JP2008531024A - Identification method of PDE10 modulator - Google Patents

Abstract

The present invention relates to novel polypeptides having the GAF _A and GAF _B domains of human phosphodiesterase 10 (PDE10) and the catalytic domain of adenylate cyclase, and the use of such polypeptides in methods for identifying PDE10 modulators.

Description

The present invention relates to a novel polypeptide having a GAF _A domain, a GAF _B domain of human phosphodiesterase 10 (PDE10), and a catalytic domain of adenylate cyclase, and uses of the polypeptide in a method for identifying a PDE10 modulator.

Prior art Phosphodiesterases (= PDEs) are eukaryotic proteins and are known as modulators of cAMP and cGMP, which are cyclic nucleotides. PDEs are classified into three classes (I, II, and III), of which only class I has 11 PDE families (called PDE 1-11) in mammals.

  The GAF domain is ubiquitous in all biological kingdoms, and the domain can be found in Aravind and Ponting (Aravind L. and Ponting CP, The GAF domain: an evolutionary linked between diverse 58, 4T ) Based on protein structure comparison and protein sequence comparison. PDE2, PDE5 and PDE6 have a so-called cGMP-binding GAF domain, which plays a role in the allosteric activation of PDEs.

  Adenylate cyclases (= ACs) catalyze the conversion of ATP to cAMP in a range of organisms (Cooper DM: Regulation and organization of adenylylcycles and cAMP. 2003, Biochem J., 375 (Pt3). ), 517-29; Tang WJ and Gilman A. G .: Construction of a sewable adenylyl cyclized activated Gsα and forskolin. 1995, Science, 268, 1769. Based on sequence comparisons and structural considerations, adenylate cyclases are classified into five classes (IV). Bacterial class III ACs derived from cyanobacteria, in particular from N. spp. PCC7120, to which CyaB1 also belongs, are of biological interest. The cyanobacterial ACs CyaB1 and CyaB2 similarly have an N-terminal GAF domain, which is structurally similar to that of PDEs, but has cAMP as an activating ligand. Nine known families of human class III ACs are all membrane-bound and are regulated via the G protein (Tang WJ and Gilman AG: Construction of a soluble adenylyl cyclase). activated by Gsα and forskolin. 1995, Science, 268, 1769-1772). The combination with the GAF domain is not known.

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

  Chimeras from human PDE10 and bacterial adenylate cyclase are not known. Similarly, the use of such chimeras in methods for identifying PDE10 modulators is not known from the prior art.

DETAILED DESCRIPTION OF THE INVENTION An object of the present invention is to provide a method for identifying a PDE10 modulator.

The problems include (a) the GAF _A domain and GAF _B domain of human phosphodiesterase 10 (PDE10) or functionally equivalent variants thereof, and (b) the catalytic domain of adenylate cyclase or functionally equivalent thereof. This is solved by providing polypeptides according to the present invention having functionally linked variants as well as using them in a method of identifying PDE10 modulators.

  Surprisingly, it was confirmed that a chimeric protein from the N-terminal human PDE10-GAF domain and the C-terminal catalytic center of adenylate cyclase is suitable as an effector molecule. In the chimeric protein, the GAF domain is an activation domain that alters its conformation upon ligand binding, thereby modulating the catalytic activity of the adenylate cyclase domain that serves as a readout.

Furthermore, it was surprisingly confirmed that cAMP selectively activates the GAF domain of PDE10 as a PDE10 agonist, particularly with an EC ₅₀ of about 50-100 μM.

  This result is particularly surprising, for example, that the PDE10 GAF domain shows only 28% identity to the CyaB1 GAF domain, and that the functional, activating ligand of the PDE10 GAF domain is now Because it was unknown until.

  The present invention allows the identification of PDE10 modulators, ie PDE10 antagonists or PDE10 agonists that act by allosteric modulation at the N-terminus of PDE10, ie in the GAF domain, without acting through binding and blocking of the catalytic center of PDE10 To.

As mentioned above, the present invention
( _A ) the GAF _A domain and GAF _B domain of human phosphodiesterase 10 (PDE10) or functionally equivalent variants thereof;
(B) relates to a polypeptide functionally linked to a catalytic domain of adenylate cyclase or a functionally equivalent variant thereof.

  Human phosphodiesterase or PDE refers to an enzyme derived from human that can convert cAMP or cGMP to the corresponding inactive 5'-monophosphate. Based on their structure and properties, PDEs are classified into various families. Human phosphodiesterase 10 or PDE10 refers to an enzyme family that is particularly a human enzyme family that can convert cAMP or cGMP to an inactive 5'-monophosphate.

According to the present invention, the PDE 10 includes the entire PDE 10 having a GAF _A domain and a GAF _B domain. Those GAF domains of PDE10 are located in the protein at the N-terminus as tandems. The GAF domain closest to the N-terminus is referred to as GAF _A and the immediate following is referred to as GAF _B. The start and end of the GAF domain can be determined based on protein sequence comparisons. SMART sequence comparison (Schultz J., Milpetz F., Bork P. and Pointing CP): SMART a simple architectural research tool: identification of signaling 58, 19-98, 19A-58, 19A. For GAF _A of PDE10A2, it was: D91-A244, and for GAF _B : A266-R422.

  Adenylate cyclase refers to an enzyme that can convert ATP to cAMP. Correspondingly, adenylate cyclase activity represents the amount of ATP converted or the amount of cAMP formed by the polypeptide according to the invention in a defined time.

  The catalytic domain of adenylate cyclase is a portion of the amino acid sequence of adenylate cyclase and is necessary for adenylate cyclase to still have its property of converting ATP to cAMP and is therefore substantially functional. And therefore represents a moiety having adenylate cyclase activity.

  The catalytic domain of adenylate cyclase can be easily determined by shortening the repetition of the amino acid sequence and subsequent measurement of adenylate cyclase activity.

The adenylate cyclase activity can be measured, for example, by measuring the conversion of radioactivity [α- ³² P] -ATP to [α- ³² P] -cAMP.

In general, adenylate cyclase activity is easily possible by measuring the cAMP produced via antibody binding. To do so, various commercially available assay kits such as cAMP [ ³ H-] or [ ¹²⁵ -I] Biotrak® cAMP SPA assay (Amersham®) or AlphaScreen® or Lance® ™ cAMP assays (PerkinElmer®) exist: all based on the principle that unlabeled cAMP is produced during the AC reaction from ATP. It competes for binding to cAMP-specific antibodies with exogenously added ³ H-labeled cAMP, ¹²⁵ I-labeled cAMP or biotin-labeled cAMP. In the case of a non-radioactive Lance® assay, Alexa® Fluor and the antibody are bound, which produces a TR-FRET signal at 665 nm by the tracer. The more unlabeled cAMP is bound, the weaker the signal caused by labeled cAMP. With a standard curve, the signal intensity can be assigned to the corresponding cAMP concentration.

