WO2002083883A2

WO2002083883A2 - Human phosphodiesterase 9a isoform

Info

Publication number: WO2002083883A2
Application number: PCT/EP2002/003898
Authority: WO
Inventors: Armin Hatzelmann; Gianluca Quintini; Wolfram Steinhilber
Original assignee: Altana Pharma Ag
Priority date: 2001-04-11
Filing date: 2002-04-09
Publication date: 2002-10-24
Also published as: WO2002083883A3; AU2002312801A1

Abstract

The invention provides human phosphodiesterase PDE9A5 and polynucleotides which encode PDE9A5.

Description

Novel human phosphodiesterase 9A isoform

The present invention provides a novel human cyclic GMP phosphodiesterase (PDE9A) isoform. It also provides nucleotide sequences encoding the same. In particular, the present invention provides three novel nucleic acid sequences all three encoding the same novel human cyclic GMP phosphodiesterase (PDE9A) isoform.

The present invention further relates to the use of the novel nucleic acid and amino acid sequences to evaluate and/or to screen for agents that can modulate phosphodiesterase activity or expression.

Background Art

Cyclic nucleotide phosphodiesterases (PDEs) are a family of enzymes that hydrolyze cyclic nucleotides (cAMP and cGMP). Cyclic nucleotides are intracellular second messengers and PDEs regulate their concentration in response to different stimuli (Beavo, Physiol. Rev., 75: 725-748, 1995).

In recent years 11 different families and many subtypes of these enzymes (isozymes) have been defined according to their distinctive catalytic and regulatory properties and according to sequence homo- logy (Fawcett et al., Proc. Natl. Acad. Sci. USA, 97:3702-3707, 2000).

Non-specific PDE inhibitors such as theophylline have been used in the treatment of asthma. Also more selective inhibitors of different PDEs have been identified and some have undergone clinical evaluation. It has been suggested that selective inhibitors of PDEs will lead to more effective therapy with fewer side effects.

Thus for some application it is desirable to have a selective inhibition of an individual type of PDE (Tenor and Schudt, Anti-Inflammatory Drugs in Asthma, edited by A.P.Simpson and M.K.Church, 1999 Birkhauser Verlag Basel). Hence the cloning and expression of novel PDEs would greatly aid the discovery of selective inhibitors.

Guipponi et al. reported in Hum Genet (1998) 103: 386-392 the identification and characterization of a novel cyclic nucleotide phosphodiesterase - PDE9A - and four different human splice variants thereof. The cDNA's of the different splice variants [PDE9A1 (GenBank accession no. AF067223), PDE9A2 (GenBank accession no. AF067224), PDE9A3 (GenBank accession no. AF067225) and PDE9A4 (GenBank accession no. AF067226)] showed open reading frames (ORFs) of 1779, 1599, 1398 and 1395 nucleotides, potentially encoding protein isoforms of 593, 533, 466 and 465 amino acids, respectively.

