WO2006092174A1

WO2006092174A1 - Method for identifying pde10 modulators

Info

Publication number: WO2006092174A1
Application number: PCT/EP2005/052271
Authority: WO
Inventors: Joachim Schultz; Jost Weber
Original assignee: Nycomed Gmbh
Priority date: 2005-02-28
Filing date: 2005-05-18
Publication date: 2006-09-08
Also published as: IL185086A0; EP1856253A1; JP2008531024A; CA2604349A1; AU2005328604A1; KR20070116015A; NO20074815L

Abstract

The invention relates to a novel polypeptide, containing the GAFA domain and GAFB domain of a human phosphodiesterase 10 (PDE10) and the catalytic domain of an adenylate cyclase and to the use of said polypeptide in a method for identifying PDE10 modulators.

Description

Method for identifying PDE10 modulators

Technical area

The present invention relates to a novel polypeptide comprising the GAF _A domain and GAF _B domain of a human phosphodiesterase 10 (PDE10) and the catalytic domain of adenylate cyclase and the use of this polypeptide in a method for the identification of PDE10 modulators.

State of the art

Phosphodiesterases (= PDEs) are eukaryotic proteins and known as modulators of the cydic nucleotides cAMP and cGMP. PDEs are divided into 3 classes (I, II and III), of which only class I with its 11 PDE families (called PDE1 to -11) occurs in mammals

GAF domains are ubiquitous in all kingdoms of life and have been compared by Aravind and Ponting (Aravind L. and Ponting CP: The GAF domain: an evolutionary link between diverse phototransducing proteins., 1997, TIBS, 22, 45 & 459) due to protein structure and sequence comparison Are defined. PDE2, PDE5 and PDE6 contain so-called cGMP-binding GAF domains, which play a role in the allosteric activation of PDEs.

Adenylate cyclases (= ACs) catalyze the conversion of ATP into cAMP in all areas of life (Cooper DM: Regulation and organization of adenylyl cyclases and cAMP, 2003, Biochem J., 375 (Pt 3), 517-29, Tang WJ Gilman AG: Construction of a soluble adenylyl cyclase activated by Gsα and forskolin, 1995, Science, 268, 1769-1772). Due to sequence comparisons and structural considerations, they are subdivided into 5 classes (I to V). Of molecular biological interest are the bacterial class III ACs from cyanobacteria, in particular from Nostoc sp. PCC 7120 which also includes CyaB1. The cyanobacterial ACs CyaB1 and CyaB2 also contain N-terminal GAF domains which are structurally similar to those of PDEs but have cAMP as the activating ligand. The 9 known families of human Class III ACs are all membrane bound and regulated by G proteins (Tang WJ and Gilman AG: Construction of a soluble adenylyl cyclase activated by Gsα and forskolin, 1995, Science, 268, 1769-1772). , A combination with GAF domains is unknown.

The construction of a chimera from the GAF domains of the rat PDE2 and the catalytic center of the adenylate cyclase CyaB1 has already been described (Kanadier T., Schultz A., Linder JU and Schultz JE: A GAF-domain-regulated adenylyl cyclase from Anabaena is a seif activated cAMP switch. 2002, EMBO J., 21, 3672-3680).

A chimer from human PDE10 and bacterial adenylate cyclase is not known. Likewise, the use of such a chimer in a method of identifying PDE10 modulators of the prior art is not known

Description of the invention

The object underlying the invention is to provide a method for identifying PDE10 modulators.

The object is achieved by providing the polypeptide of the invention comprising functionally (a) the GAF _A domain and GAF _B domain of a human phosphodiesterase 10 (PDE10) or their functionally equivalent variants and (b) the catalytic domain of an adenylate cyclase or its functionally equivalent Variants and their use in a method for identifying PDE10 modulators solved.

It has surprisingly been found that a chimeric protein of N-terminal human PDE10 GAF domains and C-terminal catalytic center of an adenylate cyclase is useful as effector molecule. In the chimeric protein, the GAF domains are the activation domains that change their conformation upon ligand binding, thereby modulating the catalytic activity of the adenylate cyclase domain, which serves as a read-out.

Furthermore, it was surprisingly found that cAMP as a PDE10 agonist selectively activates the GAF domain of PDE10, in particular with an ECs ₀ of about 50-100 μM.

These results were particularly surprising since, for example, the GAE domain of PDE10 shows only 28% identity to the GAF domain of CyaB1 and a functional, activating ligand of the GAF domain of PDE10 was unknown until now.

The present invention enables PDE10 modulators, i. To identify PDE10 antagonists or PDE10 agonists that do not act by binding and blocking the catalytic center of PDE10, but by allosteric regulation at the N-terminus of PDE10, i. at the GAF domains.

As mentioned above, the invention relates to a polypeptide comprising functionally linked (a) the GAF _A domain and GAF _B domain of a human phosphodiesterase 10 (PDE10) or their functionally equivalent variants and (b) the catalytic domain of adenylate cyclase or its functionally equivalent variants.

A human phosphodiesterase or PDE is understood to mean an enzyme of human origin which is capable of converting cAMP or cGMP into the corresponding inactive 5'-monophosphates. Due to their structure and properties, the PDEs are classified into different families. A human phosphodiesterase 10 or else PDE10 is understood in particular as meaning an enzyme family of human origin which is capable of converting cAMP or cGMP into the inactive 5'-monophosphates.

According to the invention as all PDE10 PDE10 come into question, a GAF _A domain and _B domain have GAF. The GAF domains of PDE10 are located in tandem as N-terminal protein. The GAF domain closest to the N-terminus is referred to as GAF _A , and the immediately following as GAF _B - beginning and ending of the GAF domains can be determined from protein sequence comparisons. A SMART sequence comparison (Schultz J., Milpetz F., Bork P. and Pointing CP: SMART a simple modular architecture research tool: 1998, PNAS, 95, 5857-5864) yields, for example, the isoform for GAF _A. PDE10A2: D91 to A244 and for GAF _B : A266 to R422.

By an adenylate cyclase is meant an enzyme capable of converting ATP into cAMP. Accordingly, adenylate cyclase activity is understood as meaning the amount of ATP reacted or amount of cAMP formed in a certain time by the polypeptide according to the invention.

A catalytic domain of adenylate cyclase is understood as meaning the part of the amino acid sequence of an adenylate cyclase which is necessary for the adenylate cyclase to still have the property of converting ATP into cAMP, ie it is still essentially functional and thus has adenylate cyclase activity.

By iteratively shortening the amino acid sequence and then measuring the adenylate cyclase activity, the catalytic domain of adenylate cyclase can be easily determined.

The determination of the adenylate cyclase activity can be carried out, for example, by measuring the conversion of radioactive [α- ^3Z P] -ATP into [α- ³² P] -cAMP.