  Similar to the High Efficiency Fluorescence Polarization (HEFP ™) -PDE assay based on IMAP technology from Molecular Devices, a fluorescently labeled substrate can be used instead of radioactivity. For the HEFP-PDE assay, fluorescein labeled cAMP (Fl-cAMP) is used, which is converted to fluorescein labeled 5'AMP (Fl-AMP) by PDE. Fl-AMP selectively binds to special beads, whereby the fluorescence is strongly polarized. Since Fl-cAMP does not bind to the beads, the increase in polarization is proportional to the amount of Fl-AMP produced. For corresponding AC tests, beads labeled with fluorescently labeled ATP instead of Fl-cAMP and selectively bind to Fl-cAMP instead of Fl-cAMP (eg beads loaded with cAMP antibody) ) Can be used.

  A polypeptide or domain having a defined function, ie, a “functionally equivalent variant” of a sequence fragment of a polypeptide, which is structurally different as described below but still fulfills the same function Or it represents a domain. Functionally equivalent variants of a domain can be obtained by one of skill in the art by comparing the corresponding domains of other known proteins with sequence comparisons with corresponding domains and functional tests, as described in detail below. Alternatively, it can be easily found by hybridization of the corresponding nucleic acid sequence encoding the domain with a suitable sequence from another organism.

“Functional binding” refers to binding of domains, preferably covalent bonds, leading to an arrangement in which the domains can fulfill their functions. For example, the functional binding of the GAF _A domain, the GAF _B domain, and the catalytic domain of adenylate cyclase alters its conformation upon ligand binding, eg, cAMP or PDE10 modulator binding, Represents the binding of domains leading to an arrangement in which the catalytic activity of adenylate cyclase is modulated. Furthermore, the functional coupling between the GAF _A domain and GAF _B domain, upon ligand binding as GAF domain and GAF _A domain and GAF _B domain together, for example, the conformation upon binding of cAMP or PDE10 modulator Represents domain binding leading to a changing configuration.

Advantageously, the corresponding human phosphodiesterase 10 (PDE10) for GAF domains GAF _A and GAF _B is isoform PDE10A (accession number: CAI20436 / CAB92797 / CAH72023 / Q9Y233 / NP_006652 / CAG38804 / BAA78034), PDE10A1 ( Selected from the group of accession numbers: BAB16383 / BAB92963 / AAD32595), PDE10A2 (accession number: AB026816 / BAA84467 / AAD32596) and PDE10A7 (accession number: BAB16368), or a functionally equivalent variant of each, GAF domain of type PDE10A2 (Genbank accession number: AB026816) Are preferably used according to the invention of their functionally equivalent variants.

In a preferred embodiment, the GAF _A domain of the polypeptide according to the invention is the amino acid sequence of SEQ ID NO: 6 or a sequence derived from said sequence by amino acid substitution, insertion or deletion, the amino acid level SEQ ID NO: 6 At least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96% It preferably has an amino acid sequence having a sequence having at least 97%, preferably at least 98%, preferably at least 99% identity and having the characteristics of a GAF _A domain.

Instead of SEQ ID NO 6, SEQ ID NO 16 can be used as well for all descriptions. In the case of SEQ ID NO: 16, the N-terminus of the GAF _A domain is shortened by one amino acid (D79) relative to SEQ ID NO: 6.

It is a natural functionally equivalent variant of the GAF _A domain that can be found by sequence identity comparison with another protein as described above, or an artificial starting from the sequence of SEQ ID NO: 6 Do modified, for example, substitution of an amino acid may be an artificial GAF _a domain modified by insertion or deletion.

  The term “substitution” in this application refers to the replacement of one or more amino acids by one or more amino acids. Advantageously, so-called conservative exchanges are carried out, in which the exchanged amino acids have the same properties as the original amino acids, eg Glu Asp exchange, Gln Asn exchange, Val Exchange by Ile, Exchange by Leu Ile, Exchange by Ser Thr.

  Deletion is the exchange of amino acids by direct linkage. Preferred deletion positions are the ends of the polypeptide and the junctions between the individual protein domains.

  Insertion is the introduction of an amino acid into a polypeptide chain, where formally a direct bond is exchanged by one or more amino acids.

Identity between two proteins refers to the identity of the amino acids over the entire length of each protein, particularly DNASTAR, Inc., located in Madison, Wisconsin, USA. By using the Classtal method (Higgins DG, Sharp PM. Fast and sensitive multiple sequences on a microcomputer. Computation Appl. 1989 A1; Biosci. Parameters:
Multiple alignment parameters:
Gap penalty 10
Gap length penalty 10
Pairwise alignment parameters:
K tuple 1
Gap penalty 3
Window 5
Saved diagonal 5
Represents the identity calculated by setting.

  A protein or domain having at least 90% identity at the amino acid level with the sequence of SEQ ID NO: 6 corresponds accordingly to the protein or domain, which sequence and the sequence of SEQ ID NO: 6, in particular the above-mentioned parameters Represents at least 90% identity when compared according to the program algorithm described above.

The characteristics of the GAF _A domain represent in particular its function, in particular the function of binding cAMP together with the GAF _B domain.

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

In a preferred embodiment, the GAF _B domain of the polypeptide according to the invention is the amino acid sequence of SEQ ID NO: 8 or a sequence derived from said sequence by amino acid substitution, insertion or deletion, the amino acid level SEQ ID NO: 8 At least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96% It preferably has an amino acid sequence having a sequence having at least 97%, preferably at least 98%, preferably at least 99% identity and having the characteristics of a GAF _B domain.

It is a natural functionally equivalent variant of the GAF _B domain that can be found by sequence identity comparison with another protein as described above, or an artificial starting from the sequence of SEQ ID NO: 6 Do modified, for example, substitution of an amino acid as described above, may be an artificial GAF _B domain modified by insertion or deletion.

The properties of the GAF _B domain represent in particular its function that can be responsible for dimer formation, in particular its properties that can activate PDE10 by binding cAMP together with GAF _A or its properties that can modulate PDE10 activity by binding PDE10 modulators, That is, it represents the property of increasing or decreasing the activity.

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

In a further preferred embodiment of the polypeptide according to the invention, the functionally linked GAF _A and GAF _B domains of human phosphodiesterase 10 (PDE10), ie the complete GAF domain or their functional equivalents The variant is the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 14, or a sequence derived from the sequence by amino acid substitution, insertion or deletion, and at least 70 and the sequence of SEQ ID NO: 10 or SEQ ID NO: 14 at the amino acid level. %, Preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, preferably at least 97% Preferably have at least 98% identity, preferably at least 99% identity. And an amino acid sequence having a sequence having regulatory characteristics of the GAF domain of human phosphodiesterase 10 (PDE10), wherein the amino acid sequence includes the GAF _A domain of SEQ ID NO: 6 and the GAF _B domain of SEQ ID NO: 8 By amino acid substitution, insertion or deletion, up to 10%, preferably up to 9%, preferably up to 8%, preferably up to 7%, preferably up to Only 6%, preferably at most 5%, preferably at most 4%, preferably at most 3%, preferably at most 2%, preferably at most 1%, advantageous Has been modified by a maximum of 0.5%.

