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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • G01N2333/914—Hydrolases (3)
  • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

Definitions

• the present invention relates to a novel polypeptide containing the GAF A domain and GAF B domain of a human phosphodiesterase 11 (PDE11) and the catalytic domain of adenylate cyclase and the use of this polypeptide in a method for the identification of PDE11 modulators.
• PDE11 human phosphodiesterase 11
• PDEs Phosphodiesterases
• I 1 Il and III 3 classes 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 described by Aravind and Ponting (Aravind L. and Ponting CP: The GAF domain: an evolutionary link between diverse phototransducing proteins., 1997, TIBS, 22, 458-459) due to protein structure and - defined sequence comparisons.
• PDE2, PDE5 and PDE6 contain so-called cGMP-binding GAF domains, which play a role in the allosteric activation of PDEs.
• Adenylate cyclases 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 (R 3), 517-29, Tang WJ Gilman AG: Construction of a soluble adenylyl cyclase activated by Gs ⁇ 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 Gs ⁇ and forskolin, 1995, Science, 268, 1769-1772). , A combination with GAF domains is unknown.
• a chimer from human PDE11 and bacterial adenylate cyclase is not known.
• the use of such a chimer in a method for identifying PDE 11 modulators of the prior art is not known.
• the object underlying the invention is to provide a method for identifying PDE11 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 11 (PDE11) 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 PDE11 modulators solved.
• PDE11 human phosphodiesterase 11
• a chimeric protein from N-terminal human PDE 11 -GAF domains and C-terminal catalytic center of an adenylate cyclase is useful as effector molecule.
• 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.
• cGMP selectively activates the GAF domain of PDE11 as agonist.
• the present invention makes it possible to identify PDE11 modulators, ie PDE 11 antagonists or PDE11 agonists, which do not act via the binding and blocking of the catalytic center of PDE11, but by allosteric regulation at the N-terminus of PDE11, ie the GAF Domains, act.
• PDE11 modulators ie PDE 11 antagonists or PDE11 agonists
• the invention relates to a polypeptide comprising functionally linked
• 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. Under a human phosphodiesterase 11 or PDE11 is understood in particular an enzyme family of human origin, which is able to convert cGMP into the inactive 5'-monophosphates.
• GAF A domain and B domain have GAF.
• the GAF domains of PDE11 are tandemly located in the protein N-terminal.
• the GAF domain closest to the N-terminus is termed GAFA and the immediately following GAF B.
• the beginning and end of the GAF domains can be determined by 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.
• PDE11A4 L240 to L403 (SEQ ID NO: 6) and GAF B : V425 to K591 (SEQ ID NO: 8).
• adenylate cyclase 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.
• 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 [00- 32 P] -ATP into [ ⁇ - 32 P] -cAMP.
• 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.
• HEFP TM PDE Assay based on IM AP technology, 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 1 AMP (Fl-AMP).
• FI-cAMP fluorescein-labeled cAMP
• Fl-AMP fluorescein-labeled 5 1 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.
• 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).
• polypeptides or domains ie sequence segments of the polypeptides having a particular function
• 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" be threads, preferably understood as covalent compounds of domains which leads to arrangement of the domains is that they can fulfill their function.
• ß functional binding of the GAFA- domain GAF domain and the catalytic domain of Adenylate cyclase is understood as meaning a compound of these domains which results in an arrangement of the domains such that upon ligand binding, for example, cGMP or PDE11 modulators, the GAF domains change their conformation and thereby modulate the catalytic activity of the adenylate cyclase domain functional binding of the GAF a - GAF domain and the B domain is a binding of these domains understood that a Arrangement of the domains leads to the GAF A domain and the GAF ß domain together as GAF domain in ligand binding, for example by cGMP or PDE11 modulators change their conformation.
• candidate human phosphodiesterases 11 are selected from the group of isoforms PDE11 A (Accession: NP_058649 / BAB16371), PDE11A1 (Accession: BAB62714 / CAB82573), PDE11A2 ( Accession: BAB16372), PDE11A3 (Accession: BAB62713) and PDE11A4 (Accession: BAB62712) or their respective functionally equivalent variants, particularly preferred is the use according to the invention of the GAF domains of the isoform PDE11A4 or their functionally equivalent variants.
• 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.
• SEQ. ID. NO. 6 can be used for the entire description analogous to SEQ. ID. NO. 15 are used.
• SEQ. ID. NO. Figure 15 is the N-terminus of the GAF A domain around an amino acid (L240) versus SEQ. ID. NO. 6 shortened.
• substitution refers to the replacement of one or more amino acids by one or more amino acids.
