zn inhibited) PDE9-substrate complex (ES complex) PDE9 in complex with product 5′-GMP (E + P complex) PDE9A (E406A) mutation in complex with IBMX PDE9A catalytic domain in complex with inhibitor 28 PDE9A (Q453E) in complex with IBMX Phosphodiesterase 9 in complex with inhibitor PDE9 in complex with selective inhibitor Crystal structure of PDE9A catalytic domain with (R)-BAY73-6691 Crystal structure of PDE9A catalytic domain with (S)-BAY73-6691 Crystal structure of PDE9A(Q453E) in complex with inhibitor BAY73-6691 Phosphodiesterase 9 in complex with inhibitors PDE9 in complex with selective compound PDE9 in complex with selective compound

Huai et al. (2004) Handa et al. (2007) Liu et al. (2008) Liu et al. (2008) Liu et al. (2008) Liu et al. (2008) Liu et al. (2008) Hou et al. (2011) Hou et al. (2011) Hou et al. (2011) Liu et al. (2008); Verhoest et al. (2009) Liu et al. (2008); Verhoest et al. (2009) Wang et al. (2010) Wang et al. (2010) Hou et al. (2011) Liu et al. (2008); Claffey et al. (2012) Claffey et al. (2012); Liu (2013) Claffey et al. (2012); Liu (2013)



9

Table 3 Comparative study of amino acid residues present in a 5 Å radius from cGMP in various PDEs shown by sequence alignment [http://www.pymol.org/; http://www.rcsb.org]. PDE type

PDB ID

Residues in contact with cGMP within 5 Å region in active site pocket

PDE9A

3DYL

PDE5A

1T9R 3DBA

H 252 H 613 –

H 256 H 617 –

H 292 H 653 –

D 293 D 654 –

H 296 H 657

PDE6C

F 251 Y 612 –

PDE4B

1XLZ

PDE7A

3G3N

PDE8A

3ECM

PDE1B

1TAZ

PDE2A

1Z1L

PDE3B

1SOJ

PDE10A

4DFF

Y 233 Y 211 Y 555 Y 222 Y 655 Y 736 Y 524

H 234 H 212 H 556 H 223 H 656 H 737 H 525

H 238 H 216 H 560 H 227 H 660 H 741 H 529

H 274 H 252 H 596 H 263 H 696 H 821 H 563

D 275 D 253 D 597 D 264 D 697 D 822 D 564

H 278 H 256 H 600 H 267 H 700 H 825 H 567

T 363 T 723 S 121 T 345 T 321 T 668 T 334 T 768 T 893 T 633

M 365 L 725 –

D 402 D 764 –

I 403 L 765 –

M 347 I 323 M 670 M 336 L 770 L 895 L 635

D 392 D 362 D 726 D 370 D 808 D 937 D 674

L 393 I 363 V 727 I 371 L 809 I 938 L 675

4-one), was synthesized (Wunder et al., 2005). Studies on BAY 73-6691 showed its ability to improve learning, memory, basal synaptic transmission, long-term potentiation, cytokine stimulation decrease by neutrophil adhesion, and induction of apoptosis (Wunder et al., 2005; Van der Staay et al., 2008; Miguel et al., 2011; Kroker et al., 2012; Saravani et al., 2012). BAY 73-9961 is a well-known potent PDE9A inhibitor that has been used to treat Alzheimer's disease and corpus cavernosum relaxation in mice (da Silva et al., 2013). According to Wang et al. (2010), BAY 73-9961 has two enantiomers, (R) and (S), due to the presence of a chiral center, and both forms vary in their pattern of interaction (Wang et al., 2010). There is no report confirming the completion of a clinical trial of BAY 73-9961. Another potent and selective inhibitor, PF-04447943 (6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H pyrazolo[3,4-d] pyrimidin-4-one), has been used to increase cGMP in cerebrospinal