The PDE9 isozyme family specifically hydrolizes cGMP, not cAMP (Sonderling SH, Bayuga SJ, Beavo JA: Identification and characterization of a novel family of cyclic nucleotide phosphodiesterases; J Biol Chem 273: 15553-15558, 1998; Fisher DA, Smith JF, Pillar JS, St. Denis SH, Cheng JB: Isolation and characterization of PDE9A, a novel human cGMP-specific phosphodiesterase, J Biol Chem 273: 15559-15564, 1998). Further, compared with other cGMP-specific PDE's, PDE9 apparently lacks a noncatalytic cGMP-binding domain, which is present in PDE5, PDE6, and also in PDE2 (Sonnenburg WK, Beavo JA,: Cyclic GMP and regulation of cyclic nucleotide hydrolysis, Adv Pharmacol 26: 87-114, 1994; Beavo JA: Cyclic nucleotide phosphodiesterases: Functional implications of multiple isoforms, Physiol Rev 75: 725-748, 1995; Manganiello VC, Murata T, Taira M, Belfrage P, Degerman E: Diversity in cyclic nucleotide phosphodiesterase isozyme families, Arch Biochem Biophys 322: 1-13, 1995; Thomas MK, Francis SH, CorbinJD: Substrate and kinase directed regulation of phosphorylation of a cGMP-binding phosphodiesterase by cGMP, J Biol Chem 265: 14971-14978, 1990). As for PDE7 and PDE8, the activity of PDE9 is insensitive to the nonselective inhibitor IBMX (Sonderling SH, Bayuga SJ, Beavo JA: Identification and characterization of a novel family of cyclic nucleotide phosphodiesterases; J Biol Chem 273: 15553-15558, 1998; Fisher DA, Smith JF, Pillar JS, St. Denis SH, Cheng JB: Isolation and characterization of PDE9A, a novel human cGMP-specific phosphodiesterase, J Biol Chem 273: 15559-15564, 1998) and also resistant to selective inhibitors of PDE1 , PDE2, PDE3, PDE4 and PDE5 (Sonderling SH, Bayuga SJ, Beavo JA: Identification and characterization of a novel family of cyclic nucleotide phosphodiesterases; J Biol Chem 273: 15553-15558, 1998; Wilson M, Sullivan M, Brown N, Houslay MD: Purification, characterization and analysis of rolipram inhibition of a human type 4A cyclic AMP-specific phosphodiesterase expressed in yeast; Biochem J 304: 407-415, 1994). The only compounds identified to date that inhibit the activity of PDE9 (Sonderling SH, Bayuga SJ, Beavo JA: Identification and characterization of a novel family of cyclic nucleotide phosphodiesterases; J Biol Chem 273: 15553-15558, 1998; Fisher DA, Smith JF, Pillar JS, St. Denis SH, Cheng JB: Isolation and characterization of PDE9A, a novel human cGMP-specific phosphodiesterase, J Biol Chem 273: 15559-15564, 1998) are zaprinast (IC₅₀ - 30μM), an inhibitor that was previously considered to be selective for PDE5, and even more potent, on a molar basis, SCH51866 (Sonderling SH, Bayuga SJ, Beavo JA: Identification and characterization of a novel family of cyclic nucleotide phosphodiesterases; J Biol Chem 273: 15553-15558, 1998), a compound that inhibits both PDE5 and PDE1. The mRNA encoding PDE9 is expressed in many examined human tissues, including spleen, small intestine and brain (Fisher DA, Smith JF, Pillar JS, St. Denis SH, Cheng JB: Isolation and characterization of PDE9A, a novel human cGMP-specific phosphodiesterase, J Biol Chem 273: 15559-15564, 1998).

We now have identified two further cDNAs of human origin, having 1326 respectively 1318 base pairs, both encoding for an identical PDE9A isoform with 376 amino acids.

According to the existing nomenclature we named the new human cyclic GMP phoshodiesterase isoform PDE9A5.

Aspects of the present invention According to the first aspect of the present invention, there is provided an isolated and purified amino acid sequence having the sequence presented as SEQ ID No. 1 , a derivative thereof or an altered sequence thereof.

According to the second aspect of the present invention, there are provided nucleotide sequences encoding the amino acid sequence according to the first aspect of the invention.

According to the third aspect of the present invention, there is provided an isolated and purified nucleotide sequence having the sequence presented as SEQ ID No. 2, a derivative thereof or an altered sequence thereof.

According to the fourth aspect of the present invention, there is provided an isolated and purified nucleotide sequence having the sequence presented as SEQ ID No. 3, a derivative thereof or an altered sequence thereof.

According to the fifth aspect of the present invention, there is provided an isolated and purified nucleotide sequence having the sequence presented as SEQ ID No. 4, a derivative thereof or an altered sequence thereof.