In general, the adenylate cyclase activity is easily possible by measuring the resulting cAMP via antibody binding. There are various commercial assay kits such as the cAMP ^[3 H] or ^[125 -I] BioTrak® ^® cAMP SPA assay from Amersham ^® or AlphaScreen ^® and the Lance ^® cAMP assay PerkinElmer ^® are all based on the principle that matters in the AC reaction ATP unlabeled cAMP is formed. This competes with exogenously added 3H-, 1251-, or biotin-labeled cAMP for binding to a cAMP-specific antibody. When non-radioactive Lance ^® assay Alexa ^® -Flour and the Antiköφer is bound with the tracer which generates a TR-FRET signal at 665 nm. The more unlabeled cAMP bound, the weaker is the signal triggered by labeled cAMP. With a standard curve, the signal strengths can be assigned to the corresponding cAMP concentrations.

Molecular Devices' High-Efficiency Fluorescence Polarization (HEFP ™) PDE assay based on IMAP technology also allows the use of a fluorescently labeled substrate instead of radioactivity. The HEFP-PDE assay uses fluorescein-labeled cAMP (FI-cAMP), which is converted by the PDE into fluorescein-labeled 5'AMP (Fl-AMP). The FI-AMP selectively binds to special beads and this fluoresces strongly. FI-cAMP does not bind to the beads, so that an increase in polarization is proportional to the amount of FI-AMP produced. For a corresponding AC test, fluorescently-labeled ATP can be used instead of FI-cAMP and beads that selectively bind to FI-cAMP instead of FI-cAMP (eg, beads loaded with cAMP antibody).

By "functionally equivalent variants" of polypeptides or domains, ie sequence segments of the polypeptides having a particular function, are meant polypeptides or domains which differ structurally, as described below, but which still perform the same function: functionally equivalent variants of domains the skilled person easily, as described in more detail below, find by varying and functional testing of the corresponding domains, by sequence comparisons with corresponding domains of other known proteins or by hybridization of the corresponding nucleic acid sequences encoding these domains with suitable sequences from other organisms.

A "functional linkage" is understood as meaning linkages, preferably covalent bonds of domains which lead to an arrangement of the domains in such a way that they can fulfill their function, for example, under a functional link the GAF _A domain, GAF _B domain and the catalytic domain adenylate cyclase means a junction of these domains that results in an array of domains such that upon ligand binding, for example, cAMP or PDE10 modulators, the GAF domains change conformation and thereby modulate the catalytic activity of the adenylate cyclase domain For example, a functional linkage of the GAF _A domain and the GAFβ domain is understood as meaning a compound of these domains which leads to an arrangement of the domains such that the GAF _A domain and the GAF ₈ domain together act as the GAF domain in ligands. Binding, for example, of cAMP or PDE10 modulators their Konfor change. Preferably, those for the GAF domains, GAF _A and GAF _{B |} candidate human phosphodiesterases 10 (PDE10) selected from the group of isoforms PDE 10A (Accession: CAI20436 / CAB92797 / CAH72023 / Q9Y233 / NP_006652 / CAG38804 / BAA78034), PDE10A1 (Accession: BAB16383 / BAB92963 / AAD32595), PDE10A2 (Accession: AB026816 / BAA84467 / AAD32596) and PDE10A7 (Accession: BAB16368) or their respective functionally equivalent variants, particularly preferred is the use according to the invention of the GAF domains of the isoform PDE10A2 (Genbank Accession No. AB026816) or their functionally equivalent variants.

In a preferred embodiment, the GAF _A domain of the polypeptide of the invention has an amino acid sequence containing the amino acid sequence SEQ. ID. NO. 6 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids having an identity of at least 90%, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% at the amino acid level with the sequence SEQ ID NO: 6 and has the property of a GAF _A domain.

Instead of SEQ. ID. NO. 6 can be used for the entire description analogous to SEQ. ID. NO. 16 are used. At SEQ. ID. NO. Figure 16 is the N-terminus of the GAF _A domain around an amino acid (D79) versus SEQ. ID. NO. 6 shortened.

This may be a natural functionally equivalent variant of the GAF _A domain, which, as described above, can be found by identity comparison of the sequences with other proteins or to an artificial GAF _A domain starting from the sequence SEQ ID NO : 6 was modified by artificial variation, for example by substitution, insertion or deletion of amino acids.

The term "substitution" in the description refers to the replacement of one or more amino acids by one or more amino acids. Preferably, so-called conservative exchanges are carried out in which the replaced amino acid has a similar property to the original amino acid, for example exchange of Glu by Asp, GIn by Asn, VaI by Me, Leu by Ne ₁ Ser by Thr.

Deletion is the replacement of an amino acid by a direct bond. Preferred positions for deletions are the termini of the polypeptide and the linkages between the individual protein domains. Insertions are insertions of amino acids into the polypeptide chain, formally replacing a direct bond with one or more amino acids.

Identity between two proteins is understood to mean the identity of the amino acids over the entire protein length in each case, in particular the identity which can be determined by comparison with the aid of the laser gene software from DNASTAR. Madison, Wisconsin (USA) using the Clustal method (Higgins DG, Sharp PM, Fast and sensitive multiple sequence alignments on a microcomputer, Comput Appl. BioscL 1989 Apr; 5 (2): 151-1) is calculated using the following parameters :

Multiple ahgnment parameter:

Gap penalty 10

Gap length penalty 10

Pairwise alignment parameter

K-tuple 1

Gap penalty 3

Window 5

Diagonals saved 5

Accordingly, a protein or a domain which has an identity of at least 90% at the amino acid level with the sequence SEQ ID NO: 6 is understood as meaning a protein or a domain which, when comparing its sequence with the sequence SEQ ID NO: 6 has an identity of at least 90%, in particular according to the above program logarithm with the above parameter set.

The property of a GAF _A domain is understood in particular to be its function of binding together with the GAF _B domain cAMP.

In a further preferred embodiment, the GAF _A domain of the polypeptide according to the invention has the amino acid sequence SEQ. ID. NO. 6 on.

In a preferred embodiment, the GAF _B domain of the polypeptide of the invention has an amino acid sequence containing the amino acid sequence SEQ. ID. NO. 8 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids having an identity of at least 90%, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% at the amino acid level with the sequence SEQ ID NO: 8 and has the property of a GAF _B domain.

This may be a natural functionally equivalent variant of the GAF _B domain, which, as described above, can be found by identity comparison of the sequences with other proteins or to an artificial GAF _B domain starting from the sequence SEQ ID NO : 6 was modified by artificial variation, for example by substitution, insertion or deletion of amino acids as described above.

The property of a GAF _B domain in particular its function is understood to be responsible for the dimer formation and in particular its property, together with the GAF _A domain by binding cAMP to activate the PDE10 or by binding of PDE10 modulators PDE10 Activity, ie to increase or decrease.

In a further preferred embodiment, the GAF _B domain of the polypeptide according to the invention has the amino acid sequence SEQ. ID. NO.8 on.