N 405 A 767 E 133 N 395 N 365 N 729 H 373 D 811 G 940 S 677

E 406 I 768 T 134 P 396 P 366 P 730 P 374 Q 812 P 941 V 678

V 417 A 779 V 146 T 407 S 377 A 741 T 385 A 823 T 952 A 689

L 420 V 782 H 148 I 410 V 380 I 744 L 388 I 826 I 955 I 692

Y 424 F 786 F 152 F 414 F 384 Y 748 F 392 F 830 F 959 F 696

A 452 M 816 T 176 S 442 I 412 S 777 S 420 L 858 L 987 G 725

Q 453 Q 817 –

F 456 F 820 –

Q 443 Q 413 Q 778 Q 421 Q 859 Q 988 Q 726

F 446 F 416 F 781 F 424 F 862 F 991 F 729

fluid for the treatment of Alzheimer's disease and other cognitive diseases (Wunder et al., 2005; Hutson et al., 2011; Nicholas et al., 2011; Vardigan et al., 2011; Verhoest et al., 2012). To date, PF-04447943 has completed six clinical trials, with a few more ongoing (Nicholas et al., 2011; Schwam et al., 2011; clinicaltrial.gov). The development of new inhibitors is underway. Various inhibitors have been synthesized and tested on PDE9A, but most have shown only moderate selectivity. A main reason for the lack of potency and selectivity for PDE9A is coinhibition of PDE1, which is also abundant in the brain (Meng et al., 2012). Researchers are making progress in developing specific potent inhibitors with improved inhibition capacity (Deninno et al., 2009; Wang et al., 2010; Meng et al., 2012). Table 4 lists details of PDE9Aselective inhibitors reported to date. Researchers face problems drugging PDE9A because of its structural similarity to PDE1 and PDE8. PDE9A inhibitors should be developed with consideration of PDE1

Table 4 Details of reported selective inhibitors for PDE9A. Sl. no.

Inhibitors

IC50 for PDE9A

Resolution (Å)

R-factor

References

1

BAY73-6691 (1-(2-chlorophenyl)-6-(3,3,3-trifluoro-2methylpropyl)-1H-pyrazolo[3,4-d] pyrimidine-4(5H)-one)

22 nM (R-form) 88 nM (S-form)

2.7 (R-form) 2.5 (S-form)

0.23(R-form) 0.223(S-form)

Wunder et al. (2009); Wang et al. (2010)

2

PF-4181366 4H-Pyrazolo[3,4-d]pyrimidin-4-one, 1cyclopentyl-1,5-dihydro-6-[(3S,4S)-4-methyl-1-(6-quinoxalinylmethyl)-3-pyrrolidinyl]-

1.8 nM

–

–

Verhoest et al. (2009)

3

PF-04447943 (6-[(3S,4S)-4-Methyl-1(pyrimidin-2ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2Hpyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d] pyrimidin-4-one)

10 nM

2.72

0.205

Verhoest et al. (2012)

4

Inhibitor 28 (2-((1-(2-Chlorophenyl)-4-hydroxy-1Hpyrazolo[3,4-d]pyrimidin-6-yl)amino)-N-(4methoxyphenyl)propanamide)

21 nM

2.7

0.212

Meng et al. (2012)

5

1-Cyclopentyl-6-[(1r)-1-(3-phenoxyazetidin1-Yl)ethyl]-1,5-dihydro-4h-pyrazolo[3,4-D] pyrimidin-4-One

32 nM

2.40

0.202

Claffey et al. (2012)