According to the sixth aspect of the present invention, there is provided an isolated and purified poly- nucleotide sequence which is fully complementary to the nucleotide sequence encoding the amino acid sequence according to the first aspect of the present invention, as well as an isolated and purified nucleotide sequence which is fully complementary to the nucleotide sequence of SEQ ID No: 2, 3 or 4.

According to the seventh aspect of the present invention, there is provided an expression vector comprising the nucleotide sequence according to the second, third, fourth or fifth aspect of the present invention.

According to the eigth aspect of the present invention, there is provided a host cell which has been transformed with a vector according to the seventh aspect of the present invention and which expresses the amino acid sequence according to the first aspect of the invention.

According to the ninth aspect of the present invention, there is provided a method for producing an amino acid sequence according to the first aspect of the present invention, the method comprising the steps of: a) transforming a host cell with a nucleotide sequence encoding the amino acid sequence presented as SEQ ID No. 1 , a derivative thereof or an altered sequence thereof, preferably with a nucleotide sequence selected from SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4, b) cultering the transformed host cell under conditions suitable for the expression of said protein, and c) recovering said protein from the host cell culture.

According to the tenth aspect of the present invention, there is provided an assay method for identifying an agent that can affect activity or expression of the amino acid sequence according to the first aspect of the present invention, the assay method comprising contacting an agent with the amino acid sequence according to the first aspect of the present invention or a nucleotide sequence according to the second, third, fourth or fifth aspect of the present invention, and measuring the activity or expression of the amino acid sequence, wherein a difference between a) amino acid sequence activity or expression in the absence of the agent and b) amino acid sequence activity or expression in the presence of the agent is indicative that the agent can affect amino acid sequence activity or expression.

According to the eleventh aspect of the present invention, there is provided a method of affecting in vivo the activity or expression of the amino acid sequence according to the first aspect of the present invention with an agent; wherein the agent is capable of affecting activity or expression of the amino acid sequence according to the first aspect of the present invention in an in vitro assay; wherein the in vitro assay method is the assay method of the tenth aspect of the present invention.

According to the twelfth aspect of the present invention, there is provided the use of an agent in the preparation of a pharmaceutical composition in the treatment of a disease or condition associated with the amino acid sequence according to the first aspect of the invention, the agent is capable of having an effect on the activity or expression of the amino acid sequence according to the first aspect of the invention, when assayed in vitro by the assay method of the tenth aspect of the present invention.

Brief Description of the Figures

Fig. 1 : Figure 1 shows the amino acid sequence of PDE9A5 (SEQ ID No. 1 ).

Fig. 2: Figure 2 shows the nucleotide sequence of SEQ ID No. 2 and the amino acid sequence of

PDE9A5.

Fig. 3: Figure 3 shows the nucleotide sequence of SEQ ID No. 3 and the amino acid sequence of

PDE9A5.

Fig. 4: Figure 4 shows the nucleotide sequence of SEQ ID No. 4 and the amino acid sequence of

PDE9A5.

Fig. 5: Figure 5 shows the nucleotide sequences of SEQ ID Nos. 5, 6, 7 and 8.

Fig. 6: In Figure 6 the cDNA structure (5'-region) of the nucleotides encoding PDE9A5 are graphically compared with the first 9 exons of previously published nucleotide sequences encoding PDE9A iso- forms.

Fig. 7: In Figure 7 the amino acid sequence of PDE9A5 is graphically compared with the amino acid sequences of other previously known PDE9A isoforms. Description of the invention

The invention is based on the discovery of a new isoform of human cyclic-GMP specific phosphodiesterase (PDE9A), the nucleotide sequences encoding this new isoform and the use of these compositions for the identification of agents that can affect the expression or activity of this new human phosphodiesterase isoform.