In particular, the N-terminal residue of the particularly preferred GAF domain of the particularly preferred SEQ ID No. 10 or SEQ ID No. 14 can be freely changed from its N-terminus to the GAF _A domain of SEQ ID No. 6 and can be shortened in particular. is there. Advantageously, the particularly preferred N-terminal residue of the GAF domain of SEQ ID No. 10 or SEQ ID No. 14 is only 100 amino acids, preferably only 90 amino acids, preferably only 80 amino acids, preferably only 70 amino acids, preferably Is shortened at the N-terminus by 60 amino acids, preferably only 50 amino acids, preferably only 40 amino acids, preferably only 30 amino acids, preferably only 20 amino acids, preferably only 10 amino acids, preferably 5 amino acids can do.

Maximum amino acid partial sequence of the GAF _B domain of GAF _A domain and SEQ ID NO: 8 SEQ ID NO: 6, amino acid substitutions, by insertion or deletion, without losing the functions respectively, by 10% at maximum, preferably Only 9%, preferably up to 8%, preferably up to 7%, preferably up to 6%, preferably up to 5%, preferably up to 4%, Changes can preferably be made by up to 3%, preferably up to 2%, preferably up to 1%, preferably up to 0.5%.

Advantageously, the functionally linked GAF _A and GAF _B domains of human phosphodiesterase 10 (PDE10), ie the complete GAF domain or a functionally equivalent variant thereof,
(A) N-terminal from amino acid L14 to amino acid R422 of human PDE10A2, or (b) N-terminal from amino acid M19 to amino acid R422 of human PDE10A2, or (c) Mutation from S41 of human PDE10A2. The N-terminus from amino acid M41 to amino acid R422,
(D) SEQ ID NO: 10 or (e) SEQ ID NO: 14
Having an amino acid sequence selected from the group of

  For the part of the catalytic domain of the adenylate cyclase of the polypeptide according to the invention, adenylate cyclase having a GAF domain in the native form is preferably used. A particularly preferred adenylate cyclase is a bacterial, in particular cyanobacterial adenylate cyclase, in particular having a GAF domain in its native form or a functionally equivalent variant of each.

Particularly preferred adenylate cyclase is
(A) an adenylate cyclase derived from Anabaena sp. PCC7120 or a functionally equivalent variant thereof,
(B) an adenylate cyclase derived from Anabena variabili ATTC29413 or a functionally equivalent variant thereof,
(C) Adenylate cyclase derived from Nostoc punctiforme PCC73102 or a functionally equivalent variant thereof,
(D) adenylate cyclase derived from Trichodesmium erythraeum IMS101 or a functionally equivalent variant thereof,
(E) an adenylate cyclase derived from Bdellovibrio bacteriovorus HD100 or a functionally equivalent variant thereof,
(F) selected from the group of adenylate cyclase derived from the species MC-1 of the genus Magnetococcus or functionally equivalent variants thereof. A particularly preferred adenylate cyclase is an adenylate cyclase of isoform CyaB1 or CyaB2, in particular CyaB1 (accession number: NP — 486306, D89623) from Anabena sp.

  In one preferred embodiment, the catalytic domain of adenylate cyclase or a functionally equivalent variant thereof is the amino acid sequence of SEQ ID NO: 12 or a sequence derived from said sequence by amino acid substitution, insertion or deletion. At least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95% with the sequence of SEQ ID NO: 12 at the amino acid level Has an amino acid sequence having a sequence with at least 96% identity, preferably at least 97%, preferably at least 98%, preferably at least 99% and having the catalytic properties of adenylate cyclase.

  It is a natural functionally equivalent variant of the catalytic domain of adenylate cyclase that can be found by sequence identity comparison with another adenylate cyclase as described above, or from the sequence of SEQ ID NO: 12 It may be a catalytic domain of an artificial adenylate cyclase that has been altered starting from an artificial modification, for example an amino acid substitution, insertion or deletion as described above.

  The property of the catalytic domain of adenylate cyclase refers to the aforementioned catalytic properties of adenylate cyclase, in particular the ability to convert ATP to cAMP.

Advantageously, the catalytic domain of adenylate cyclase or a functionally equivalent variant thereof is
(A) C-terminal from amino acids L386 to K859 of CyaB1, wherein L386 of CyaB1 is replaced by V386, or (b) SEQ ID NO: 12
Having an amino acid sequence selected from the group of

In a particularly preferred embodiment, the polypeptide according to the invention is the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4 or a sequence derived from said sequence by amino acid substitutions, insertions or deletions, at the amino acid level. 1 or SEQ ID NO: 4 with at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably Having a sequence having at least 95%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity and having the regulatory properties of the GAF domain of human phosphodiesterase 10 (PDE10). In this case, the GAF _A domain of SEQ ID NO: 6, the GAF _B domain of SEQ ID NO: 8, and the sequence of SEQ ID NO: 12 The amino acid sequence contained in the catalytic domain of denyl cyclase has been altered by up to 10% by amino acid substitution, insertion or deletion.

In particular, in particular N-terminal residue of the polypeptide according to the invention preferably SEQ ID NO: 1 and SEQ ID NO: 4, can be made freely change from its N-terminus to the GAF _A domain of SEQ ID NO: 6, and in particular can be shortened It is. Advantageously, the N-terminal residue of the polypeptide according to the invention of the particularly preferred SEQ ID No. 1 or SEQ ID No. 4 is only 100 amino acids, preferably only 90 amino acids, preferably only 80 amino acids, preferably only 70 amino acids , Preferably only 60 amino acids, preferably only 50 amino acids, preferably only 40 amino acids, preferably only 30 amino acids, preferably only 20 amino acids, preferably only 10 amino acids, preferably only 5 amino acids N Can be shortened at the end.

The amino acid partial sequences of the GAF _A domain of SEQ ID NO: 6, the GAF _B domain of SEQ ID NO: 8, and the catalytic domain of adenylate cyclase of SEQ ID NO: 12 are lost without losing the above functions by amino acid substitution, insertion or deletion, respectively. Up to 10%, preferably up to 9%, preferably up to 8%, preferably up to 7%, preferably up to 6%, preferably up to 5% Only, preferably up to 4%, preferably up to 3%, preferably up to 2%, preferably up to 1%, preferably up to 0.5% Can be added.