Structure


10


through comparative computational and experimental studies (Meng et al., 2012). TYR424 in PDE9A (as shown in Table 3) can be targeted for inhibitor design because it is a distinctive amino acid residue present in PDE9 and PDE8, while other phosphodiesterases contain a corresponding phenylalanine. The polar hydroxyl group of tyrosine could form hydrophilic interactions with inhibitors, providing good selectivity over PDE1 (Wang et al., 2010; Meng et al., 2012). The most unique feature of PDE9A is the presence of GLU406, which is involved in fixing the orientation of the side chain of the invariant GLN453 by forming an intra-molecular hydrogen bond. This interaction enhances selectivity towards cGMP and potential inhibitors. Consequently, if GLU406 is targeted for inhibitor development, it will provide better specificity towards PDE9A. Conclusion The second messengers cAMP and cGMP are substrates for a large superfamily of phosphodiesterases that regulate cell signaling. PDE9A has the highest affinity of all PDEs for cGMP. Hence, it is an attractive target for inhibitors designed to maintain cGMP levels. In pathophysiological conditions, most cellular functions associated with cGMP signaling are hampered by the consistent lowering of cGMP levels by PDE9A. Hence, inhibition of PDE9A is needed for consistent cGMP-dependent cell signaling. This review has covered details of the structure, function, mode of action, tissue distribution, genetics, therapeutic role, and current status of drugs developed for PDE9A. Genetic studies have revealed more than 20 splice forms of the pde9A gene. Because PDE9A is the most highly expressed PDE in the brain, its inhibition may play a crucial role in treating various neurodegenerative diseases. Crystallography has revealed the enzyme's mechanism of action, but the substrate selection criteria are not yet clear. This could be one reason for the scarcity of successful PDE9A inhibitors. Another reason is the moderate specificity of inhibitors, which also inhibit PDE1, another PDE that is highly expressed in the brain. Aligning the 3D structures of all PDEs has divulged residues crucial to substrate/inhibitor selectivity. One unique feature of PDE9A compared to other PDEs is the presence of GLU406, which stabilizes the side chain of the invariant GLN453. Another residue unique to PDE9A and PDE8A is TYR424. TYR424 separates PDE9A from all other PDEs, except PDE8A. In targeting brain diseases, TYR424 should also be considered in drug design as PDE8A is much less expressed in the brain. Thus, selective inhibitor design may be achieved by targeting these residues. Conflict of interest statement “The authors declare that there is no conflict of interest.”

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Structure of bone morphogenetic protein 9 procomplex.

Ischemic stroke risk in a southeastern Chinese population: Insights from 5-lipoxygenase activating protein and phosphodiesterase 4D single-nucleotide polymorphisms.

Phosphodiesterase from Vipera lebetina venom - structure and characterization.

Phosphodiesterase inhibitors as therapeutics for traumatic brain injury.

From transcription to translation: new insights in the structure and function of Argonaute protein.

Potential for naturally derived therapeutics: the Caribbean as a model - insights from the conference on therapeutics and functional genomics.

Insights into bacteriophage T5 structure from analysis of its morphogenesis genes and protein components.

Transendothelial Transport and Its Role in Therapeutics.

Non-protein therapeutics.

Editorial: Insights into disease pathogenesis and novel therapeutics.

Structural conversion of the transformer protein RfaH: new insights derived from protein structure prediction and molecular dynamics simulations.

C: from cellular function to diseases and therapeutics.

(±)-Torreyunlignans A-D, rare 8-9' linked neolignan enantiomers as phosphodiesterase-9A inhibitors from Torreya yunnanensis.

New insights into the pathogenesis and therapeutics of kidney fibrosis.

Insights from the Structure of Mycobacterium tuberculosis Topoisomerase I with a Novel Protein Fold.

Insights into RNA structure and function from genome-wide studies.

Emerging Role of Phosphodiesterase 2A in Hypertension.

Escalating role of piezosurgery in dental therapeutics.

Dynamic protein ligand interactions--insights from MS.

Effects of Single α-to-β Residue Replacements on Structure and Stability in a Small Protein: Insights from Quasiracemic Crystallization.

Crystal structure of the C-terminal 2',5'-phosphodiesterase domain of group A rotavirus protein VP3.

The human ribosomal protein S6 gene: isolation, primary structure and location in chromosome 9.

Role of Phosphodiesterase 5 and Cyclic GMP in Hypertension.

deuterium exchange mass spectrometry.