• conservative exchanges are carried out in which the replaced amino acid has a property similar to that of the original amino acid, for example exchanging GIu with Asp, GIn with Asn, VaI with He, Leu with He, Ser with 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 links 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. Biosci 1989 Apr; 5 (2): 151-1) calculated with the following parameters becomes:
• 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 accordingly understood as meaning a protein or a domain which, when comparing its sequence with the sequence SEQ ID NO: 6, in particular according to the above program logarithm with the above parameter set has an identity of at least 90%.
• GAF A domain The property of a GAF A domain is understood in particular to be its function, in particular to bind together with the GAF B domain cGMP.
• the GAF A domain of the polypeptide according to the invention has the amino acid sequence SEQ. ID. NO. 6 on.
• 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 the property of a GAF B domain aul Anlagen.
• GAF B domain in particular its function to be responsible for the dimer formation and in particular its property, together with the GAF A domain by binding of cGMP to activate the PDE11 or by binding of PDE11 modulators PDE11 Activity, ie to increase or decrease.
• the GAF B domain of the polypeptide according to the invention has the amino acid sequence SEQ. ID. NO. 8 on.
• the functionally linked GAF A domain and GAF B domain, ie the complete GAF domain, of a human phosphodiesterase 11 (PDE11) or their functionally equivalent variants have an amino acid sequence containing the amino acid sequence SEQ. ID. NO.
• 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 and the regulatory property of the GAF domain of a human phosphodiesterase 11 (PDE11), wherein the amino acid sequences contained the GAF A domain, SEQ. ID. NO. 6 and the GAF B domain, SEQ. ID. NO.
• the N-terminal residue of the particularly preferred GAF domain SEQ. ID. NO. 10 is from the N-terminus to the GAF A domain SEQ. ID. NO. 6 freely variable and in particular shortened.
• the N-terminal residue of the most preferred GAF domain is SEQ. ID. NO. 10 by 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 by 20 amino acids, more preferably by 10 amino acids, more preferably by 5 amino acids N-terminal shortened.
• 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.
• the functionally linked GAF A domain and GAF B domain ie the complete GAF domain, of a human phosphodiesterase 11 (PDE11) or its functionally equivalent variants has an amino acid sequence selected from the group
• 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.
• adenylate cyclases are selected from the group consisting of:
• 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.
• 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.
• amino acids 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.
• a catalytic domain of adenylate cyclase is understood to be the catalytic property of an adenylate cyclase described above, in particular the ability to convert ATP into cAMP.
• the catalytic domain of an adenylate cyclase or its functionally equivalent variants has an amino acid sequence selected from the group
• 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 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 11 (PDE11) and the catalytic properties 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,
• SEQ. ID. NO. 4 can be used for the entire description analogous to SEQ. ID. NO. 13 are used. At SEQ. ID. NO. 13 is missing compared to SEQ. ID. NO. 4 the amino acid A1020.
• 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.
• 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.
• 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 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 at most 2%, more preferably at most 1, more preferably at most 0.5%, without causing the loss of the respective function described above.
• the chimeric polypeptide according to the invention contains the N-terminus of the human PDE11A4 N-terminally from M24 to K591 (Accession: BAB62712). 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).
• polypeptide according to the invention comprising the amino acid sequence SEQ. ID. NO. 1 or SEQ. ID. NO. 4th
• Very particularly preferred polypeptides according to the invention are polypeptides having the amino acid sequence SEQ. ID. NO. 1 or SEQ. ID. NO. 4th
• the invention further relates to polynucleotides, also referred to below as nucleic acids, encoding one of the polypeptides according to the invention described above.
• 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
• 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
• 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.
• 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, 3M sodium citrate, 3M sodium chloride, pH 7.0).
• 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.
• 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.
• a particularly preferred polynucleotide according to the invention encoding a polypeptide according to the invention contains the nucleic acid sequence SEQ. ID. NO.2.
• a very particularly preferred polynucleotide according to the invention encoding a polypeptide according to the invention has the nucleic acid sequence SEQ. ID. NO. 2 on.
• polypeptides according to the invention can preferably be prepared by transforming 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 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
• PUMVC ⁇ a Aldevron
• pSP64T pSP64TS
• pT7TS pCro7
• pKJE7 Takara
• pKM260 pYes260
• pGEM-T Easy PUMVC ⁇ a (Aldevron)
• pSP64T pSP64TS
• pT7TS pCro7
• pKJE7 Takara
• pKM260 pYes260
• pGEM-T Easy 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.
• prokaryotic cells such as E. coli, Corynebacteria, yeasts, streptomycetes
• 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.
• transformed host cells expressing the polypeptide of the present invention are also particularly useful for performing methods described below for identifying PDE11 modulators in a cellular assay.
• it may furthermore be advantageous to immobilize the corresponding host cells on solid supports and / or to carry out a corresponding screening procedure in high-throughput scale (high-throughput screening).