  Particularly preferably, the chimeric polypeptide according to the present invention comprises a human PDE10A2 N-terminus (activator) at the N-terminus from L14, preferably from M19, particularly preferably from M41 mutated from S41 to R422. Session number: NP_006652). There follows from V386 mutated from L386 by insertion of a cloning cleavage site at the C terminus to C859 at the C terminus of CyaB1 (accession number: NP — 486306).

  In particular, the polypeptide according to the present invention comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4 is preferred.

  A particularly preferred polypeptide according to the present invention is a polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4.

  In a further embodiment, the present invention further relates to a polynucleotide (hereinafter also referred to as a nucleic acid) encoding one of the aforementioned polypeptides according to the present invention.

  All polynucleotides or nucleic acids mentioned in the detailed description of the invention may be, for example, RNA sequences, DNA sequences or cDNA sequences.

A particularly preferred polynucleotide according to the present invention comprises (a) SEQ ID NO: 5 as a partial sequence, or a nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequence of SEQ ID NO: 5,
(B) SEQ ID NO: 7 or a nucleic acid sequence that hybridizes with the nucleic acid sequence of SEQ ID NO: 7 under stringent conditions;
(C) SEQ ID NO: 11 or a nucleic acid sequence that hybridizes with the nucleic acid sequence of SEQ ID NO: 11 under stringent conditions.

SEQ ID NO: 5 represents a particularly preferred partial nucleic acid sequence encoding a particularly preferred GAF _A domain of SEQ ID NO: 6.

SEQ ID NO: 7 represents a particularly preferred partial nucleic acid sequence encoding a particularly preferred GAF _B domain of SEQ ID NO: 8.

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

  Other natural examples for nucleic acids or partial nucleic acids encoding said domains are further given starting from said partial nucleic acid sequences, in particular SEQ ID Nos. 5, 7 from various organisms whose genomic sequences are not known. Alternatively, starting from 11 sequences, it can be easily found as known per se by hybridization techniques.

  Hybridization can be carried out under moderate (low stringency) or advantageously stringent (high stringency) conditions.

  Such hybridization conditions are described in, for example, Sambrook, J. et al. , Fritsch, E .; F. Maniatis, T .; Molecular Cloning (A Laboratory Manual) (2nd edition), Cold Spring Harbor Laboratory Press, 1989, pages 931 to 9.57, or Current Protocols in Molecular Biology, John & John S. Y. (1989), 6.3.1-6.3.6.

  For example, the conditions during the washing step are low stringency (2 × SSC, 50 ° C.) and high stringency (0.2 × SSC, 50 ° C., preferably 65 ° C.) (20 × SSC: It can be selected from a range of conditions delimited by conditions with 0.3 M sodium citrate, 3 M sodium chloride, pH 7.0).

  Furthermore, the temperature during the washing step can be increased from moderate conditions at room temperature, ie 22 ° C., to stringent conditions at 65 ° C.

  Both parameters, salt concentration and temperature can be changed simultaneously, or one of both parameters can be kept constant and only the other can be changed. During hybridization, denaturing agents such as formamide or SDS may be used. In the presence of 50% formamide, the hybridization is advantageously performed at 42 ° C.

Some exemplary conditions for the hybridization and washing steps are shown below:
(1) Hybridization conditions, for example: (i) 4 × SSC at 65 ° C. or (ii) 6 × SSC at 45 ° C. or (iii) 6 × SSC at 68 ° C. Fish sperm DNA, or (iv) 6 × SSC, 0.5% SDS, 100 mg / ml denatured and fragmented salmon sperm DNA at 68 ° C. or (v) 6 × SSC, 0.5 % SDS, 100 mg / ml denatured and fragmented salmon sperm DNA, 50% formamide at 42 ° C., or (vi) 50% formamide, 4 × SSC at 42 ° C., or (vii) 50% (volume / volume) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 50 mM NaCl, 75 mM sodium citrate at 42 ° C. or (viii) 2 × or 4 × SSC at 50 ° C. (medium conditions) or (ix) 30-40% formamide, 2 × Or 4x SSC at 42 ° C (medium conditions)
(2) Washing step for 10 minutes each, for example (i) 0.015 M NaCl / 0.0015 M sodium citrate / 0.1% SDS at 50 ° C. or (ii) 0.1 × SSC At 65 ° C. or (iii) 0.1 × SSC, 0.5% SDS at 68 ° C. or (iv) 0.1 × SSC, 0.5% SDS, 50% formamide at 42 ° C. Or (v) 0.2 × SSC, 0.1% SDS at 42 ° C., or 2 × SSC at 65 ° C. (medium conditions).

  A particularly preferred polynucleotide according to the present invention encoding a polypeptide according to the present invention has the nucleic acid sequence of SEQ ID NO: 2.

  A particularly preferred polynucleotide according to the invention which encodes a polypeptide according to the invention has the nucleic acid sequence of SEQ ID NO: 2.

  The polypeptide according to the present invention advantageously has the above-mentioned polynucleotide encoding the polypeptide according to the present invention cloned into a suitable expression vector, transformed into a host cell and allowed to express the host cell. Can be produced by expressing a polypeptide according to the invention and subsequently isolating the protein according to the invention.

  The invention therefore relates to a method for producing a polypeptide according to the invention by culturing recombinant host cells, expressing and isolating the polypeptide according to the invention.

  Transformation methods are known to those skilled in the art and are described, for example, in Sambrook, J. et al. Fritsch, E .; F. Maniatis, T .; It is described in the book Molecular Cloning (A Laboratory Manual) (2nd edition), Cold Spring Harbor Laboratory Press, 1989, pages 9.31 to 9.57.

  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 type of 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 those skilled in the art.

  Preferred expression vectors are PQE30 (Quiagen), pQE60 (Quiagen) pMAL (NEB) pIRES. PIVEX 2.4a (ROCHE), PIVEX 2.4b (ROCHE), PIVEX 2.4c (ROCHE), pUMVC1 (Aldevron), pUMVC2 (Aldevron), PUMVC3 (Aldevron), PUMVC4n (PUdevV4) Aldevron), pUMVC7 (Aldevron), PUMVC6a (Aldevron), pSP64T, pSP64TS, pT7TS, pCro7 (Takara), pKJE7 (Takara), pKM260, pYes260, pGEM-T Easy.

  Furthermore, the present invention relates to a recombinant host cell having a plasmid vector according to the present invention. Said recombinant host cell can advantageously express a polypeptide according to the invention.

  The type of host cell is not critical. Both prokaryotic and eukaryotic host cells are suitable. Any host cell capable of expressing the desired polypeptide with the corresponding expression vector can be used. Suitable expression systems from expression vectors and host cells are known to those skilled in the art.