PCR-amplification using whole brain cDNA (Clontech Laboratories Inc., Palo Alto; California) as template using S'-ATGGACGCATTCAGAAGCACTCCG-S' (SEQ ID No. 5) and δ'-CCTCAGGCACAGTCTCCTTCACTGTT-S' (SEQ ID No. 6) as primers for amplification resulted in a product of 1318 base pairs. PCR with primers 5'-ATGGGATCCGGCTCCTCCAGCTACCG-3' (SEQ. ID No. 7) and 5'-CCTCAGGCACAGTCTCCTTCACTGTT-3' (SEQ ID No. 6) resulted in a product of 1326 base pairs.

The PCR products were directly cloned into plasmid pCR2.1-TOPO (Invitrogen; Carlsbad; California) and subsequently sequenced. The sequences were compared with published phosphodiesterase genes accessible in the public gene bank from NCBI via BLAST (Basic Local Alignment Search Tool).

Nucleotide sequence SEQ ID No. 2 and SEQ ID No. 3 of this invention are previously unidentified phosphodiesterase-cDNAs derived via alternative splicing from the gene encoding PDE9A.

The cDNA structure (5'-region) of the nucleotides encoding PDE9A5 are graphically compared in Fig. 6 with the first 9 exons of previously published nucleotide sequences encoding PDE9A isoforms.

The amino acid sequence of PDE9A5 is graphically compared with the amino acid sequences of other previously known PDE9A isoforms in Fig. 7.

The deletion of exon 8 in SEQ ID No. 2 and SEQ ID No. 3 is new and was not described before. The deletion of exon 4 in SEQ ID No. 2 is also an unique part of this invention. This deletion affects only the mRNA nucleotide sequence, but not the amino acid sequence of the new PDE9A isoform (PDE9A5) described herein.

As a consequence of the deletion of exon 8, the two cDNAs encode for a 376 amino acid long peptide whose first methionine is new to the invention and not present in the amino acid sequences of previously published PDE9As.

Amplification via PCR of SEQ ID No. 3 [or alternative SEQ ID No. 2, or the whole brain cDNA (Clontech Laboratories Inc., Palo Alto; California)] using as primers the oligonucleotides having the sequence 5'-ATGACCAACTGCCCCTGTAAGTAC-3' (SEQ ID No. 8) and 5'-CCTCAGGCACAGTCTCCTTCACTGTT-3' (SEQ ID No. 6) resulted in a further nucleotide sequence (SEQ ID No. 4) encoding for PDE9A5. The ATG codon in SEQ. ID No. 8 results from the deletion of exon 8 and is therefore part of this invention.

Definitions

The term "amino acid sequence" as used herein refers to a naturally occuring or synthetic peptide or protein.

The term "derivative", as used herein, refers to the chemical modification of the nucleic acid sequences encoding PDE9A5 or the encoded PDE9A5. In connection with nucleic acid sequences such modifications include, for example, replacement of hydrogen by an alkyl, acyl or amino group. A nucleic acid sequence derivative encodes an amino acid sequence which retains the biological or immunological function of the natural amino acid sequence. A derivative of an amino acid sequence is one which is modified by PEG or by glycosylation, or any similar process which retains the biological or immunological function of the amino acid sequence from which it is derived.

The term "altered sequence", as used herein, refers to a nucleotide sequence with deletions, insertions, or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functional equivalent PDE9A5. It also refers to deletions, insertions or substitution in the amino acid sequence resulting in a functionally equivalent PDE9A5.

The term "an agent that can affect activity or expression of the amino acid sequence" means that the said agent can affect (1 ) the expression of the encoding nucleotide sequence, (2) its translation into the amino acid sequence or (3) the activity of the encoded amino acid sequence.

The term "agent", as used herein, refers to a molecule that affects the activity or expression of PDE9A5. For example, it includes agonists and antagonists of the amino acid sequence shown as SEQ ID No. 1 , as well as nucleotide sequences that are antisense to all or part of the sequences shown as SEQ ID No. 2, 3 or 4.