• 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 invention further relates to a method of identifying a modulator of a human phosphodiesterase 11 (PDE11) comprising the steps
• step (b) determining if the possible modulator alters the adenylate cyclase activity of the polypeptide of the invention compared to the absence of the possible modulator.
• step (a) in addition to the possible modulator of a human phosphodiesterase 11 (PDE11), cGMP is contacted with a polypeptide according to the invention.
• PDE11 human phosphodiesterase 11
• the potential PDE11 modulator preferably in vitro, is incubated with the preferably purified polypeptide according to the invention and particularly preferably with cGMP, and the change in the adenylate cyclase activity of the polypeptide according to the invention is measured against a test batch without PDE11 modulator.
• the alteration of adenylate cyclase activity, after addition of the potential PDE11 modulator, can be measured to a pilot preparation containing the polypeptide of the invention and optionally cGMP.
• the adenylate cyclase activity of the PDE11 / CyaB1 chimera is determined by reaction of a defined amount of ATP in cAMP, as described in more detail below.
• PDE11 modulator By a modulator of a human phosphodiesterase 11 (PDE11), hereinafter also PDE11 modulator is meant a substance capable of modulating PDE11 activity via binding to the GAF domains of PDE11, i. here, measured by the change in adenylate cyclase activity.
• a PDE 11 modulator thus acts via the allosteric center of PDE11 and not or not alone via the catalytic center of PDE11.
• the modulator may be an agonist by increasing the enzymatic activity of PDE11 (PDE11 agonist) or an antagonist by lowering the enzymatic activity of RDE11 (PDE11 antagonist).
• cGMP is a PDE11 agonist.
• Preferred PDE11 modulators are furthermore, for example, peptides, peptidomimetics, proteins, in particular antibodies, in particular monoclonal antibodies directed against GAF domains, amino acids, amino acid analogs, 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 oranometallic compounds having a molecular weight of less than 1000 g / mol, in particular having a molecular weight of from 200 to 800 g / mol, more preferably having a molecular weight of from 300 to 600 g / mol.
• a PDE 11 modulator preferably binds to the GAF domains in the polypeptide according to the invention (PDE11 / CyaB1 chimera) and leads either directly to a change in the adenylate cyclase activity of the polypeptide according to the invention (PDE11 / CyaB1-). Chimera) or to alter the adenylate cyclase activity of the PDE11 / CyaB1 chimera by displacing cGMP from the PDE11 / CyaB1 chimera.
• the dose-response curve according to FIG. 5 results.
• the PDE 11 A4 / CyaB1 chimera is activated about 4 times by 1 mM cGMP. This corresponds to a% basal value of 400 and shows that cGMP is a PDE11 A4 GAF agonist.
• cAMP does not activate at 1mM and has a% basal value of about 150, i. it is neither a GAF agonist nor an antagonist.
• the modulation, ie the change, ie the increase or decrease of the adenylate cyclase activity by the PDE11 modulator in a test batch without cGMP is calculated as% basic value according to the following formula:
• the% basal value is less than 50 using 100 ⁇ M of the potential PDE11 modulator, this indicates a PDE 11 antagonist binding to the GAF domains in the PDE11 / CyaB1 chimera, while a% basal value greater than 200 suggesting PDE11 agonists.
• 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 to the absence of the modulator and the modulator represents a PDE11 antagonist.
• the invention relates to a particularly preferred method according to the invention after an increase in adenylate cyclase activity is measured in the presence of the modulator compared to the absence of the modulator and the modulator is a PDE11 agonist.
• the determination of the adenylate cyclase activity is carried out by measuring the conversion of radioactively or fluorescently labeled ATP.
• the measurement of the adenylate cyclase activity of the polypeptide according to the invention, the PDE11 / CyaB1 chimera, can be carried out by measuring the conversion of radioactive [ ⁇ - 32 P] -ATP into [Ct- 32 P] -CAMP.
• 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.
• HEFP TM 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 1 AMP (Fl-AMP).
• FI-cAMP fluorescein-labeled cAMP
• Fl-AMP fluorescein-labeled 5 1 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.
• 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).
• a counterscreen is additionally performed.
• the invention therefore furthermore relates to a preferred process according to the invention in which, in order to exclude direct modulators of the catalytic domain of adenylate cyclase, a process according to the invention is carried out using a polypeptide which has the catalytic domain of an adenylate cyclase and no functional GAF domain of a human phosphodiesterase 11 (PDE11). having.
• PDE11 human phosphodiesterase 11
• the% base value is determined analogously to the method described above, preferably instead of with the PDE11 / 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.