  Preferred host cells are, for example, prokaryotic cells such as E. coli, corynebacteria, yeast, Streptomyces bacteria or eukaryotic cells such as CHO, HEK293 or insect cell lines such as SF9, SF21, Xenopus oocytes.

  The culture conditions of 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 selected.

  Polypeptide isolation and purification can be performed according to standard methods, for example, as described in “The QueaExpressionists®, 5th edition, June 2003”.

  Said transformed host cells expressing a polypeptide according to the invention are also suitable for carrying out the method for identifying PDE10 modulators in particular in the cellular assays described below. For this purpose, it may be further preferable to immobilize corresponding host cells on a solid support and / or to perform a corresponding screening method from a high throughput viewpoint (high throughput screening).

  All of the above nucleic acid sequences can be excised from known nucleic acid sequences known by those skilled in the art, for example by enzymatic methods, and can be newly assembled and produced together with known nucleic acid sequences. Furthermore, all the nucleic acids mentioned above can be produced from nucleotide building blocks by chemical synthesis in a manner known per se, for example by fragment condensation of individual overlapping complementary nucleic acid building blocks of a double helix. The oligonucleotide can be chemically synthesized, for example, according to the phosphoramidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897) in a known manner. Using the Klenow fragment of DNA polymerase, ligation reaction and addition of synthetic oligonucleotides by general cloning methods and gap filling are described in Sambrook et al. (1989), Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.

Furthermore, the present invention provides a method for identifying a modulator of human phosphodiesterase 10 (PDE10),
(A) contacting a potential modulator of human phosphodiesterase 10 (PDE10) with a polypeptide according to the invention;
(B) determining whether the potential modulator alters the adenylate cyclase activity of the polypeptide according to the invention compared to when the potential modulator is absent;
Relates to a method comprising:

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

  In the method according to the invention, a potential PDE10 modulator is advantageously incubated in vitro with a polypeptide according to the invention, which is preferably purified, particularly preferably with cAMP, and the polypeptide according to the invention The change in the adenylate cyclase activity is measured relative to the test batch in the absence of the PDE10 modulator.

  Alternatively, the change in adenylate cyclase activity can be measured after adding a potential PDE10 modulator to a test batch containing a polypeptide according to the invention and optionally cAMP. The adenylate cyclase activity of the PDE10 / CyaB1 chimera is measured through the conversion of a defined amount of ATP to cAMP, as described in detail below.

  A modulator of human phosphodiesterase 10 (PDE10) (hereinafter also referred to as a PDE10 modulator) refers to a substance that can modulate, ie alter, PDE10 activity through binding to the GAF domain of PDE10. Here, the measurement is performed via a change in adenylate cyclase activity. Thus, the PDE10 modulator acts through the allosteric center of PDE10 and does not act through the catalytic center of PDE10 or never. The modulator may be an agonist that increases the catalytic activity of PDE10 (PDE10 agonist) or an agonist that decreases the catalytic activity of PDE10 (PDE10 antagonist).

  For example, the method according to the present invention was able to show that cAMP is a PDE10 agonist, as explained below.

  Furthermore, preferred PDE10 modulators are for example peptides, peptidomimetics, proteins, in particular antibodies, in particular monoclonal antibodies against the GAF domain, amino acids, amino acid analogues, nucleotides, nucleotide analogues, polynucleotides, in particular oligonucleotides, particularly preferably so-called “Small molecule” or SMOLs. Preferred SMOLs are organic or inorganic compounds, such as heteroorganic compounds or organometallic compounds, with a molecular mass of 1000 g / mol, in particular a molecular mass of 200 to 800 g / mol, particularly preferably of 300 to 600 g / mol. A compound having a molecular mass.

  According to the invention, the PDE10 modulator advantageously binds to the GAF domain in the polypeptide according to the invention (PDE10 / CyaB1 chimera) and of the adenylate cyclase activity of the polypeptide according to the invention (PDE10 / CyaB1 chimera). The change is brought directly, or the elimination of cAMP from the PDE10 / CyaB1 chimera results in a change in the adenylate cyclase activity of the PDE10 / CyaB1 chimera.

The substances according to the invention are tested as cGMP, cAMP or various other nucleotide derivatives such as 7-CH-cAMP, 2-Cl-cAMP, cPuMP, 6-Cl-cPuMP or 2-NH ₂ -cAMP. 5 and without using a PDE10 modulator, the dose response curve according to FIG. 5 is obtained. The PDE10 / CyaB1 chimera is activated approximately 50-fold by 100 μM cAMP. It corresponds to a% reference value of 5000 and indicates that cAMP is a PDE10-GAF agonist. cGMP is not activated at 100 μM and has a% reference value of about 200, ie it is neither a PDE10 agonist nor a PDE10 antagonist.

Modulation of adenylate cyclase activity, and hence change or increase or decrease, by PDE10 modulator in a test batch without cGMP is calculated according to the following formula as% reference value:

  Refers to a PDE10 antagonist that binds to the GAF domain in the PDE10 / CyaB1 chimera when the% reference value is less than 50 when a potential PDE10 modulator of 100 μM is used, but the% reference value is higher than 400 Refers to a PDE10 agonist.

  The present invention therefore relates to a particularly preferred method according to the invention, wherein in the presence of a modulator, a decrease in adenylate cyclase activity is measured compared to 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, wherein in the presence of a modulator, the increase in adenylate cyclase activity is measured compared to the absence of the modulator and the modulator represents a PDE10 agonist.

  In one particularly preferred embodiment of the method according to the invention, the measurement of adenylate cyclase activity is carried out by measuring the conversion of radioactively labeled ATP.

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

Polypeptide according to the invention, i.e. measurement of PDE10 / CyaB1 chimera of adenylate cyclase activity can be performed by measuring the conversion of radioactivity from [α- ³² P] -ATP into [α- ³² P] -cAMP .

In general, adenylate cyclase activity is easily possible by measuring the cAMP produced via antibody binding. To do so, various commercially available assay kits such as cAMP [ ³ H-] or [ ¹²⁵ -I] Biotrak® cAMP SPA assay (Amersham®) or AlphaScreen® or Lance® ™ cAMP assays (PerkinElmer®) exist: all based on the principle that unlabeled cAMP is produced during the AC reaction from ATP. It competes for binding to cAMP-specific antibodies with exogenously added ³ H-labeled cAMP, ¹²⁵ I-labeled cAMP or biotin-labeled cAMP. In the case of a non-radioactive Lance® assay, Alexa® Fluor and the antibody are bound, which produces a TR-FRET signal at 665 nm by the tracer. The more unlabeled cAMP is bound, the weaker the signal caused by labeled cAMP. With a standard curve, the signal intensity can be assigned to the corresponding cAMP concentration.