The term "complementary" refers to the natural binding of nucleotide sequences under permissive salt and temperature conditions by base pairing. For example, the sequence 5'-AGT-3' binds to the complementary sequence 5'-ACT-3'.

As used herein, the terms "purified" and "isolated" refer to molecules, either nucleotide or amino acid sequences, that are removed from their natural environment and isolated or separated from at least one other component with which they are naturally associated. Nucleotide sequences of the present invention

Nucleotide sequences according to the present invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.

In general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques to accomplish this are readily available in the art. Longer nucleotide sequences will generally be produced using recombinant techniques, e. g. polymerase chain reaction (PCR). This method will involve the usage of(1 ) at-least one pair of primers (e.g. of about 15-30 nucleotides) binding to a region of the nucleotide sequence which is desired to clone and (2) mRNA or cDNA obtained from eukaryontic or prokaryontic cells as template for nucleotide amplification, performing PCR under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.

The nucleotide sequences of the present invention include nucleotide sequences encoding the amino acid sequences of the present invention. It will be appreciated that a range of different nucleotide sequences encode a given amino acid sequence as a consequence of the degeneracy of the genetic code.

A nucleotide sequence ^"(SEQ^" ID No. 4) encoding the amino acid sequence of the present invention may be produced recombinantly for example by using in a PCR reaction [starting from SEQ ID No. 2 or 3, the whole brain cDNA from Clontech or any other tissue which contains nucleotide sequences encoding for human PDE9A] S'-ATGACCAACTGCCCCTGTAAGTAC-S' (SEQ ID No. 8) as a sense primer and 5'-CCTCAGGCACAGTCTCCTTCACTGTT-3' (SEQ ID No. 6) as an antisense primer.

Vectors/Host Cells

In order to express a biologically active PDE9A5, the nucleotide sequences encoding PDE9A5, preferably the sequences ID No. 2, 3 or 4, may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.

Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding PDE9A5 and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook and Russell (2001 ) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y..

A variety of expression vector/host systems may be utilized to contain and express sequences encoding PDE9A5. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. The invention is not limited by. the host.cell employed..

The "control elements" or "regulatory sequences" are those non-translated regions of the vector - enhancers, promoters, 5'- and 3'- untranslated regions - which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phage- mid (Stratagene, LaJolla, Calif.) or pSPORTI plasmid (Gibco BRL; Rockville Maryland) and the like may be used. The baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO; and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters -from mammalian genes or from mammalian viruses are preferable.

Amino acid sequences of the present invention

The production of the amino acid sequence of the present invention can be affected by the culturing of suitable expression hosts, for example insect cells, which have been transformed with one or more nucleotide sequences of the present invention, in a conventional nutrient fermentation medium. The selection of the appropiate medium may be based on the choice of expression hosts and/ or based on the regulatory requirements of the expression construct. Such media are well known to those skilled in the art.

Thus, the present invention also provides a method for producing a peptide having the amino acid sequence presented as SEQ ID No. 1 , or a derivative thereof or an altered sequence thereof, the method comprising the steps of a) transforming a host cell with a nucleotide sequence encoding the amino acid sequence presented as SEQ ID No. 1 , or a derivative thereof or an altered sequence thereof, preferably with a nucleotide sequence selected from SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4, b) cultering the transformed host cell under conditions suitable for the expression of said peptide; and c) recovering said peptide from the host cell culture.

In addition, or in the alternative, the peptide itself could be produced using chemical methods to synthesize a PDE amino acid sequence, in whole or in part. For example, peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing.

Assays / Identification Methods

The ability of an agent to affect PDE9A5 activity may be, for example, determined by measuring cyclic nucleotide levels.