In a further preferred embodiment of the polypeptide according to the invention, the functionally linked GAF _A domain and GAF _B domain, ie the complete GAF domain, of a human phosphodiesterase 10 (PDE10) or its functionally equivalent variants have an amino acid sequence containing the amino acid sequence SEQ. ID. NO. 10 or SEQ. ID. NO. 14 or one of these sequences derived by substitution, insertion or deletion of amino acids having an identity of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 93%, more preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% at the amino acid level with the sequence SEQ ID NO: 10 or SEQ. ID. NO. 14 and the regulatory property of the GAF domain of a human phosphodiesterase 10 (PDE10), wherein the amino acid sequences contained the GAF _A domain, SEQ. ID. NO. 6 and the GAF _B domain, SEQ. ID. NO. 8 by substitution, insertion or deletion of amino acids by a maximum of 10%, more preferably not more than 9%, more preferably not more than 8%, more preferably not more than 7%, more preferably not more than 6%, more preferably not more than 5%, more preferably not more than 4%, more preferably not more than 3% not more than 2%, more preferably not more than 1, more preferably not more than 0.5%.

In particular, the N-terminal residue of the particularly preferred GAF domains SEQ. ID. NO. 10 or SEQ. ID. NO. 14 is from the N-terminus to the GAF _A domain SEQ. ID. NO. 6 freely variable and in particular shortened. Preferably, the N-terminal residue of the most preferred GAF domains is SEQ. ID. NO. 10 or SEQ. ID. NO. 14 by 100 amino acids, more preferably by 90 amino acids, more preferably by 80 amino acids, more preferably by 70 amino acids, more preferably by 60 amino acids, more preferably by 50 amino acids, more preferably by 40 amino acids, more preferably by 30 amino acids, more preferably by 20 amino acids, more preferably by 10 amino acids, more preferably by 5 amino acids N-terminus shortened.

The amino acid partial sequences of the GAF _A domain SEQ. ID. NO. 6 and the GAF _B domain, SEQ. ID. NO. 8 can be replaced by substitution, insertion or deletion of amino acids by not more than 10%, more preferably not more than 9%, more preferably not more than 8%, more preferably not more than 7%, more preferably not more than 6%, more preferably not more than 5%, more preferably not more than 4%, more preferably not more than 3% , more preferably not more than 2%, more preferably not more than 1, more preferably not more than 0.5%, without the loss of the respective functions described above.

Preferably, the functionally linked GAF _A domain and _B domain GAF, the complete GAF domain of a human phosphodiesterase 10 (PDE10) or their functionally equivalent variants ie an amino acid sequence selected from the group

(a) N-terminus of human PDE10A2 from amino acid L14 to amino acid R422 or

(b) N-terminus of human PDE10A2 from amino acid M19 to amino acid R422 or

(c) N-terminus of human PDE10A2 from amino acid M41 mutated from S41 to amino acid R422,

(d) SEQ. ID. NO. 10 or (e) SEQ. ID. NO. 14th

For the part of the catalytic domain of an adenylate cyclase of the polypeptide according to the invention, preference is given to using adenylate cyclases which have a GAF domain in their natural form. Particularly preferred adenylate cyclases are adenylate cyclases of bacterial origin, in particular of cyanobacteria which have a GAF domain in their natural form or their respective functionally equivalent variants.

Particularly preferred adenylate cyclases are selected from the group

(a) Adenylate cyclase from Anabaena sp. PCC 7120 or its functionally equivalent variant,

(b) adenylate cyclase from Anabaena variabili ATTC 29413 or its functionally equivalent variant,

(c) adenylate cyclase from Nostoc punctiforme PCC 73102 or its functionally equivalent variant,

(d) adenylate cyclase from Trichodesmium erythraeum IMS101 or its functionally equivalent variant,

(e) adenylate cyclase from Bdellovibrio bacteriovorus HD100 or its functionally equivalent variant,

(f) adenylate cyclase from Magnetococcus sp. MC-1 or its functionally equivalent variant, Very particularly preferred adenylate cyclases are adenylate cyclases from Anabaena sp. PCC 7120 of the isoform CyaB1 or CyaB2, in particular CyaB1 (Accession: NP_486306, D89623) or their functionally equivalent variant.

In a preferred embodiment, the catalytic domain of an adenylate cyclase or its functionally equivalent variants has an amino acid sequence containing the amino acid sequence SEQ. ID. NO. 12 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids having an identity of at least 90%, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% at the amino acid level with the sequence SEQ ID NO: 12 and has the catalytic property of an adenylate cyclase.

This may be a natural functionally equivalent variant of the catalytic domain of an adenylate cyclase, which, as described above, can be found by identity comparison of the sequences with other adenylate cyclases or an artificial catalytic domain of adenylate cyclase starting from the sequence SEQ ID NO : 12 was modified by artificial variation, for example, by substitution, insertion or deletion of amino acids as described above.

By the property of a catalytic domain of adenylate cyclase is meant the above-described catalytic property of an adenylate cyclase, in particular the ability to convert ATP to cAMP.

Preferably, the catalytic domain of an adenylate cyclase or its functionally equivalent variants has an amino acid sequence selected from the group

(a) C-terminus of CyaB1 from amino acid L386 to K859, where L386 of CyaB1 is replaced by V386 or

(b) SEQ. ID. NO. 12

In a particularly preferred embodiment, the polypeptide according to the invention comprises the amino acid sequence SEQ. ID. NO. 1 or SEQ. ID. NO.4 or one of these sequences derived by substitution, insertion or deletion of amino acids derived sequence having an identity of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 93 %, more preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% at the amino acid level with the sequence SEQ ID NO: 1 or 4 and the regulatory properties of the GAF domain of a human phosphodiesterase 10 (PDE10) and the catalytic properties of an adenylate cyclase, wherein the amino acid sequences contained the GAF _A domain, SEQ. ID. NO. 6, the GAF _B domain, SEQ. ID. NO. 8 and the catalytic domain of adenylate cyclase, SEQ. ID. NO. 12 by substitution, insertion or deletion of amino acids vary by a maximum of 10%.

In particular, the N-terminal residue of the particularly preferred polypeptides according to the invention SEQ. ID. NO. 1 and SEQ. ID. NO. 4 is from the N-terminus to the GAF _A domain SEQ. ID. NO. 6 freely variable and in particular shortened. Preferably, the N-terminal residue of the particularly preferred polypeptides of the invention SEQ. ID. NO. 1 or SEQ. ID. NO.4 is 100 amino acids, more preferably 90 amino acids, more preferably 80 amino acids, more preferably 70 amino acids, more preferably 60 amino acids, more preferably 50 amino acids, more preferably 40 amino acids, more preferably 30 amino acids, more preferably 20 amino acids, even more preferably 10 Amino acids, more preferably by 5 amino acids N-terminal shortened.