  Similar to the High Efficiency Fluorescence Polarization (HEFP ™) -PDE assay based on IMAP technology from Molecular Devices, a fluorescently labeled substrate can be used instead of radioactivity. For the HEFP-PDE assay, fluorescein labeled cAMP (Fl-cAMP) is used, which is converted to fluorescein labeled 5'AMP (Fl-AMP) by PDE. Fl-AMP selectively binds to special beads, whereby the fluorescence is strongly polarized. Since Fl-cAMP does not bind to the beads, the increase in polarization is proportional to the amount of Fl-AMP produced. For corresponding AC tests, beads labeled with fluorescently labeled ATP instead of Fl-cAMP and selectively bind to Fl-cAMP instead of Fl-cAMP (eg beads loaded with cAMP antibody) ) Can be used.

  In a further preferred embodiment of the method according to the invention, in order to distinguish whether the changed% reference value is caused by the GAF modulator effect of the substance or by direct modulation of the AC catalyst center, it is further countered. Perform screening.

  In addition, the present invention provides for the elimination of a direct modulator of the catalytic domain of adenylate cyclase but a poly having the catalytic domain of adenylate cyclase but not the functional GAF domain of human phosphodiesterase 10 (PDE10). It relates to a preferred method according to the invention carried out using peptides.

Advantageously, for that purpose, the% reference value is advantageously a) having an AC catalytic center, or b) GAF function, instead of preferably with a PDE10 / CyaB1 chimera, as in the previous method. Measured only by proteins that have mutations in essential amino acids or c) are truncated by the GAF domain at the N-terminus.

  An example for a) is a polypeptide having the amino acid of SEQ ID NO: 1 but lacking L2-L617 at the N-terminus.

  An example for b) is a polypeptide having the amino acid of SEQ ID NO 1 but with the mutation D385A.

  An example for c) is a polypeptide having the amino acid sequence of SEQ ID NO: 1 but lacking the partial sequence of D79-R410.

  When 100 μM of the material produces a% reference value of less than 50 with a protein modified by a, b or c, there is inhibition of the AC catalytic center and pure GAF antagonism can be eliminated.

  In a further preferred embodiment according to the invention, the method is carried out as a cellular assay in the presence of a host cell according to the invention as described above, wherein the increase in adenylate cyclase activity is relative to the absence of modulator. The modulator is a PDE10 agonist.

  An assay system according to the invention, which, based on detection, distinguishes catalytically converted cAMP from cAMP added for chimera activation, for example of the cellular assay described below. In some cases, it is advantageously only suitable for finding PDE10 agonists.

  As such, the resulting cAMP can also be measured in cellular assays as a measure of adenylate cyclase activity, see, for example, Johnston, P. et al. A. In Cellular assays in HTS, Methods Mol Biol. 190, 107-16. (2002) and Johnston, P.M. A. And Johnston, P .; A. Cellular platforms for HTS: three case studies. Drug Discov Today. 7,353-63. (2002).

  To that end, the polypeptide according to the invention, ie the cDNA of the PDE10 / CyaB1 chimera, is advantageously inserted into the transfection vector via a suitable cleavage site and, depending on the resulting vector construct, suitable cells such as CHO cells or HEK283 cells are transfected. A cell clone that stably expresses the polypeptide according to the invention is selected.

  The intracellular cAMP concentration of the transfected cell clone is critically influenced by the adenylate cyclase activity of the polypeptide according to the invention. GAF antagonists cause a decrease in intracellular cAMP by inhibiting adenylate cyclase activity, and GAF agonists cause an increase in intracellular cAMP.

  The amount of cAMP can be measured by the method described above (BioTrak®, Alphascreen® or HEFP®) after cell lysis, or directly in the cell. To that end, in this cell line, a reporter gene is advantageously coupled to CRE (cAMP response element) (Johnston, P. Cellular assays in HTS, Methods Mol Biol. 190, 107-16. (2002). )). Increased cAMP concentration results in increased binding of CREB (cAMP response element binding protein) to the CRE regulator and thus increased transcription of the reporter gene. As the reporter gene, for example, green fluorescent protein, β-galactosidase or luciferase can be used, and its expression level can be measured fluorometrically, photometrically or luminometrically, for example, Greer, L. et al. F. And Szalay, A .; A. Imaging of light emission from the expression of living cells and organisms: a review. Luminescence 17, 43-72 (2002) or Hill, S .; Other reporter-gene systems for the study of G-protein coupled receptors. Curr. Opin. Pharmacol. 1,526-532 (2001).

  In a particularly preferred embodiment, the method according to the invention as described above is used from a high throughput perspective, in particular as a cellular assay.

  The following examples illustrate the present invention, but the present invention is not limited thereto.

Example 1
Production of recombinant DNA encoding the PDE10 / CyaB1 chimera Cloning was performed according to standard methods. An original clone carrying the gene for human PDE10A2 (Genbank accession number: AB026816) was provided in the vector by ALTANAPHARMA. By PCR, Kanadaier et al. This was performed in the same manner as the cloning of the PDE2-GAF chimera described in 2002. Using specific primers, to amplify the gene fragment hPDE10A4 _41-283, fragment encodes a portion of the N-terminus of the PDE10 with GAF _A domain, and has a BamHI cleavage site at the N-terminus, and C It has an XbaI cleavage site at the end. At that time, S41 of PDE10A2 was mutated to M. Similarly, the gene fragment hPDE10A2 _284-432 is amplified, which encodes the GAF _B domain, has an XbaI cleavage site at the N-terminus, and a SalI cleavage site at the C-terminus. Both fragments were linked to hPDE10A2 _41-432 via the XbaI cleavage site in the cloning vector pBluescriptII SK (−) via a subcloning step. Gene fragment hPDE10A2 _41-432 was _ligated with the gene domain CyaB1 _386-859 generated by PCR at the C-terminus via the SalI cleavage site with the catalytic domain of adenylate cyclase CyaB1 (Genbank accession number: D89623). At this time, the N-terminal SalI cleavage site hPDE10A23 _41-432, and cloned into the C-terminal XhoI cleavage site CyaB1 _386-859, and the L386 of CyaB1 was mutated to V. All cloning steps were performed in E. coli Xl1blue MRF '.

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

Example 2
Polypeptide Expression and Purification The pQE30 vector carrying the gene for the PDE10-GAF chimera was retransformed into E. coli BL21 cells. The protein was expressed and purified in the same manner as “The QiaExpressionist (registered trademark)”, fifth edition, June 2003. In this case, the optimal protein yield was achieved by expression conditions: induction with 25 μM IPTG, incubation at 16 ° C., followed by French press treatment of E. coli.