The present invention provides a method of identifying an agent which is capable of affecting the activity of the amino acid sequence presented as SEQ ID No. 1 , or a derivative thereof or an altered sequence thereof, the method comprising the steps of: a) contacting the agent with the amino acid sequence presented as SEQ ID No. 1 , or a derivative thereof or an altered sequence thereof, "b^") " incubating the mixture of step a) with a cyclic nucleotide under conditions^" suitable for the^" hydrolysis of the cyclic nucleotide, c) measuring the amount of cyclic nucleotide hydrolysis, and d) comparing the amount of cyclic nucleotide hydrolysis of step c) with the amount of cyclic nucleotide hydrolysis obtained with SEQ ID No. 1 , or a derivative thereof or an altered sequence thereof, not incubated with the agent, thereby determining whether the agent affects (such as stimulates or inhibits) cyclic nucleotide hydrolysis.

There are also commercially available immunoassay kits for the measurement of cAMP and cGMP (eg Amersham International, Arlington Heights, IL and DuPont Boston, MA).

The present invention also relates to a method of identifying an agent that is capable of affecting the expression of the amino acid sequence presented as SEQ ID No. 1 , or a derivative thereof, or an altered sequence thereof, the method comprising the steps of: a) measuring the expression of the amino acid sequence presented as SEQ ID No. 1 , or a derivative thereof, or an altered sequence thereof, in the presence of the agent or after the addition of the a- gent in a suitable cell line into which has been transformed a vector comprising one of the nucleo- tide sequences presented as SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4, or a derivative thereof, or an altered sequence thereof b) measuring the expression of the amino acid sequence presented as SEQ ID No. 1 or a derivative thereof, or an altered sequence thereof, in the same cell line without the presence of the agent c) comparing the amount of expression measured in step a) and b).

A variety of protocols for detecting and measuring the expression of PDE amino acid sequences, such as by using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).

The present invention also provides a method of- screening an agent for specific-binding affinity. with the amino acid sequence of SEQ ID No. 1 , or a derivative thereof, or an altered sequence thereof, or the nucleotide sequences of SEQ ID No. 2, 3 or 4, or a derivative thereof, or an altered sequence thereof, the method comprising the steps of : a) providing an (candidate) agent; b) combining the amino acid sequence of SEQ ID No. 1 , or a derivative thereof, or an altered sequence thereof, or the nucleotide sequences of SEQ ID No. 2, 3 or 4, or a derivative thereof, or an altered sequence thereof, with the agent for a time sufficient to allow binding under suitable conditions; and c) detecting binding of the agent to the amino acid sequence of SEQ ID No. 1 , or a derivative thereof, or an altered sequence thereof, or the nucleotide sequences of SEQ ID No. 2, 3 or 4, or a derivative thereof, or an altered sequence thereof.

Furthermore, the nucleotide sequences of the present invention coding for the amino acid sequence presented as SEQ ID No. 1 , or the nucleotide sequences that are complementary thereto may be used in assays to detect the presence of PDE9A coding sequences in human cells. These assays would provide information regarding the tissue distribution of this enzyme isoform and its biological relevance to particular disease states.

Therapeutics

Expressed PDE9A has been found in various tissues, with the most significant expression in colon, small intestine, brain, spleen, ovary and testis. PDE9A is also expressed in cancer and tissues associated with inflammation and the immune response. PDE9A appears therefore to play a role in cancer and immune disorders. PDE9A is furthermore expressed in Crohn's disease.

The exact biological role of PDE9A is not yet known and it will be therefore of interest to have specific inhibitors of PDE9A to investigate which cGMP-driven processes are regulated by this enzyme. Methods and Materials

a) Cloning of the nucleotide sequences encoding for PDE9A5 via PCR

Human whole brain cDNA was purchased from Clontech (Human Brain QUICK-Clone™ cDNA, constructed from whole brains obtained from two Caucasian males; 43- and a 47- year old). 1 μl was used for PCR reaction. PCR was carried out in a Stratagene Robocycler 40 with the Expand Long Template PCR System from Roche in Buffer 3 plus 0.75 mM MgCI₂, 0.3 μM each primer, 500 μM dNTPs. Amplification conditions were (1 ) 1 minute denaturation at 94°C, (2).35 cycles of-denaturation. at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 68°C for 3 minutes and (3) a final extension step at 68°C for 10 minutes.

