EP1749093A2

EP1749093A2 - Method for identifying pde5-modulators

Info

Publication number: EP1749093A2
Application number: EP05743032A
Authority: EP
Inventors: Tobias Kanacher; Juergen Linder; Joachim Schultz
Original assignee: Altana Pharma AG
Current assignee: Takeda GmbH
Priority date: 2004-05-28
Filing date: 2005-05-18
Publication date: 2007-02-07
Also published as: NZ551240A; AU2005248079A1; KR20070030221A; IL179057A0; WO2005116195A3; JP2008500824A; WO2005116195A2; NO20065850L; CA2590553A1; US20090087871A1

Abstract

The invention relates to a novel polypeptide containing the GAFA-domain and GAFB-domain of a human phosphodiesterase 5 (PDE5) and the catalytic domain of an adenylate cyclase, and to the use of this polypeptide in a method for identifying PDE5-modulators.

Description

Method of identifying PDE5 odulators

Technical field

The present invention relates to a new polypeptide comprising the GAF _A domain and GAF _B domain of a human phosphodiesterase 5 (PDE5) and the catalytic domain of an adenylate cyclase and the use of this polypeptide in a method for identifying PDE5 modulators.

State of the art

Phosphodiesterases (= PDEs) are eukaryotic proteins and are known as modulators of the cydic nucleotides cAMP and cGMP. PDEs are divided into 3 classes (I, II and III), of which only class I with its 11 PDE families (called PDE1 to 11) occurs in mammals. PDE5 plays a role in the relaxation and contraction of smooth muscles and in the survival of neurons. A number of PDE5 inhibitors are known and three (Sildenafii, Tadalafil and Vardenafil) are used to treat erectile dysfunction or pulmonary hypertension.

GAF domains are ubiquitous in all realms of life and were developed 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 - sequence comparison defined. PDE2, PDE5 and PDE6 contain so-called cGMP-binding GAF domains, which play a role in the allosteric activation of the PDEs.

High affinity cGMP binding has been demonstrated for the GAF _A domain of the PDE5, while the GAF _B domain is more used for dimerization (Mc-Allister Lucas LM, Haik TL, Colbran JL, Sonnenburg WK, Seger D., Turko IV, Beavo JA , Francis SH and Corbin JD: An essential aspartic acid at each of the allosteric cGMP-binding sites of a cGMP-specific phosphodiesterase. 1995, JBC, 270, 30671-30679; Turko IV, Haik TL, McAllister-Lucas LM, Bums F ., Francis SH and Corbin JD: Identification of key amino acids in a conserved cGMP-binding site of cGMP-binding. 1996, JBC, 271, 22240-22244). According to Rybalkin et al. (Rybalkin SD, Rybalkina IG. Shimizu-Albergine M., Tang XB and Beavo JA: PDE5 is converted to an activated state upon cGMP binding to the GAF A domain, 2003, EMBO J. 22, 469-78) can the GAF- In tandem with cGMP binding increase the activity of PDE5 tenfold.

Adenylate cyclases (= ACs) catalyze the conversion of ATP to cAMP in all areas of life (Cooper DM: Regulation and organizaüon of adenylyl cyclases and cAMP. 2003, Biochem J., 375 (Pt 3), 517-29; Tang WJ and Gilman AG: Construction of a soluble adenylyl cyclase activated by Gsα and forskolin. 1995, Sdence, 268, 1769-1772). Based on sequence comparisons and structural considerations, they are divided into 5 classes (I to V). The bacterial class III ACs from cyanobacteria, in particular from Nostoc sp., Are of molecular biological interest. PCC 7120 which also includes CyaB1. The cyanobacterial ACs CyaB1 and CyaB2 also contain N-terminal GAF domains that are structurally similar to those of the PDEs, but have cAMP as the activating ligand. The 9 known families of human class III ACs are all membrane-bound and regulated by G proteins (Tang WJ and Gilman AG: Construction of a soluble adenylyl cyclase activated by Gsα and forskolin. 1995, Science, 268, 1769-1772). A combination with GAF domains is not known.

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

A chimer from human PDE5 and bacterial adenylate cyanase is not known. Likewise, the use of such a chimer in a method for identifying PDE5 modulators is not known from the prior art.