Example 3
Performing the assay The adenylate cyclase activity of the PDE10 / CyaB1 chimera is measured with and without the substance to be investigated. In so doing, adenylate cyclase activity is measured by the conversion of a defined amount of ATP to cAMP, and the chromatographic separation is determined according to Salomon et al. Through two column steps. For detection of conversion, a radioactivity tracer [α- ³² P] -AMP is used and the amount of [α- ³² P] -cAMP produced is measured. ³ H-cAMP is used as an internal standard for recovery. The incubation time is preferably 1 to 120 minutes, the incubation temperature is preferably 20 to 45 ° C., and the Mg ²⁺ -cofactor concentration is 1 to 20 mM (with a corresponding amount of Mn ²⁺ as a cofactor). The ATP concentration is preferably 0.5 μM to 5 mM. An increase in conversion with the substance relative to the absence of the substance indicates GAF-antagonism. When conversion is inhibited by the addition of a substance, it exhibits GAF antagonism of the substance. GAF antagonism can also be measured by cAMP, the original GAF ligand, through blocking the activation of the PDE10 / CyaB1 chimera. For this purpose, the conversion is measured with and without the substance while increasing the cAMP concentration. When the conversion rate using a substance is lower than when no substance is used, the substance exhibits GAF antagonism.

The reaction batch is:
50 μl AC test cocktail (glycerin 43.5% (V / V), 0.1 M Tris / HCl (pH 7.5), 20 mM MgCl ₂ )
40-x μl enzyme dilution (containing 0.1-0.3 μg PDE10 / CyaB1-chimera in 0.1% (W / V) aqueous BSA solution depending on activity)
• xμl of material • Contains 10 μl of 750 μM ATP starting solution containing 16-30 kBq [α- ³² P] -ATP.

The protein sample and the cocktail are mixed on ice in a 1.5 ml reaction vessel, the reaction is started with ATP and incubated at 37 ° C. for 10 minutes. The reaction was quenched by 150μl of AC stop buffer, place the reaction vessel in ice, adding 20mM of cAMP 10μl containing [2,8- ³ H] -cAMP and 750μl of water 100 Bq.

Perform twice for each test batch. As a blank, a test batch with water instead of enzyme was used. Enzyme reference activity is measured using test batches without material and cGMP. For separation of ATP and cAMP, each sample is placed in a glass column with 1.2 g Dowex-50WX4-400 and washed with 3-4 ml water after infiltration. Subsequently, 5 ml of water was used to elute into an aluminum oxide column (9 × 1 cm glass column with 1.0 g AL ₂ O ₃ , 90 activity, neutral) and this was 4 ml of 0.1 M TRIS / HCl (pH 7.5) was used to elute into a scintillation vessel provided with 4 ml of scintillator Ultima XR Gold. After careful mixing, they were counted in a liquid scintillation counter. The amount of radiolabeled cAMP and ATP used is counted directly in 5 ml elution buffer and 4 ml scintillator as a total of ³ H- and ³² P-.

に従って計算される。 Conversion is expressed as the enzyme activity:

Calculated according to

に従って計算される。 The inhibition or activation of the enzyme by the substance is expressed as a% reference value according to

Calculated according to

  If the% reference value is less than 50 for a 100 μM substance, eliminating the inhibition of the AC catalytic center indicates a GAF antagonist, while a% reference value greater than 400 indicates a GAF agonist.

  In test batches with 100 μM cAMP, if 100 μM of the substance to be investigated is used, a GAF antagonist is present if the% reference value is less than 90.

The column was regenerated after use as follows:
Dowex column: 5 ml 2N HCl, 2 × 5 ml aluminum hydroxide column: 2 × 5 ml 0.1 M TRIS / HCl, pH 7.5
The substances according to the invention are tested as cGMP, cAMP or various other nucleotide derivatives such as 7-CH-cAMP, 2-Cl-cAMP, cPuMP, 6-Cl-cPuMP or 2-NH ₂ -cAMP. 5 and without using a PDE10 modulator, the dose response curve according to FIG. 5 is obtained. The PDE10 / CyaB1 chimera is activated approximately 50-fold by 100 μM cAMP. It corresponds to a% reference value of 5000 and indicates that cAMP is a PDE10-GAF agonist. cGMP is not activated at 100 μM and has a% reference value of about 200, ie it is neither a PDE10 agonist nor a PDE10 antagonist.

FIG. 1 shows the amino acid sequence of the PDE10 / CyaB1 chimera. FIG. 2 shows the cDNA sequence of the PDE10 / CyaB1 chimera. FIG. 3 shows the protein sequence of PDE10 / CyaB1 chimera after purification. N-terminal to C-terminal: Italics = purification tag from expression vector (PQE30 from Quiagen); bold = N-terminal with PDE10-GAF domain; bold underlined = GAF _A and GAF _B domains; V386 Mutated from L386 for insertion of a cloning cleavage site; underlined = CyaB1 C-terminus with catalytic domain. FIG. 4 shows a schematic diagram of the chimeric PDE10 / CyaB1-polypeptide. FIG. 5 shows activation of the PDE10 / CyaB1 chimera by cyclic nucleotides.

Claims (32)