The amino acid partial sequences of the GAF _A domain SEQ. ID. NO. 6, the GAF _B domain, SEQ. ID. NO. 8 and the catalytic domain of adenylate cyclase, SEQ. ID. NO. 12 can be replaced by substitution, insertion or deletion of amino acids by a maximum of 10%, more preferably not more than 9%, more preferably not more than 8%, more preferably not more than 7%, more preferably not more than 6%, more preferably not more than 5%, more preferably not more than 4%, more preferably not more than 3% , more preferably not more than 2%, more preferably not more than 1, more preferably not more than 0.5%, without causing any of the above-described function.

Particularly preferably, the chimeric polypeptide according to the invention contains N-terminally of L14, preferably of M19 particularly preferably of M41, which was mutated from S41 to R422 the N-terminus of human PDE10A2 (Accession: NP_006652). This is followed by C-terminal of V386, which mutated from L386 upon insertion of the cloning site, to K859, the C-terminus of CyaB1 (Accession: NP_486306).

Particularly preferred is a polypeptide according to the invention comprising the amino acid sequence SEQ. ID. NO. 1 or SEQ. ID. NO.4.

Very particularly preferred polypeptides according to the invention are polypeptides having the amino acid sequence SEQ. ID. NO. 1 or SEQ. ID. NO.4.

In a further embodiment, the invention further relates to polynucleotides, also referred to below as nucleic acids, coding one of the above-described invention Polypeptides.

All polynucleotides or nucleic acids mentioned in the description can be, for example, an RNA, DNA or cDNA sequence.

Particularly preferred polynucleotides according to the invention contain as partial sequences

(a) SEQ. ID. NO. 5 or a nucleic acid sequence which corresponds to the nucleic acid sequence SEQ. ID. NO. 5 hybridized under stringent conditions and

(b) SEQ. ID. NO. 7 or a nucleic acid sequence which corresponds to the nucleic acid sequence SEQ. ID. NO. 7 hybridized under stringent conditions and

(c) SEQ. ID. NO. 11 or a nucleic acid sequence which corresponds to the nucleic acid sequence SEQ. ID. NO. 11 hybridized under stringent conditions.

SEQ. ID. NO. Figure 5 represents a particularly preferred partial nucleic acid sequence encoding the most preferred GAF _A domain SEQ. ID. NO. 6th

SEQ. ID. NO. Figure 7 represents a particularly preferred partial nucleic acid sequence encoding the most preferred GAF _B domain SEQ. ID. NO. 8th.

SEQ. ID. NO. Figure 11 represents a particularly preferred partial nucleic acid sequence encoding the most preferred catalytic domain of an adenylate cyclase SEQ. ID. NO. 12th

Further natural examples of nucleic acids or partial nucleic acids encoding the domains described above can furthermore be obtained starting from the partial nucleic acid sequences described above, in particular starting from the sequences SEQ ID NO: 5, 7 or 11 from various organisms whose genomic sequence is unknown, by hybridization techniques easy to find in a conventional manner.

Hybridization may be under moderate (low stringency) or preferably under stringent (high stringency) conditions.

Such hybridization conditions are described, for example, in Sambrook, J., Fritsch, EF, Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2nd Ed., CoId Spring Harbor Laboratory Press, 1989, pp. 9.31-9.57 or in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6.

By way of example, the conditions during the washing step can be selected from the range of conditions limited by those with low stringency (with 2X SSC at 50_C) and those with high stringency (with 0.2X SSC at 50_C, preferably at 65_C) (2OX SSC: 0, 3 m Sodium citrate, 3 M sodium chloride, pH 7.0).

In addition, during the washing step, the temperature may be raised from moderate conditions at room temperature, 22_C, to stringent conditions at 65_C.

Both parameters, salt concentration and temperature, can be varied at the same time, also one of the two parameters can be kept constant and only the other can be varied. During hybridization, denaturing agents such as formamide or SDS may also be used. In the presence of 50% formamide, hybridization is preferably carried out at 42_C.

Some exemplary conditions for hybridization and washing step are given as follows:

(1) hybridization conditions with, for example

(1) 4X SSC at 65_C, or (ii) 6X SSC at 45_C, or

(iii) 6X SSC at 68_C, 100 mg / ml denatured fish sperm DNA, or

(iv) 6X SSC, 0.5% SDS, 100 mg / ml denatured, fragmented salmon sperm DNA at 68_C, or

(v) 6XSSC, 0.5% SDS, 100 mg / ml denatured, fragmented salmon sperm DNA, 50% formamide at 42_C, or

(vi) 50% formamide, 4X SSC at 42_C, or

(vii) 50% (vol / vol) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinyl pyrrolidone, 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 75 mM sodium citrate at 42 ° C, or

(viii) 2X or 4X SSC at 50_C (moderate conditions), or

(ix) 30 to 40% formamide, 2X or 4X SSC at 42_ (moderate conditions).

(2) Wash for 10 minutes each with, for example

(i) 0.015M NaCl / 0.0015M sodium citrate / 0.1% SDS at 50_C, or (ii) 0.1X SSC at 65_C, or (iii) 0.1X SSC ₁ 0.5% SDS at 68_C, or (iv) 0.1X SSC, 0.5 % SDS, 50% formamide at 42_C, or (v) 0.2X SSC, 0.1% SDS at 42_C, or 2X SSC at 65_C (moderate conditions). A particularly preferred polynucleotide according to the invention encoding a polypeptide according to the invention contains the nucleic acid sequence SEQ. ID. NO. Second

A very particularly preferred polynucleotide according to the invention which codes for a polypeptide according to the invention comprises the nucleic acid sequence SEQ. ID. NO. 2 on.

The polypeptides according to the invention can preferably be prepared by cloning a polynucleotide described above, encoding a polypeptide according to the invention, into a suitable expression vector, transforming a host cell with this expression vector, expressing this host cell expressing the polypeptide according to the invention and then isolating the protein according to the invention becomes.

The invention therefore relates to a method for producing a polypeptide according to the invention by cultivating a recombinant host cell, expression and isolation of the polypeptide according to the invention.

The transformation methods are known to the person skilled in the art and are described, for example, in Sambrook, J., Fritsch, EF, Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2nd edition, CoId Spring Harbor Laboratory Press, 1989, pages 9.31-9.57 described.

The invention further relates to a recombinant plasmid vector, in particular an expression vector, comprising a polynucleotide according to the invention encoding a polypeptide according to the invention.

The nature of the expression vector is not critical. Any expression vector capable of expressing the desired polypeptide in a corresponding host cell can be used. Suitable expression systems are known to the person skilled in the art.