Description of the invention

The object on which the invention is based is to provide a method for identifying PDE5 modulators.

The object is achieved by providing the polypeptide according to the invention comprising functionally linked (a) the GAF _A domain and GAF _B domain of a human phosphodiesterase 5 (PDE5) or its functionally equivalent variants and (b) the catalytic domain of an adenylate cyanase or its functionally equivalent Variants and its use in a method for identifying PDE5 modulators solved.

It was surprisingly found that a chimeric protein from N-terminal human PDE5-GAF domains and C-terminal catalytic center of an adenylate cyclase is suitable as an effector molecule. In the chimeric protein, the GAF domains are the activation domains that change their conformation upon ligand binding and thereby modulate the catalytic activity of the adenylate cyclase domain, which serves as a read-out.

The present invention enables PDE5 modulators, ie PDE5 antagonists or PDE5 agonists, to be identified that do not have the binding and blocking of the catalytic center the PDE5 act, but act through allosteric regulation at the N-terminus of the PDE5, ie on the GAF domains.

As mentioned above, the invention relates to a polypeptide which is functionally linked

(a) the GAF _A domain and GAF _B domain of a human phosphodiesterase 5 (PDE5) or its functionally equivalent variants and

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

A human phosphodiesterase or PDE is understood to mean an enzyme of human origin which is capable of converting cAMP or cGMP into the corresponding inactive 5'-monophosphates. Based on their structure and properties, the PDEs are classified into different families. A human phosphodiesterase 5 or PDE5 is understood to mean in particular an enzyme family of human origin which is able to convert cGMP into the inactive 5'-monophosphate.

According to the invention, PDE5 can be any PDE5 which has a GAF _A domain and GAFß domain. The GAF domains of PDE5 are located as tandem N-terminal in the protein. The GAF domain that is closest to the N-terminus is referred to as GAF _A and the immediately following one as GAF _B. The beginning and end of the GAF domains can be determined using protein sequence comparisons. A SMART sequence comparison (Schultz J., Milpetz F., Bork P. and Pointing CP: SMART a simple modular architecture research tool: identification of signaling domains. 1998, PNAS, 95, 5857-5864) shows, for example, the isoform for GAF _A. PDE5A1: D164 to L324 and for GAF _B : S346 to E513. ' ^!

An adenylate cyclase is an enzyme that is able to convert ATP to cAMP. Accordingly, adenylate cyclase activity is understood to mean the amount of ATP or amount of cAMP converted by the polypeptide according to the invention in a certain time.

A catalytic domain of an adenylate cyclase is understood to mean that part of the amino acid sequence of an adenylate cyclase which is necessary so that the adenylate cyclase still has the property of converting ATP to cAMP, that is to say is still essentially functional and thus has an adenylate cyclase activity.

The catalytic domain of an adenylate cyanase can be easily determined by iteratively shortening the amino acid sequence and then measuring the adenylate cyclase activity. The adenylate cyanase activity can be determined, for example, by measuring the conversion of radioactive [α- ³² P] -ATP in [α- ³² P] -cAMP.

In general, adenylate cyanase activity is easily possible by measuring the resulting cAMP via antibody binding. There are various commercially available assay kits such as the cAMP [ ³ H-] or [ ¹²⁵ -l] Biotrak ^® cAMP SPA assays from Amersham ^® or the AlphaScreen ^® or the Lance ^® cAMP assay from PerkinElmer ^® : They are all based on the principle that arises in the AC reaction from ATP unlabeled cAMP. This competes with exogenously added 3H, 1251, or biotin-labeled cAMP for binding to a cAMP-specific antibody. In the non-radioactive Lance ^® assay, Alexa ^® flour is bound to the antibody, which generates a TR-FRET signal at 665 nm with the tracer. The more unmarked cAMP is bound, the weaker is the signal triggered by the marked cAMP. The signal strengths can be assigned to the corresponding cAMP concentrations using a standard curve.