Amplification using primers 5'-ATGGACGCATTCAGAAGCACTCCG-3' (SEQ ID No. 5) and 5'-CCTCAGGCACAGTCTCCTTCACTGTT-3' (SEQ ID No. 6) resulted in a product of 1318 base pairs, PCR with primers 5'-ATGGGATCCGGCTCCTCCAGCTACCG-3' (SEQ ID No. 7) and 5'-CCTCAGGCACAGTCTCCTTCACTGTT-3' (SEQ ID No. 6) resulted in a product of 1326 base pairs.

The PCR products were directly cloned into plasmid pCR2.1-TOPO (Invitrogen; Carlsbad; California) and sequenced. The sequences were compared with published phosphodiesterases and genes accessible in the public gene bank from NCBI via BLAST.

b) Expression in baculovirus

A variety of expression vector/host systems, as described above, may be utilized to contain and express sequences encoding the new PDE9A isoform.

For example an insect system was used. A 1.1 kb fragment (= SEQ ID No. 4) of SEQ ID No. 3 encoding the full length protein (SEQ ID No. 1 ) was PCR-amplified with the 5' primer having the SEQ ID No. 8 matching 100% to SEQ ID No. 2 and No. 3 of this invention and the 3' antisense primer 5'-CCTCAGGCACAGTCTCCTTCACTGTT-3' (SEQ ID No. 6), direct cloned into plasmid pCR2.1- TOPO (Invitrogen; Carlsbad; California) and transferred with EcoRI in the baculovirus transfer vector pBacPakθ (Clontech Laboratories Inc., Palo Alto; California). The expression vector was cotransfected in SFVL cells with Baculo-Gold DNA (Pharmingen; San Diego California) using CellFECTIN (Life Technologies; Basel, Switzerland). Virus was amplified and titer determined by plaque assay according to Pharmigen's instruction manual. To obtain recombinant protein, Sf-21 cells were infected at a multiplicity of infection >1. Transfected cells were typically harvested after 48 hours and were suspended in ice-cold (4°C) homogenization buffer (20 mM Tris, pH 8.2, containing 140 mM NaCI, 3.8 mM KCI, 1 mM EGTA, 1 mM MgCI₂, 1 mM β-mercaptoethanol, 2 mM benzamidine, 0.4 mM Pefabloc, 10 μM leu- peptin, 10 μM pepstatin A and 5 μM trypsin inhibitor) at a cell concentration of about 10⁷ cells/ml and broken by sonication. Afterwards, the homogenate was centrifuged for 10 min at 1 ,000xg, and the supernatant was stored at -80°C until further use (see below).

Demonstration of PDE9A5 activity

For determination of K_m values, PDE activity was determined as described by Thompson et al. (Adv Cycl Nucl Res 10: 69-92, 1979) with some modifications (Bauer and Schwabe, Naunyn- Schmiedeberg^'s Arch Pharmacol 311 : 193-198, 1980). At a final assay volume of 200 μl (96well MTPs, microtiter plates) the assay mixture contained 60 mM Tris (pH 7.4), 5 mM MgCI₂, varying concentrations of cAMP (1-100-μM) or cGMP (0=01-10-μM)r[^?H]cAMP or [³H.cGMP]-(about-30,000-cpm/assay) and an aliquot of recombinant enzyme (1 ,000xg supernatant, see above).