Preferred expression vectors are PQE30 (Quiagen), pQE60 (Quiagen) pMAL (NEB) pIRES. PIVEX2.4a (ROCHE), PIVEX2.4b (ROCHE), PIVEX2.4c (ROCHE), pUMVCI (Aldevron), pUMVC2 (Aldevron) ,. PUMVC3 (Aldevron) ,. PUMVC4a (Aldevron) ,. PUMVC4b (Aldevron), pUMVC7 (Aldevron) ,. PUMVCβa (Aldevron), pSP64T, pSP64TS, pT7TS, pCro7 (Takara), pKJE7 (Takara), pKM260, pYes260, pGEMT Easy.

The invention further relates to a recombinant host cell comprising a plasmid vector according to the invention. This transformed host cell is preferably able to express the polypeptide of the invention. The type of host cell is not critical. Both prokaryotic host cells and eukaryotic host cells are suitable. Any host cell capable of expressing the desired polypeptide with a corresponding expression vector can be used. Suitable expression systems from expression vectors and host cells are known in the art.

Preferred host cells are, for example, prokaryotic cells such as E. coli, Corynebacteria, yeasts, streptomycetes or eukaryotic cells such as CHO, HEK293 or insect cell lines such as SF9, SF21, Xenopus oocytes.

The culturing conditions of the transformed host cells, such as culture medium composition and fermentation conditions, are known to those skilled in the art and depend on the type of host cell chosen.

Isolation and purification of the polypeptide may be by standard methods, for example as described in "The QuiaExpressionist®, Fifth Edition, June2003.

The above-described transformed host cells expressing the polypeptide of the present invention are also particularly useful for performing methods described below for identifying PDE10 modulators in a cellular assay. For this purpose, it may furthermore be advantageous to immobilize the corresponding host cells on solid carriers and / or to carry out a corresponding screening procedure in high-throughput scale (high-throughput screening).

All of the abovementioned nucleic acid sequences can be cut out from known nucleic acid sequences by methods known, for example enzymatic, and reassembled with known nucleic acid sequences and thus produced. Furthermore, all of the abovementioned nucleic acids can be prepared in a manner known per se by chemical synthesis from the nucleotide units, for example by fragment condensation of individual overlapping, complementary nucleic acid units of the double helix. The chemical synthesis of oligonucleotides can be carried out, for example, in a known manner by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, p. 896-897). The attachment of synthetic oligonucleotides and filling in of gaps using the Wenow fragment of the DNA polymerase and ligation reactions and general cloning methods are described in Sambrook et al. (1989), Molecular cloning: A laboratory manual, Col. Spring Harbor Laboratory Press.

The invention further relates to a method for identifying a modulator of a human

Phosphodiesterase 10 (PDE10) comprising the steps

(a) contacting a possible modulator of a human phosphodiesterase 10 (PDE10) with a polypeptide of the invention and

(b) determining if the possible modulator is the adenylate cyclase activity of the invention

Polypeptide compared to the absence of the possible modulator changed.

In a preferred embodiment of the method according to the invention, in step (a), in addition to the possible modulator of a human phosphodiesterase 10 (PDE10), cAMP is contacted with a polypeptide according to the invention.

In the method according to the invention, the potential PDE10 modulator, preferably in vitro, is incubated with the preferably purified polypeptide according to the invention and particularly preferably with cAMP, and the change in the adenylate cyclase activity of the polypeptide according to the invention is measured against a test batch without PDE10 modulator.

Alternatively, the change in adenylate cyclase activity, after addition of the potential PDE10 modulator, can be measured to a pilot preparation containing the polypeptide of the invention and optionally cAMP. The adenylate cyclase activity of the PDE10 / CyaB1 chimera is determined by reaction of a defined amount of ATP in cAMP, as described in more detail below.

By a modulator of a human phosphodiesterase 10 (PDE10), hereinafter also PDE10 modulator is meant a substance capable of modulating PDE1O activity via binding to the GAF domains of PDE10, i. here, measured by the change in adenylate cyclase activity. A PDE10 modulator thus acts via the allosteric center of PDE10 and not or not alone via the catalytic center of PDE10. The modulator may be an agonist by increasing the enzymatic activity of PDE10 (PDE10 agonist) or an antagonist by lowering the enzymatic activity of PDE10 (PDE10 antagonist).

For example, with the method of the invention, as explained below, it could be shown that cAMP is a PDE10 agonist.

Preferred PDE10 modulators are furthermore, for example, peptides, peptidomimetics, proteins, in particular antibodies, in particular monoclonal antibodies directed against GAF domains, amino acids, amino acids analogues, nucleotides, nucleotide analogs, polynucleotides, in particular oligonucleotides and particularly preferably so-called "small molecules" or SMOLs Preferred SMOLs are organic or inorganic compounds, including heteroorganic compounds or organometallic compounds having a molecular weight of less than 1000 g / mol, in particular having a molecular weight of from 200 to 800 g / mol, particularly preferably having a molecular weight of from 300 to 600 g / mol. According to the present invention, a PDE 10 modulator preferentially binds to the GAF domains in the polypeptide of the invention (PDE10 / CyaB1 chimera) and either directly leads to a change in the adenylate cyclase activity of the polypeptide of the invention (PDE10 / CyaB1 chimera) or to a change adenylate cyclase activity of the PDE10 / CyaB1 chimera by displacing cAMP from the PDE10 / CyaB1 chimera.

If the inventive method only with cGMP, cAMP or various other nucleotide derivatives, such as 7-CH-cAMP, 2-CI-cAMP, cPuMP, 6-CI-cPuMP or 2-NH ₂ - cAMP and without PDE10 modulator than to be tested Substances, the dose response curve of FIG. 5. The PDE10 / CyaB1 chimera is activated by 100 uM cAMP about 50 times. This corresponds to a% basal value of 5000 and shows that cAMP is a PDE10 GAF agonist. cGMP is non-activating at 100 μM and has a% basal value of about 200, ie it is neither a PDE10 agonist nor a PDE10 antagonist.

The modulation, ie the change, ie the increase or decrease of the adenylate cyclase activity by the PDE10 modulator in a test batch without cGMP is calculated as% basic value according to the following formula:

Sales with substance

% Basic value = 100 x turnover without substance

If the% basal value is less than 50 using 100 μM of the potential PDE10 modulator, this indicates a PDE10 antagonist binding to the GAF domains in the PDE10 / CyaB1 chimera, while a% basal value greater than 400 PDE10 agonists suggest.

The invention therefore relates to a particularly preferred method according to the invention in which, in the presence of the modulator, a decrease in the adenylate cyclase activity is measured in comparison with the absence of the modulator and the modulator represents a PDE10 antagonist.

Furthermore, the invention relates to a particularly preferred method according to the invention in which, in the presence of the modulator, an increase in adenylate cyclase activity is measured in comparison with the absence of the modulator and the modulator is a PDE10 agonist.

In a particularly preferred embodiment of the method according to the invention takes place Determination of adenylate cyclase activity by measuring the conversion of radioactively labeled ATP.