Analogous to the High-Efficiency Fluorescence Polarization (HEFP ™) PDA assay from Molecular Devices, which is based on IMAP technology, fluorescence-labeled substrates can also be used instead of radioactive. The HEFP-PDE assay uses fluorescein-labeled cAMP (FI-cAMP), which the PDE converts to fluorescein-labeled 5'AMP (Fl-AMP). The FI-AMP binds selectively to special beads and the fluorescence is strongly polarized. FI-cAMP does not bind to the beads, so an increase in polarization is proportional to the amount of FI-AMP produced. For a corresponding AC test, fluorescence-labeled ATP can be used instead of FI-cAMP and beads which bind selectively to FI-cAMP instead of FI-cAMP (e.g. beads which are loaded with cAMP antibodies).

“Functionally equivalent variants” of polypeptides or domains, ie sequence sections of the polypeptides with a specific function, are understood to mean polypeptides or domains which differ structurally, as described below, but still perform the same function. Functions equivalent variants of domains can the person skilled in the art can easily find these domains with suitable sequences from other organisms by coding 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.

A “functional linkage” is understood to mean links, preferably covalent connections, of domains which leads to an arrangement of the domains such that they can fulfill their function. For example, a functional linkage of the GAF _A domain, GAF _β domain and the catalytic domain the adenylate cyanase understood a connection of these domains, which leads to an arrangement of the domains, so that the GAF domains with ligand binding, for example of cGMP or PDE5 modulators Change conformation and thereby modulate the catalytic activity of the adenylate cyclase domain. Furthermore, a functional link between the GAF _A domain and the GAFa domain is understood to mean a connection of these domains, which leads to an arrangement of the domains such that the GAF _A domain and the GAFß domain together as a GAF domain in league Binding, for example cGMP or PDE5 modulators change their conformation.

The human phosphodiesterases 5 (PDE5) which are suitable for the GAF domains, GAF _A and GAF _B , are preferably selected from the group of isoforms PDE5A1 (Accession: NP_001074), PDE5A2 (Accession: NP_236914), PDE5A3 (Accession: NP_246273) and PDE5A4 (Accession: NP_237223) or their respective functionally equivalent variants, particularly preferred is the use according to the invention of the GAF domains of the isoform PDE5A1 or their functionally equivalent variants.

In a preferred embodiment, the GAF _A domain of the polypeptide according to 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, which has 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.

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

In the description, the term “substitution” is to be understood as meaning the replacement of one or more amino acids by one or more amino acids. So-called conservative exchanges are preferably carried out, in which the replaced amino acid has a similar property to the original amino acid, for example exchange of Glu by Asp, Gin by Asn, Val by He, Leu by He, Ser by Thr.

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

Identity between two proteins is understood to mean the identity of the amino acids over the respective total protein length, in particular the identity obtained by comparison with the aid of the laser genes software from DNASTAR, ine. Madison, Wisconsin (USA) using the Clustal method (Higgins DG, Shaφ PM. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosd. 1989 Apr; 5 (2): 151-1) using the following parameters becomes:

Multiple alignment parameters:

Gap penalty 10

Gap length penalty 10

Pairwise alignment parameter.

K-tuple 1

Gap penalty 3

Diagonals saved 5

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 to mean a protein or a domain which, when its sequence is compared 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%.

The property of a GAF _A domain means in particular its function to bind cGMP.

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

In a preferred embodiment, the GAF _B domain of the polypeptide according to 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, which has an identity of at least 90%, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% at the amino acid level with the sequence SEQ ID NO: 8 and has the property of a GAF _B domain.

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

The property of a GAF _B domain is understood to mean, in particular, its function of being responsible for the formation of dimers and, in particular, its property of activating the PDE5 together with the GAF _A domain by binding cGMP or the PDE5 by binding PDE5 modulators -Modulate activity, ie increase or decrease.

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

In a further preferred embodiment of the polypeptide according to the invention, the functionally linked GAF _A domain and GAF _B domain, ie the complete GAF domain, of a human phosphodiesterase 5 (PDE5) or its functionally equivalent variants have an amino acid sequence containing the amino acid sequence SEQ. ID. NO. 10 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has 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 5 (PDE5), the amino acid sequences contained the GAF _A domain, SEQ. ID. NO. 6 and the GAFß domain, SEQ. ID. NO. 8 by substitution, insertion or deletion of amino acids by at most 10%, more preferably at most 9%, more preferably at most 8%, more preferably at most 7%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, more preferably vary at most 2%, more preferably at most 1, more preferably at most 0.5%.