• a) is a polypeptide having the amino acid sequence SEQ. ID. NO. 1, with the proviso that N-terminal A2 to L775 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 D355A.
• c) is a polypeptide having the amino acid sequence SEQ. ID. NO. 1, with the proviso that the partial sequence from L240 to K568 is missing.
• the method is carried out as a cellular assay in the presence of an above-described host cell according to the invention.
• 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. Biol. 190, 107-16. (2002) and Johnston, P.A. and Johnston, P.A. Cellular platforms for HTS: three case studies.Drag Discov Today. 7, 353-63. (2002).
• cDNA of the polypeptides of the invention, the PDE11 / CyaB1 chimera, inserted via suitable interfaces in a transfection vector and with the resulting , preferably cDNA of the polypeptides of the invention, the PDE11 / CyaB1 chimera, inserted via suitable interfaces in a transfection vector and with the resulting ,.
• Vector construct suitable cells such as CHO or HEK293 cells transfected. 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 of 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.
• 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.
• cAMP response element binding protein cAMP response element binding protein
• CRE regulator cAMP response element binding protein
• ß-galactosidase or luciferase whose expression level can be determined by fluorometric, photometric or luminometric, as in Greer, LF and Szalay, AA Imaging of light emission from the expression of luciferase in living cells and organisms: a review. Luminescence 17, 43-72 (2002) or Hill, S. et al. Reporter gene system for the study of G-protein coupled receptors. Curr. Opin. Pharmacol. 1, 526-532 (2001).
• the above-described inventive method in particular as a cellular assay in high-throughput scale is applied.
• the cloning was carried out according to standard methods.
• the original clone with the human PDE11A4 gene (Genbank Accession No. BAB62712) was provided in a vector.
• a gene fragment hPDE11A4 1 _ 391 was amplified, which encodes the PDE11 A4 N-terminus with the GAF A domain and N-terminally contains a BglII and C-terminal an XbaI site.
• the N-terminal SalI site of hPDE11 A4 ⁇ 69 was cloned onto the C-terminal Xhol site of CyaBi 3 ⁇ 6-859 and L386 was mutated from CyaB1 to V. All cloning steps were carried out in E. coli XHblueMRF.
• the gene for the PDE11-GAF chimera was recloned into the expression vector pQE30 (from Quiagen).
• the pQE30 vector with the gene for the PDE11 -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.
• the optimal protein yield under the expression conditions induction with 25 ⁇ M IPTG, 16h incubation at 16 ° C and subsequent French Press treatment of E. coli.
• the adenylate cyclase activity of the PDE 11 A4 / CyaB1 chimera is measured with and without substance to be investigated.
• 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 [ ⁇ - 32 P] -ATP and the resulting amount of [ ⁇ - 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 0 C, the Mg 2+ cofactor concentration between 1 and 20 mM (it can also be appropriate amounts of Mn 2+ used as cofactor) and the ATP concentration between 0.5 ⁇ M 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 PDE11 A4 / CyaB1 chimera by the native GAF ligand cAMP. For this purpose, the conversion is measured with increasing cAMP concentrations with and without substance. If the sales with substance are below those without substance, this indicates a GAF antagonism of the substance.
• a reaction mixture contains:
• the protein samples and the cocktail are mixed in 1.5 ml reaction vessels on ice, the reaction started with ATP and incubated at 37 0 C for 10 min. The reaction is stopped with 150 ⁇ l of AC stop buffer, the reaction vessels are placed on ice and 10 ⁇ l of 20 mM cAMP including 100 Bq [2,8- 3 H] -CAMP and 750 ⁇ l of water are added.
• each test batch is duplicated.
• the blank used was a test batch with water instead of enzyme.
• the enzyme basal activity is determined.
• 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.
• the inhibition or activation of the enzyme by the substance is calculated as% basic value according to the following formula:
• a GAF antagonist is present in a test batch with 100 ⁇ M cGMP when the% base value is less than 90 using 100 ⁇ M of the substance to be investigated.
• Fig. 1 Amino acid sequence of the PDE11 / CyaB1 chimera
• Fig. 2 cDNA sequence of the PDE11 / CyaB1 chimera
• Fig. 4 Schematic representation of the chimeric PDE11 / CYAB1 polypeptide
• FIG. 5 Activation of the PDE11 / CyaB1 chimera by cyclic nucleotides If the assay with cGMP or cAMP is carried out as substances to be tested, the dose response curve according to FIG. 5 results.
• the PDE 11A4 / CyaB1 chimera is activated about 4 times by 1 mM cGMP , This corresponds to a% baseline value of 400 and shows that cGMP is a PDE11 A4 GAF agonist.
• cAMP does not activate at 1mM and has a% basal value of about 150, i. it is neither a GAF agonist nor an antagonist.

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