In a particularly preferred embodiment of the method according to the invention, the determination of the adenylate cyclase activity is carried out by measuring the conversion of fluorescence-labeled ATP.

The measurement of the adenylate cyclase activity of the polypeptide according to the invention, the PDE10 / CyaB1 chimera, can be carried out by measuring the conversion of radioactive [α- ³² P] -ATP into [Ct- ³² P] -AMP.

In general, the adenylate cyclase activity is easily possible by measuring the resulting cAMP via antibody binding. There are various commercial assay kits such as the cAMP ^[3 H] or ^[125 -I] BioTrak® ^® cAMP SPA assay from Amersham ^® or AlphaScreen ^® or ^® Lance cAMP assay PerkinElmer ^® are all based on the Principle that arises in the AC reaction from ATP unlabeled cAMP. This competes with exogenously added 3H-, 1251-, or biotin-labeled cAMP for binding to a cAMP-specific antibody. When non-radioactive Lance ^® assay Alexa ^® -Flour and the antibody is bound, which produced with the tracer, a TR-FRET signal at 665 nm. The more unlabeled cAMP bound, the weaker is the signal triggered by labeled cAMP. With a standard curve, the signal strengths can be assigned to the corresponding cAMP concentrations.

Molecular Devices' High-Efficiency Fluorescence Polarization (HEFP ™) PDE assay based on IMAP technology also allows the use of a fluorescently labeled substrate instead of radioactivity. The HEFP-PDE assay uses fluorescein-labeled cAMP (FI-cAMP), which is converted by the PDE into fluorescein-labeled 5'AMP (Fl-AMP). The FI-AMP selectively binds to special beads and this fluoresces strongly. FI-cAMP does not bind to the beads, so that an increase in polarization is proportional to the amount of FI-AMP produced. For a corresponding AC assay, fluorescently-labeled ATP can be used instead of FI-cAMP and beads that selectively bind to FI-cAMP instead of FI-cAMP (eg, beads loaded with cAMP antibody).

In a further preferred embodiment of the method according to the invention, in order to differentiate whether the altered% base value is due to a GAF-modulatory effect of the substance or due to the direct modulation of the AC-catalytic center, a counterscreen is additionally performed.

The invention therefore furthermore relates to a preferred process according to the invention in which one excludes direct modulators of the catalytic domain of adenylate cyclase method according to the invention using a polypeptide which has the catalytic domain of an adenylate cyclase and has no functional GAF domain of a human phosphodiesterase 10 (PDE10).

Preferably, the% basic value is determined analogously to the method described above, preferably instead of the PDE10 / CayB1 chimera with a protein which preferably contains only a) the AC catalytic center or b) mutations to amino acids essential for GAF function or c) is truncated N-terminal to the GAF domains.

An example of a) is a polypeptide having the amino acid sequence SEQ. ID. NO. 1, with the proviso that N-terminal L2 to L617 are missing.

An example of b) is a polypeptide having the amino acid sequence SEQ. ID. NO. 1, with the proviso that it contains the mutation D385A.

An example of c) is a polypeptide having the amino acid sequence SEQ. ID. NO. 1, with the proviso that the partial sequence from D79 to R410 is missing.

If 100 μM of a substance with the protein modified according to a, b or c has a% basal value of less than 50, there is an inhibition of the AC catalytic center and pure GAF antagonism can be ruled out.

In a further preferred embodiment of the method according to the invention, the method is carried out as a cellular assay in the presence of a host cell according to the invention described above, wherein an increase in the adenylate cyclase activity is measured in comparison to the absence of the modulator and the modulator is a PDE 10 agonist.

Assay systems according to the invention in which catalytically reacted cAMP can not be distinguished from the added cAMP for activation of the chimera as a result of the detection, as for example in the cellular assay described below, are preferably only suitable for searching for PDE10 agonists.

For this purpose, the resulting cAMP, as a measure of the adenylate cyclase activity, can also be determined in cellular assays, as for example in Johnston, P. Cellular assays in HTS, Methods Mol BbI. 190, 107-16. (2002) and Johnston, PA and Johnston, PA Cellular platforms for HTS: three case studies.Dπvg Discov Today. 7, 353-63. (2002). For this purpose, preferably cDNA of the polypeptides of the invention, the PDE10 / CyaB1 chimera, inserted via suitable interfaces in a transfection vector and transfected with the resulting vector construct suitable cells, such as CHO or HEK293IIellen. The cell clones are selected which stably express the polypeptides according to the invention.

The intracellular cAMP level of the transfected cell clones is significantly influenced by the adenylate cyclase activity of the polypeptides according to the invention. GAF antagonists cause a decrease in adenylate cyclase activity and GAF agonists cause an increase in intracellular cAMP.

The amount of cAMP can be measured either after lysis of the cells with the methods described above (BioTrak ^®, Alpha Screen ^® or HEFP ^®), or directly into the cell. For this purpose, a reporter gene is preferably coupled to a CRE (cAMP response element) in the cell line (Johnston, P. Cellular assays in HTS, Methods Mol Biol. 190, 107-16 (2002).) An increased cAMP level leads to increased binding of CREB (cAMP response element binding protein) to the CRE regulator and thus to increased transcription of the reporter gene can serve as Reportergen greβn fluorescent protein, ß-galactosidase or luciferase whose expression level can be determined by fluorometric, photometric or luminometric, as in Greer, LF and Szalay, AA Luminscscencβ 17, 43-72 (2002) or Hill, S. et al., Reporter gene Systems for the study of G protein coupled receptors, Curr. Opin. Pharmacol., 1, 526-532 (2001).

In a particularly preferred embodiment, the above-described inventive method, in particular as a cellular assay in high-throughput scale is applied

The following examples illustrate the present invention without limiting it to these examples:

example 1

Preparation of the recombinant DNA encoding a PDE10 / CyaB1 chimera

The cloning was carried out according to standard methods. The original human PDE10A2 gene clone (Genbank Accession No. AB026816) was provided by ALTANAPHARMA in a suitable vector. The cloning of the PDE2-GAF chimera described by Kanadier et al., EMBO J. 2002, was carried out by PCR. With specific primers was a gene fragment hPDE10A2 _41st ₂₈₃ amplifies that encodes part of the PDE10 N-terminus with the GAF A domain and N-terminally contains a BamHI and C-terminal an XbaI site. In this case, S41 of the PDE10A2 was mutated to M. Analog became a gene fragment hPDE10A2 ₂₈ Φ ₄₃₂ , which encodes the GAF-B domain and amplifies N-terminal Xbal and C-terminal Sall. The two fragments were assembled via subcloning steps in the cloning vector pBluescriptII SK (-) via the XbaI interface to hPDE10A24i-4 ₃ 2. To the gene fragment hPDEIOAZn ^ interface has been C-terminally CyaB1 ₃ β ₆ a generated by PCR gene fragment on the SalI - ₈₅₉ attached to the catalytic domain of the adenylate cyclase CyaB1 (Genbank Accession No. D89623). The N-terminal SalI site of hPDE10A2 ₄₁ w _{t32 was cloned} onto the C-terminal Xhol site of CyaB1 ₃₈₆₄₅₉ , and L386 was mutated from CyaB1 to V. All cloning steps were carried out in E. coli XHbluθMRF '.