In particular, 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 shortenable. The N-terminal residue of the particularly preferred GAF domain is preferably SEQ. ID. NO. 10 by 100 amino acids, more preferably by 90 amino acids, more preferably by 80 amino acids, more preferably by 70 amino acids, more preferably by 60 amino acids, more preferably by 50 amino acids, more preferably by 40 amino acids, more preferably by 30 amino acids, more preferably by 20 amino acids, more preferably by 10 amino acids, more preferably 5 amino acids N-terminal shortened.

The partial amino acid 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 at most 10%, more preferably at most 9%, more preferably at most 8%, more preferably at most 7%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3% , more preferably at most 2%, more preferably at most 1, more preferably at most 0.5%, without the respective functions described above being lost.

The functionally linked GAF _A domain and GAFβ domain, ie the complete GAF domain, of a human phosphodiesterase 5 (PDE5) or its functionally equivalent variants preferably have an amino acid sequence selected from the group

(a) N-terminus from human PDE5A1 to amino acid E513 or (b) SEQ. ID. NO. 10

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

Particularly preferred adenylate cyclases are selected from the group

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

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

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

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

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

(f) Magnetococcus sp. adenylate cyanase. MC-1 or its functionally equivalent variant,

Adenylate cyclases from Anabaena sp. PCC 7120 of the isoform CyaB1 or CyaB2, in particular CyaB1 (Accession: NP_486306, D89623) or its functionally equivalent variant. In a preferred embodiment, the catalytic domain of an adenylate cyclase or its functionally equivalent variants has an amino acid sequence containing the amino acid sequence SEQ. ID. NO. 12 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids and 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 having the catalytic property of an adenylate cyclase.

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

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

The catalytic domain of an adenylate cyanase or its functionally equivalent variants preferably has an amino acid sequence selected from the group

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

(b) SEQ. ID. NO. 12

In a particularly preferred embodiment, the polypeptide according to the invention comprises the amino acid sequence SEQ. ID. NO. 1 or SEQ. ID. NO.4 or a sequence derived from these sequences by substitution, insertion or deletion of amino acids, which have 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 5 (PDE5) and the has catalytic properties of an adenylate cyclase, the amino acid sequences contained in the GAF _A domain, SEQ. ID. NO. 6, the GAF _B domain, SEQ. ID. NO. 8 and the catalytic domain of adenylate cyclase, SEQ. ID. NO. 12 by substitution, insertion or deletion of amino acids vary by a maximum of 10%.

In particular, the N-terminal residue of the particularly preferred polypeptides SEQ according to the invention. 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 shortenable. The N-terminal residue of the particularly preferred polypeptides according to the invention is preferably SEQ. ID. NO. 1 or SEQ. ID. NO. 4 by 100 amino acids, more preferably by 90 amino acids, more preferably by 80 amino acids, more preferably by 70 amino acids, more preferably by 60 amino acids, more preferably by 50 amino acids, more preferably by 40 amino acids, more preferably by 30 amino acids, more preferably by 20 amino acids, more preferably by 10 amino acids, more preferably shortened by 5 amino acids at the N-terminal.

The partial amino acid sequences of the GAF _A domain SEQ. ID. NO. 6, the GAF _B domain, SEQ. ID. NO. 8 and the catalytic domain of adenylate cyanase, SEQ. ID. NO. 12 can be replaced by substitution, insertion or deletion of amino acids by at most 10%, more preferably at most 9%, more preferably at most 8%, more preferably at most 7%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3% , more preferably at most 2%, more preferably at most 1, more preferably at most 0.5%, without the respective function described above being lost.

The chimeric polypeptide according to the invention particularly preferably contains the N-terminus to E513 up to the N-terminus of the human PDE5A1 (Accession: NP_001074). This is followed by the C-terminal of V386, which was mutated from L386 when the cloning interface was inserted, to K859, the C-terminus of CyaBI (Accession: NP_486306).