After preincubation for 5 min at 37°C, the reaction was started by the addition of substrate (cAMP or cGMP) and the assays were incubated for further 30 min at 37°C. 50 μl of 0.2 N HCI was added to stop the reaction and the assays were left on ice for about 10 min. Following incubation with 25 μg 5'- nucleotidase (Crotalus atrox snake venom) for 10 min at 37°C, the assays were loaded on QAE Sephadex A-25 (1 ml bed volume). The columns were eluted with 2 ml of 30 mM ammonium formiate (pH 6.0) and the eluate was counted for radioactivity. Results were corrected for blank values (measured in the presence of denatured protein) which were below 5 % of total radioactivity. The amount of cyclic nucleotides hydrolyzed did not exceed 30 % of the original substrate concentration. K_m values were calculated by linear regression analysis plotting V/S (x-axis) versus V (y-axis), with S being the substrate concentration and V being the corresponding specific activity (nmolxmin^"1xmg^"1).

The inhibition of PDE9A5 activity by the compounds indicated was performed in a modified SPA test (scintillation proximity assay) from Amersham Pharmacia Biotech (see protocol "Phosphodiesterase [³H]cAMP SPA enzyme assay, code TRKQ 7090") in 96well MTPs. The assay volume was 100 μl and contained 20 mM Tris buffer (pH 7.4), 0.1 mg/ml bovine serum albumine, 5 mM Mg²⁺, 0.5 μM cGMP, [³H]cGMP (about 50,000 cpm), 1 μl of the compound in DMSO as well as recombinant enzyme (1 ,000xg supernatant, see above) to guarantee a cGMP hydrolysis of about 10-20 %. After preincubation for 5 min at 37°C the reaction was started by addition of substrate (cGMP), incubated for further 15 min and then stopped by the addition of SPA beads (50 μl). SPA beads had been resuspended in water before, according to the manufacturers instructions, then diluted 1 :3 (v/v) and IBMX (3 mM) was added. After sedimentation of the beads (> 30 min) the MTPs were counted, and corresponding IC₅₀ values of the compounds for inhibition of PDE9A5 activity were calculated from concentration- inhibition-curves by non-linear regression analysis.

PDE9A5 has - just as the other isoforms of PDE9A - a very high affinity for cGMP with a Km of 110 nM and a very low affinity for cAMP (Km = 238 μM). The effects of various known PDE inhibitors on the activity of PDE9A5 are shown in Table 1 : Table 1 :

The data confirms previous findings for other isoforms of PDE9A, showing that of the tested compounds only SCH51866 (PDE5 and PDE1 inhibitor) and Zaprinast (PDE5 inhibitor) inhibit the activity of the PDE9A5 enzyme.

Claims

Patent Claims

1. An isolated and purified amino acid sequence having the sequence presented as SEQ ID No. 1 , a derivative thereof or an altered sequence thereof.

2. An isolated and purified amino acid sequence having the sequence presented as SEQ ID No. 1.

3. An isolated and purified nucleotide sequence encoding an amino acid sequence as defined in claim 1.

4. An isolated and purified nucleotide sequence having the sequence presented as SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4, a derivative thereof or an altered sequence thereof.

5. An isolated and purified nucleotide sequence having the sequence presented as SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4.

6. An isolated and purified nucleotide sequence which is fully complementary to one of the nucleotide sequences according to any one of claims 3 or 4.

7. A vector comprising a nucleotide sequence according to any one of claims 3 or 4.

8. A host cell transformed with a vector according to claim 7 expressing the amino acid sequence according to claim 1.

9. An assay method for identifying an agent that can affect activity or expression of the amino acid sequence according to claim 1 , the assay method comprising contacting an agent with the amino acid sequence according to claim 1 , or a nucleotide sequence according to claim 3 or 4, and measuring the activity or expression of the amino acid sequence, wherein a difference between a) amino acid sequence acvtivity or expression in the absence of the agent and b) a- mino acid sequence activity or expression in the presence of the agent is indicative that the a- gent can affect amino acid sequence activity or expression.

10. Use of an agent in the preparation of a pharmaceutical composition in the treatment of a disease or condition associated with the amino acid sequence according to claim 1 , the agent is capable of having an effect on the activity or expression of the amino acid sequence according to claim 1 , when assayed in vitro by the assay method of claim 9.