The gene for the PDE10-GAF chimera was recloned into the expression vector pQE30 (from Quiagen).

Example 2

Expression and purification of the polypeptide

The pQE30 vector with the gene for the PDE10-GAF chimera was retransformed into E. coli BL21 cells. The expression and purification of the protein was analogous to "The QIAexpressionist ^®", Fifth Edition, June 2003. In this case, the optimal protein yield in the expression conditions of induction with 25 uM IPTG ₁ 16h incubation at 16 ° C and subsequent French Press Treatment of E. coli

Example 3

Carrying out the assay

The adenylate cyclase activity of the PDE10 / CyaB1 chimera is measured with and without substance to be tested. In this case, the adenylate cyclase activity by reacting a defined amount of ATP in cAMP and its chromatographic separation via 2 column steps according to Salomon et al. certainly. For the detection of the conversion is used as a radioactive tracer [α- ³² P] -ATP and the resulting amount of [α- ³² P] -cAMP measured. ³ H-CAMP serves as an internal standard for the recovery rate. The incubation time should be between 1 and 120 min, the incubation temperature between 20 and 45 ° C, the Mg ²⁺ cofactor concentration between 1 and 20 mM (it is also possible to use corresponding amounts of Mn ²⁺ as cofactor) and the ATP concentration between 0.5 μM and 5 mM. An increase of the substance with respect to the substance without substance indicates a GAF-agonistic effect. If the conversion is inhibited by substance addition, this indicates a GAF-antagonistic effect of the substance. GAF antagonism can also be measured by blocking the activation of the PDE10 / CyaB1 chimera by the native GAF ligand cAMP. For this purpose, the conversion is measured with increasing cAMP concentrations with and without substance. Are the sales with Substance below those without substance, this indicates a GAF antagonism of the substance.

A reaction mixture contains:

• 50 μl AC test cocktail (glycerol 43.5% (v / v), 0.1 M Tris / HCl pH 7.5, 20 mM MgCl ₂ )

• 40-x μl of enzyme dilution (contains 0.1-0.3 μg PDE10 / CyaB1 chimera in 0.1% (w / v) aqueous BSA solution depending on activity)

• x μl of substance

• 10 μl 750 μM ATP Start Solution incl. 16-30 kBq [- ³² P] ATP.

The protein samples and the cocktail are mixed in 1.5 ml reaction vessels on ice, the reaction is started with ATP and incubated at 37 ^° C. for 10 min. The reaction is stopped with 150 μl of AC stop buffer, the reaction vessels are placed on ice and 10 μl of 20 mM cAMP including 100 Bq [2,8- ³ H] -cAMP and 750 μl of water are added.

Each test batch is duplicated. The blank used was a test batch with water instead of enzyme. With a test batch without substance and cGMP the enzyme basal activity is determined. For the separation of ATP and cAMP, each sample is placed on glass columns with 1.2 g of Dowex-50WX4-400 and, after infiltration with 3-4 ml of water. It was then eluted with 5 ml of water on aluminum oxide columns (9 × 1 cm glass columns with 1.0 g of AL ₂ O ₃ 90 active, neutral) and this with 4 ml of 0.1 M TRIS / HCl, pH 7.5 in scintillation vials eluted with 4 ml of scintillator Ultima XR Gold. After thorough mixing, counted in Liquid Scintillation Countβr The amounts of radioactively labeled cAMP and ATP are counted as ³ H and ³² P-fofa / s directly in 5 ml Elutiorϊspuffer and 4 ml scintillator.

The conversion is calculated as enzyme activity according to the following formula:

pmol [cAMP] substrate [μM] 10 ⁵ mg [protein] x min time [[min] protein amount [μg]

The inhibition or activation of the enzyme by the substance is calculated as% basic value according to the following formula:

_n , _n , _<ΛΛ turnover with substance

% Basic value = 100 x

Sales without substance If% basal at 100 μM substance is less than 50, this would indicate GAF antagonist if inhibition of the AC catalytic site is excluded, while% baseline greater than 400 indicates GAF agonist.

In a test batch with 100 μM cAMP, a GAF antagonist is present when the% base value is less than 90 using 100 μM of the substance to be investigated

The columns were regenerated after use as follows: Dowex columns: 5 ml 2N HCl, 2x 5 ml water

Aluminum oxide columns: 2x 5 ml 0.1 M TRIS / HCl, pH 7.5

figure description

Fig. 1: Amino acid sequence of the PDE10 / CyaB1 chimera

Fig. 2: cDNA sequence of the PDE10 / CyaB1 chimera

FIG. 3: Protein sequence of the PDE10 / CYAB1 chimera after purification. From N-terminus to C-terminus: italic = purification tag from the expression vector (PQE30 from Quiagen); M41 was mutated from S41; bold = N-terminus with PDE-10 GAF domains; bold and underlined = GAFA domain and GAFB domain; V386 was mutated to insert the cloning interface from L386; underlined = C-terminus of catalytic domain CyaB1.

Figure 4: Schematic representation of the chimeric PDE10 / CYAB1 polypeptide

FIG. 5 Activation of the PDE10 / CyaB1 Chimera by Cyclic Nucleotides If the method according to the invention is used only with cGMP, cAMP or various other nucleotide derivatives, for example 7-CH-cAMP, 2-CI-cAMP, cPuMP, 6-CI-cPuMP or 2 -NH ₂ -cAMP and without PDE10 modulator as substances to be tested, the dose response curve according to FIG. 5 results. The PDE10 / CyaB1 chimera is activated by 100 μM cAMP approximately 50 times. This corresponds to a% basal value of 5000 and shows that cAMP is a PDE10 GAF agonist. cGMP is non-activating at 100 μM and has a% basal value of about 200, ie it is neither a PDE10 agonist nor a PDE10 antagonist.

Claims

claims

1. Functionally linked polypeptide

(a) the GAF _A domain and GAF _B domain of a human phosphodiesterase 10 (PDE10) or their functionally equivalent variants and

(b) the catalytic domain of adenylate cyclase or its functionally equivalent variants.

2. Polypeptide according to claim 1, characterized in that the phosphodiesterase 10 (PDE10) is selected from the group PDE10A, PDE10A1, PDE10A2 or PDE10A7 or their respective functionally equivalent variants.

3. Polypeptide according to claim 1 or 2, characterized in that the phosphodiesterase 10 (PDE10) has the isoform PDE10A2.