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

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

In a further embodiment, the invention further relates to polynucleotides, also referred to below as nucleic acids, 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 SEQ as partial sequences (a). ID. NO. 5 or a nucleic acid sequence with the nucleic acid sequence SEQ. ID. NO. 5 hybridized under constant conditions and

(b) SEQ. ID. NO. 7 or a nucleic acid sequence with the nucleic acid sequence SEQ. ID. NO. 7 hybridized under constant conditions and

(c) SEQ. ID. NO. 11 or a nucleic acid sequence with the nucleic acid sequence SEQ. ID. NO. 11 hybridized under constant conditions.

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

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

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

Further natural examples of nucleic acids or partial nucleic acids encoding the domains described above can furthermore be derived from the partial nucleic acid sequences described above, in particular from the sequences SEQ ID NO: 5, 7 or 11 from different organisms, the genomic sequence of which is not known, by hybridization techniques easy to find in a manner known per se.

The hybridization can take place under moderate (low stringency) or preferably under constant (high stringency) conditions.

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

For example, the conditions during the washing step can be selected from the range of conditions limited by those with low stringency (with 2X SSC at 50_C) and those with high stringency (with 0.2X SSC at 50_C, preferably at 65_C) (20X SSC: 0, 3 M sodium dtrate, 3 M sodium chloride, pH 7.0).

In addition, the temperature during the washing step can 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 simultaneously, one of the two parameters can be kept constant and only the other can be varied. While In the hybridization, denaturing agents such as formamide or SDS can also be used. In the presence of 50% formamide, the hybridization is preferably carried out at 42_C.

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

(1) Hybridization conditions with, for example

(i) 4X SSC at 65_C, or

(ii) 6X SSC at 45_C, or

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

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

(v) 6XSSC, 0.5% SDS, 100 mg / ml denatured, fragmented salmon sperm DNA, 50% formamide at 42 C, or (vi) 50% formamide, 4X SSC at 42_C, or (vii) 50% (vol / vol ) Formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCI, 75 mM sodium citrate at 42_C, or (viii) 2X or 4X SSC at 50_C (moderate conditions), or (ix) 30 to 40% formamide, 2X or 4X SSC at 42_ (moderate conditions).

(2) washing steps for 10 minutes each with, for example, (i) 0.015 M NaCI / 0.0015 M sodium citrate / 0.1% SDS at 50_C, or (ii) 0.1X SSC at 65_C, or (iii) 0.1X SSC, 0.5% SDS at 68_C, or (iv) 0.1X SSC, 0.5% SDS, 50% formamide at 42_C, or (v) 0.2X SSC, 0.1% SDS at 42_C, or 2X SSC at 65_C (moderate conditions).

A particularly preferred polynucleotide according to the invention encoding a polypeptide according to the invention contains the nucleic acid sequence SEQ. ID. NO. Second

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

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

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

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

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

The type of expression vector is not critical. Any expression vector which is able to express 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 (RÖCHE), PIVEX2.4b (RÖCHE), PIVEX2.4c (RÖCHE), pUMVCI (Aldevron), pUMVC2 (Aldevron) ,. PUMVC3 (Aldevron) ,. PUMVC4a (Aldevron) ,. PUMVC4b (Aldevron), pUMVC7 (Aldevron) ,. PUMVCβa (Aldevron), .pSP64T, pSP64TS, pT7TS, pCro7 (Takara), pKJE7 (Takara), pKM260, pYes260, pGEMTeasy.

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 according to 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 an appropriate expression vector can be used. Suitable expression systems from expression vectors and host cells are known to the person skilled in the art.

Preferred host cells are, for example, prokaryotic cells such as E. coli, Corynebacteria, yeast, streptomycetes or eukaryotic cells such as CHO, HEK293 or insect cell lines such as SF9, SF21, Xenopus oocytes. The cultivation conditions of the transformed host cells, such as, for example, culture medium composition and fermentation conditions, are known to the person skilled in the art and depend on the type of host cell chosen.

The polypeptide can be isolated and purified by standard methods, for example as described in “The QuiaExpressionist®, Fifth Edition, June 2003”.

Transformed host cells described above, which express the polypeptide according to the invention are also particularly suitable for carrying out the methods described below for identifying PDE5 modulators in a cellular assay. To this end, it can further be advantageous to immobilize the corresponding host cells on solid supports and / or to carry out a corresponding screening process on a high-throughput scale (high-through put screening).