A polypeptide comprising:
( _A ) the GAF _A domain and GAF _B domain of human phosphodiesterase 10 (PDE10) or functionally equivalent variants thereof;
(B) A polypeptide functionally linked to the catalytic domain of adenylate cyclase or a functionally equivalent variant thereof.   2. The polypeptide according to claim 1, wherein the phosphodiesterase 10 (PDE10) is selected from the group of PDE10A, PDE10A1, PDE10A2 or PDE10A7 or their functionally equivalent variants.   3. The polypeptide according to claim 1, wherein the phosphodiesterase 10 (PDE10) has an isoform of PDE10A2. The polypeptide according to any one of claims 1 to 3, wherein the GAF _A domain is the amino acid sequence of SEQ ID NO: 6 or a sequence derived from the sequence by amino acid substitution, insertion, or deletion. _A polypeptide comprising an amino acid sequence having a sequence having at least 90% identity with the sequence of SEQ ID NO: 6 at the amino acid level and having the characteristics of a GAFA domain. 5. The polypeptide according to claim 4, wherein the GAF _A domain has an amino acid sequence having the amino acid sequence of SEQ ID NO: 6. The polypeptide according to any one of claims 1 to 5, wherein the GAF _B domain is the amino acid sequence of SEQ ID NO: 8, or a sequence derived from the sequence by amino acid substitution, insertion, or deletion. A polypeptide comprising an amino acid sequence having a sequence having at least 90% identity to the sequence of SEQ ID NO: 8 at the amino acid level and having the characteristics of a GAF _B domain. 7. The polypeptide according to claim 6, wherein the GAF _B domain has an amino acid sequence having the amino acid sequence of SEQ ID NO: 8. A polypeptide according to any one of claims 1 to 7, wherein the functionally linked GAF _A domain and GAF _B domain of human phosphodiesterase 10 (PDE10) or a functionally equivalent variant thereof is provided. An amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 14, or a sequence derived from the sequence by amino acid substitution, insertion or deletion, and at least 70% identical to the sequence of SEQ ID NO: 10 or SEQ ID NO: 14 at the amino acid level And an amino acid sequence having a sequence having the regulatory characteristics of the GAF domain of human phosphodiesterase 10 (PDE10), including the GAF _A domain of SEQ ID NO: 6 and the GAF _B domain of SEQ ID NO: 8 Change by up to 10% due to amino acid substitution, insertion or deletion Polypeptide, wherein being applied. A polypeptide according to any one of claims 1 to 8, wherein the functionally linked GAF _A domain and GAF _B domain of human phosphodiesterase 10 (PDE10) or a functionally equivalent variant thereof is provided. ,
(A) N-terminal from amino acid L14 to amino acid R422 of human PDE10A2, or (b) N-terminal from amino acid M19 to amino acid R422 of human PDE10A2, or (c) Mutation from S41 of human PDE10A2. The N-terminus from amino acid M41 to amino acid R422,
(D) SEQ ID NO: 10 or (e) SEQ ID NO: 14
A polypeptide having an amino acid sequence selected from the group consisting of:   The polypeptide according to any one of claims 1 to 9, wherein the adenylate cyclase is a GAF domain-containing adenylate cyclase derived from a bacterium or a functionally equivalent variant thereof. Polypeptide to be. The polypeptide according to any one of claims 1 to 10, wherein adenylate cyclase is
(A) an adenylate cyclase derived from Anabaena sp. PCC7120 or a functionally equivalent variant thereof,
(B) Adenylate cyclase derived from Anavena variabili ATTC29413 or a functionally equivalent variant thereof,
(C) an adenylate cyclase derived from Nostok punchiforme PCC73102 or a functionally equivalent variant thereof,
(D) Trichodesnium erythraeum IMS101-derived adenylate cyclase or a functionally equivalent variant thereof,
(E) Adenylate cyclase derived from Buderobibrio bacteriobora HD100 or a functionally equivalent variant thereof,
(F) A polypeptide characterized by being an adenylate cyclase selected from the group of adenylate cyclase derived from species MC-1 of the genus Magnetococcus or functionally equivalent variants thereof.   12. The polypeptide according to any one of claims 1 to 11, wherein the adenylate cyclase is CyaB1 or CyaB2 of the isoform of adenylate cyclase derived from the species Anabena sp. PCC7120 or a functionally equivalent variant thereof. A polypeptide characterized in that   The polypeptide according to any one of claims 1 to 12, wherein the catalytic domain of adenylate cyclase or a functionally equivalent variant thereof is an amino acid sequence of SEQ ID NO: 12 or an amino acid substitution from the sequence. An amino acid sequence having a sequence derived by insertion or deletion, the sequence having at least 90% identity with the sequence of SEQ ID NO: 12 at the amino acid level and having the catalytic properties of adenylate cyclase Characteristic polypeptide. The polypeptide according to any one of claims 1 to 13, wherein the catalytic domain of adenylate cyclase or a functionally equivalent variant thereof,
(A) C-terminal from amino acids L386 to K859 of CyaB1, wherein L386 of CyaB1 is replaced by V386, or (b) SEQ ID NO: 12
A polypeptide having an amino acid sequence selected from the group consisting of: The polypeptide according to any one of claims 1 to 14, wherein the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4 or a sequence derived from the sequence by amino acid substitution, insertion or deletion, A sequence having at least 70% identity with the sequence of SEQ ID NO: 1 or SEQ ID NO: 4 at the amino acid level and having the regulatory properties of the GAF domain of human phosphodiesterase 10 (PDE10), wherein SEQ ID NO: 6 The amino acid sequence contained in the GAF _A domain, the GAF _B domain of SEQ ID NO: 8 and the catalytic domain of adenylate cyclase of SEQ ID NO: 12 has been altered by up to 10% by amino acid substitution, insertion or deletion Polypeptide.   The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4.   A polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4.   A polynucleotide encoding one of the polypeptides of any one of claims 1-17. The polynucleotide of claim 18, wherein (a) as a partial sequence, SEQ ID NO: 5 or a nucleic acid sequence that hybridizes with the nucleic acid sequence of SEQ ID NO: 5 under stringent conditions;
(B) SEQ ID NO: 7 or a nucleic acid sequence that hybridizes with the nucleic acid sequence of SEQ ID NO: 7 under stringent conditions;
(C) A polynucleotide having SEQ ID NO: 11 or a nucleic acid sequence that hybridizes with the nucleic acid sequence of SEQ ID NO: 11 under stringent conditions.   A polynucleotide having the nucleic acid sequence of SEQ ID NO: 2.   A polynucleotide of the nucleic acid sequence of SEQ ID NO: 2.   A recombinant plasmid vector comprising the polynucleotide according to any one of claims 18 to 22.   A recombinant host cell comprising the plasmid vector of claim 22.   A method for producing the polypeptide according to any one of claims 1 to 17, wherein the recombinant host cell according to claim 23 is cultured, and the polypeptide according to any one of claims 1 to 17 is obtained. Production method by expression and isolation. In a method for identifying a modulator of human phosphodiesterase 10 (PDE10),
(A) contacting a potential modulator of human phosphodiesterase 10 (PDE10) with the polypeptide of any one of claims 1 to 17;
(B) whether or not the potential modulator alters the adenylate cyclase activity of the polypeptide of any one of claims 1 to 17 as compared to the absence of the potential modulator. Measuring the
Including methods.   26. The method according to claim 25, wherein, in step (a), in addition to a possible modulator of human phosphodiesterase 10 (PDE10), cAMP and the polypeptide of any one of claims 1 to 17 are used. How to contact.   27. The method according to claim 25 or 26, wherein the measurement of adenylate cyclase activity is carried out by measuring the conversion of radioactively labeled ATP or fluorescently labeled ATP.   28. The method according to any one of claims 25 to 27, wherein in the presence of a modulator, a decrease in adenylate cyclase activity is measured relative to the absence of the modulator, and the modulator represents a PDE10 antagonist. Feature method.   29. A method according to any one of claims 25 to 28, wherein in the presence of a modulator, an increase in adenylate cyclase activity is measured relative to the absence of the modulator, and said modulator represents a PDE10 agonist. Feature method.   30. The method according to any one of claims 25 to 29, wherein for the elimination of a direct modulator of the catalytic domain of adenylate cyclase, the method according to any one of claims 25 to 27 is adenylated. A method characterized in that it is carried out using a polypeptide having the catalytic domain of acid cyclase but not the functional GAF domain of human phosphodiesterase 10 (PDE10).   A method according to any one of claims 25 to 30, characterized in that it is carried out in the presence of a host cell according to claim 23 to find a PDE10 agonist as a cellular assay. .   32. The method of claim 31, wherein the method is used from a high throughput perspective.

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