4. Polypeptide according to one of claims 1 to 3, characterized in that the GAF _A - domain has an amino acid sequence containing the amino acid sequence SEQ. ID. NO. 6 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 90% at the amino acid level with the sequence SEQ ID NO: 6 and the property of a GAF _A domain.

5. Polypeptide according to claim 4, characterized in that the GAF _A domain has an amino acid sequence containing the amino acid sequence SEQ. ID. NO. 6th

6. Polypeptide according to one of claims 1 to 5, characterized in that the GAF _B - domain has an amino acid sequence containing the amino acid sequence SEQ. ID. NO. 8 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids which has an identity of at least 90% at the amino acid level with the sequence SEQ ID NO: 8 and the property of a GAF _B domain.

7. Polypeptide according to claim 6, characterized in that the GAF _B domain has an amino acid sequence containing the amino acid sequence SEQ. ID. NO. 8th.

8. Polypeptide according to one of claims 1 to 7, characterized in that the functionally linked GAF _A domain and GAF _B domain of a human phosphodiesterase 10 (PDE10) or their functionally equivalent variants having an amino acid sequence containing the amino acid sequence SEQ. ID. NO. 10 or SEQ. ID. NO. 14 or one of these sequences derived by substitution, insertion or deletion of amino acids sequence having an identity of at least 70% at the amino acid level with the sequence SEQ ID NO: 10 or SEQ. ID. NO. 14 and the regulatory property of the GAF domain of a human phosphodiesterase 10 (PDE10), wherein the amino acid sequences contained the GAF _A domain, SEQ. ID. NO. 6 and the GAF _B domain, SEQ. ID. NO. 8 by substitution, insertion or deletion of amino acids vary by a maximum of 10%.

9. Polypeptide according to one of claims 1 to 8, characterized in that the functionally linked GAF _A domain and GAF _B domain of a human phosphodiesterase 10 (PDE10) or their functionally equivalent variants has an amino acid sequence selected from the group

(a) N-terminus of human PDE10A2 from amino acid L14 to amino acid R422 or

(b) N-terminus of human PDE10A2 from amino acid M19 to amino acid R422 or

(c) N-terminus of human PDE10A2 from amino acid M41 mutated from S41 to amino acid R422 or

(d) SEQ. ID. NO. 10 or

(e) SEQ. ID. NO. 14th

10. Polypeptide according to one of claims 1 to 9, characterized in that the adenylate cyclase represents a GAF domains containing adenylate cyclase of bacterial origin or their respective functionally equivalent variants.

11. Polypeptide according to one of claims 1 to 10, characterized in that the adenylate cyclase represents an adenylate cyclase selected from the group

(d) adenylate cyclase from Trichodesmium erythraeum IMS101 or its functionally equivalent variant

(f) adenylate cyclase from Magnetococcus sp. MC-1 or its functionally equivalent variant

12. Polypeptide according to one of claims 1 to 11, characterized in that the adenylate cyclase an adenylate cyclase from Anabaena sp. PCC 7120 is the isoform CyaB1 or CyaB2 or their functionally equivalent variant

13. Polypeptide according to one of claims 1 to 12, characterized in that the catalytic domain of adenylate cyclase or its functionally equivalent variants has an amino acid sequence containing the amino acid sequence SEQ. ID. NO. 12 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids which has an identity of at least 90% at the amino acid level with the sequence SEQ ID NO: 12 and the catalytic property of an adenylate cyclase.

14. Polypeptide according to one of claims 1 to 13, characterized in that the catalytic domain of adenylate cyclase or their functionally equivalent variants has an amino acid sequence selected from the group

(b) SEQ. ID. NO. 12

15. Polypeptide according to one of claims 1 to 14, containing the amino acid sequence SEQ. ID. NO. 1 or SEQ. ID. NO. 4 or one of these sequences derived by substitution, insertion or deletion of amino acids having an identity of at least 70% at the amino acid level with the sequence SEQ ID NO: 1 or 4 and the regulatory properties of the GAF domain of a human phosphodiesterase 10 (PDE10 ) and the catalytic properties of an adenylate cyclase, wherein the amino acid sequences contained in the GAF _A domain, SEQ. ID. NO. 6, the GAF ₈ domain, SEQ. ID. NO. 8 and the catalytic domain of adenylate cyclase, SEQ. ID. NO. 12 by substitution, insertion or deletion of amino acids vary by a maximum of 10%.

16. Polypeptide according to claim 1, comprising the amino acid sequence SEQ. ID. NO. 1 or SEQ. ID. NO. 4th

17. Polypeptide with the amino acid sequence SEQ. ID. NO. 1 or SEQ. ID. NO.4.

18. polynucleotide encoding one of the polypeptides according to one of claims 1 to 17.

19. Polynucleotide according to claim 18, containing as partial sequences

20. Polynucleotide containing the nucleic acid sequence SEQ. ID. NO. Second

21. Polynucleotide of the nucleic acid sequence SEQ. ID. NO. Second

22. A recombinant plasmid vector comprising a polynucleotide according to any one of claims 18 to 22.

23. A recombinant host cell comprising a plasmid vector according to claim 22.

24. A method for producing a polypeptide according to any one of claims 1 to 17 by culturing a recombinant host cell according to claim 23, expression and isolation of the polypeptide according to any one of claims 1 to 17.

25. A method of identifying a modulator of a human phosphodiesterase 10 (PDE10) comprising the steps

(A) contacting a possible modulator of a human phosphodiesterase 10 (PDE10) with a polypeptide according to any one of claims 1 to 17 and

(b) Determining whether the possible modulator alters the adenylate cyclase activity of the polypeptide of any one of claims 1 to 17 compared to the absence of the possible modulator.

26. The method of claim 25, wherein in step (a) in addition to the possible modulator of a human phosphodiesterase 10 (PDE 10) cAMP is contacted with a polypeptide according to any one of claims 1 to 17.

27. The method according to claim 25 or 26, characterized in that the determination of the adenylate cyclase activity is carried out by measuring the conversion of radioactively or fluorescently labeled ATP.

28. The method according to claim 25 to 27, characterized in that in the presence of the modulator, a decrease in the adenylate cyclase activity compared to the absence the modulator is measured and the modulator represents a PDE10 antagonist.

29. The method according to claim 25 to 28, characterized in that in the presence of the modulator an increase in the adenylate cyclase activity in comparison to the absence of the modulator is measured and the modulator is a PDE10 agonist.

30. The method according to any one of claims 25 to 29, characterized in that to exclude direct modulators of the catalytic domain of adenylate cyclase, a method according to any one of claims 25 to 27 is performed using a polypeptide having the catalytic domain of adenylate cyclase and no functional GAF domain of a human phosphodiesterase 10 (PDE 10).

31. The method according to any one of claims 25 to 30, characterized in that the method is carried out as a cellular assay for finding PDE10 agonists in the presence of a host cell according to claim 23.

32. The method according to claim 31, characterized in that the method is applied in high throughput scale.