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

The invention further relates to a method for identifying a modulator of a human phosphodiesterase 5 (PDE5) comprising the steps

(a) contacting a possible modulator of a human phosphodiesterase 5 (PDE5) with a polypeptide according to the invention and

(b) Determining whether the possible modulator changes the adenylate cyclase activity of the polypeptide according to the invention compared to the absence of the possible modulator.

In a preferred embodiment of the method according to the invention, in step (a), in addition to the possible modulator of a human phosphodiesterase 5 (PDE5), cGMP is contacted with a polypeptide according to the invention.

In the method according to the invention, the possible PDE5 modulator is used, preferably in vitro the preferably purified polypeptide according to the invention and particularly preferably incubated with cGMP and the change in the adenylate cyanase activity of the polypeptide according to the invention compared to a test batch without PDE5 modulator measured.

As an alternative to this, the change in the adenylate cyclase activity can be measured after adding the possible PDE5 modulator to a test batch which contains the polypeptide according to the invention and optionally cGMP. The adenylate cyanase activity of the PDE5 / CyaB1 chimera is determined, as described in more detail below, by converting a defined amount of ATP into cAMP.

A modulator of a human phosphodiesterase 5 (PDE5), hereinafter also referred to as a PDE5 modulator, is understood to mean a substance which is able to modulate PDE5 activity by binding to the GAF domains of PDE5, i.e. to change, measured here via the change in the adenylate cyclase activity. A PDE5 modulator thus acts via the Allostensian center of the PDE5 and not or not alone via the catalytic center of the PDE5. The modulator can be an agonist by increasing the enzymatic activity of PDE5 (PDE5 agonist) or an antagonist by reducing the enzymatic activity of PDE5 (PDE5 antagonist).

For example, the method according to the invention, as explained below, has shown that cGMP is a PDE5 agonist.

Preferred PDE5 modulators are furthermore, for example, peptides, peptidomimetics, proteins, in particular antibodies, in particular monoclonal antibodies directed against GAF domains, amino acids, amino acids 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 organometallic compounds with a molecular weight of less than 1000 g / mol, in particular with a molecular weight of 200 to 800 g / mol, particularly preferably with a molecular weight of 300 to 600 g / mol.

According to the present invention, a PDE5 modulator preferably binds to the GAF domains in the polypeptide according to the invention (PDE5 / CyaB1 chimeric) and leads either directly to a change in the adenylate cyclase activity of the polypeptide according to the invention (PDE5 / CyaB1 chimeric) or to a change in the Adenylate cyclase activity of the PDE5 / CyaB1 chimer by displacement of cGMP from the PDE5 / CyaB1 chimer.

If the method according to the invention is carried out only with cGMP or cAMP and without a PDE5 modulator as substances to be tested, the dose-response curve according to FIG. 5 results PDE5 / CyaB1 chimera is activated approximately 7.8 times by 100 μM cGMP. This corresponds to a% basal value of 780 and shows that cGMP is a PDE5-GAF agonist. cAMP has no activating effect and has a% basal value of approximately 100, ie it is neither a PDE5 agonist nor a PDE5 antagonist.

The modulation, i.e. the change, i.e. the increase or decrease in the adenylate cyclase activity by the PDE5 modulator in a test batch without cGMP, is calculated as a% basal value using the following formula:

Turnover with substance% basal value = 100 x turnover without substance

If the% basal value when using 100 μM of the possible PDE5 modulator is less than 50, this indicates a PDE5 antagonist that binds to the GAF domains in the PDE5 / CyaB1 chimera, while a% basal value greater than 200 indicates PDE5 agonists suggest.

The invention therefore relates to a particularly preferred method according to the invention after a decrease in adenylate cyanase activity is measured in the presence of the modulator compared to the absence of the modulator and the modulator is a PDE5 antagonist.

Furthermore, 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 PDE5 agonist

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

The adenylate cyclase activity of the polypeptide according to the invention, the PDE5 / CyaB1 chimeric, can be measured by measuring the conversion of radioactive [α- ³² P] -ATP in [α- ³² P] -cAMP.

In general, the adenylate cyanase activity is easily possible by measuring the resulting cAMP via antibody binding. There are various commercially available assay kits such as the cAMP [ ³ H-] or [ ¹²⁵ -l] Biotrak ^® cAMP SPA assays from Amersham ^® or the AlphaScreen ^® or the Lance ^® cAMP assay from PerkinElmer ^® : They are all based on the principle that arises in the AC reaction from ATP unlabeled cAMP. This competes with exogenously added 3H, 1251, or Biotin-labeled cAMP for binding to a cAMP-specific antibody. In the non-radioactive Lance ^® assay, Alexa ^® flow is bound to the antibody, which generates a TR-FRET signal at 665 nm with the tracer. The more unmarked cAMP is bound, the weaker is the signal triggered by the marked cAMP. The signal strengths can be assigned to the corresponding cAMP concentrations using a standard curve.

Analogous to the High-Efficiency Fluorescence Polarization (HEFP ™) PDA assay from Molecular Devices, which is based on IM AP technology, fluorescence-labeled substrates can also be used instead of radioactive. The HEFP-PDE assay uses fluorescein-labeled cAMP (FI-cAMP), which the PDE converts to fluorescein-labeled 5'AMP (FI-AMP). The FI-AMP binds selectively to special beads and the fluorescence is strongly polarized. FI-cAMP does not bind to the beads, so an increase in polarization is proportional to the amount of FI-AMP produced. For a corresponding AC test, fluorescence-labeled ATP can be used instead of FI-cAMP and beads which bind selectively to FI-cAMP instead of FI-cAMP (e.g. beads which are loaded with cAMP antibodies).

In a further preferred embodiment of the method according to the invention, a counter screen is additionally carried out in order to differentiate whether the changed% basal value is caused by a GAF-modulatory effect of the substance or by the direct modulation of the AC catalytic center.

The invention therefore further relates to a preferred method according to the invention in which, to exclude direct modulators of the catalytic domain of adenylate cyclase, a method according to the invention is carried out using a polypeptide which has the catalytic domain of an adenylate cyclase and does not have a functional GAF domain of a human phosphodiesterase 5 (PDE5) having.

For this purpose, the% basal value is preferably determined analogously to the method described above, preferably instead of using the PDE5 / CayB1 chimera with a protein which preferably only contains a) the AC catalytic center or b) mutations in amino acids essential for the GAF function contains, or c) N-terminal is shortened by the GAF domains.

An example of a) is a polypeptide with the amino acid sequence SEQ. ID. NO. 1, with the proviso that N-terminal E2 to L720 are missing.

An example of b) is a polypeptide with the amino acid sequence SEQ. ID. NO. 1, provided that it contains the D299A mutation. An example of c) is a polypeptide with the amino acid sequence SEQ. ID. NO. 1, with the proviso that the partial sequence from D164 to E513 is missing.

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

In a further preferred embodiment of the method according to the invention, the method is carried out as a cellular assay in the presence of a host cell according to the invention described above.

For this purpose, the resulting cAMP, as a measure of the adenylate cyclase activity, can also be determined in cellular assays, such as, for example, in Johnston, P. Cellular assays in HTS, Mβthods Mol Biol. 190, 107-16. (2002) and Johnston, P.A. and Johnston, P.A. Cellular platforms for HTS: three case studies.Dπ / g Discov Today. 7, 353-63. (2002).

For this purpose, cDNA of the polypeptides according to the invention, the PDE5 / CyaB1 chimera, is preferably inserted into a transfection vector via suitable interfaces and suitable cells, such as CHO or HEK293 cells, are transfected with the resulting vector construct. The cell clones are selected which stably express the polypeptides according to the invention.

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

The amount of cAMP can be measured either after lysis of the cells with the methods described above (BioTrak ^®, Alpha Screen ^® or HEFP ^®), or directly into the cell. For this purpose, a reporter gene is preferably coupled to a CRE (cAMP response element) in the cell line (Johnston, P. Cellular assays in HTS, Mthods Mol Biol. 190, 107-16. (2002). An increased cAMP level leads to increased binding from CREB (cAMP response element binding protein) to the CRE regulator and thus to increased transcription of the reporter gene. The reporter gene used can be, for example, large fluorβscent protθin, β-galactosidase or luciferase, the expression level of which can be determined flourometrically, photometrically or luminometrically, as in Greer, LF and Szalay, AA Imaging of light emission from the expression of luciferase in living cells and organisms: a review.Luminβscβnce 17, 43-72 (2002) or Hill, S. et al. Reporter-gene Systems for the study of G-protein coupled receptors. Curr. Opin. Pharmacol. 1, 526-532 (2001). In a particularly preferred embodiment, the method according to the invention described above is used, in particular as a cellular assay on a high-throughput scale

The present invention further relates to the following embodiments, which are described in the following 15 paragraphs:

The object on which the invention is based is to provide a method for identifying PDE5 antagonists.

The object is achieved by the polypeptide according to claim A as described below and by the method according to claim Q as described below. Preferred embodiments are formulated in the subclaims below.

It was surprisingly found that a chimeric protein from N-terminal human PDE5-GAF domains and preferably C-terminal catalytic center of the adenylate cyanase (AC) CyaBI from Anabaena or Nostoc sp. PCC 7120 is suitable as an effector molecule. The chimeric protein preferably contains N-terminal to E513 the N-terminus of human PDE5A1 (Accession: NP_001074). This is preferably followed by the C-terminal of V386, which was mutated from L386 when the cloning interface was inserted, to K859, the C-terminus of CyaBI (Accession: NP_486306). In the chimeric protein, the GAF domains are preferably the activation domains, which change their conformation upon ligand binding and thereby increase the catalytic activity of the AC domain, which serves as a read-out.

The present method makes it possible to identify antagonists which do not act via the binding and blocking of the catalytic center of the PDE5, but by allostene regulation at the N-terminus of the PDE5, i.e. on the GAF domains.

For this purpose, the possible PDE5 antagonist is preferably incubated in vitro with the purified polypeptide according to the invention and preferably with cGMP, and the reduction in the adenylate cyclase activity of the polypeptide according to the invention is preferably measured compared to a test batch without a PDE5 antagonist. Alternatively, the reduction in adenylate cyclase activity can be measured after adding the possible PDE5 antagonist to a test batch which contains the polypeptide according to the invention and cGMP. The adenylate cyanase activity of the PDE5 / CyaB1 chimera is preferably determined by converting a defined amount of ATP into cAMP.

According to the present invention, a PDE5 antagonist (antagonist against PDE5) preferably binds to the GAF domains in the PDE5 / CyaB1 chimer and preferably either delivers directly a reduction in the adenylate cyanase activity of the PDE5 / CyaB1 chimeric or a reduction in the adenylate cyanase activity of the PDE5 / CyaB1 chimeric by displacing cGMP from the PDE5 / CyaB1 chimeric.

If the method according to the invention is carried out only with cGMP or cAMP and without a PDE5 antagonist as the substances to be tested, the dose-response curve according to FIG. 5 results. The PDE5 / CyaB1 chimera is activated approximately 7.8 times by 100 μM cGMP. This corresponds to a% basal value of 7800 and shows that cGMP is a PDE5-GAF agonist. cAMP has no activating effect and has a% basal value of approximately 100, i.e. it is neither a GAF agonist nor an antagonist.

The inhibition of adenylate cyanase by the PDE5 antagonist in a test batch without cGMP is calculated as a% basal value according to the following formula:

% Basal value =

If the% basal value when using 100 μM of the possible PDE5 antagonist is less than 50, this indicates a PDE5 antagonist that binds to the GAF domains in the PDE5 / CyaB1 chimera, while a% basal value greater than 200 indicates PDE5 agonists suggest.

For expression, the PDE5 / CyaB1 chimera was inserted into the PQE30 expression vector of Quiagen via the BamHI and Sall restriction enzyme interface of the MCS. Expression can take place in prokaryotic and eukaryotic cells. The protein is purified using standard methods, see above. eg according to The QiaExpressionist ^® ", Fifth Edition, June 2003.

The adenylate cyclase activity of the PDE5 / CyaB1 chimer can be detected by measuring the conversion of radioactive [α- ³² P] -ATP into tα- ³² P] -cAMP.

In general, the adenylate cyanase activity is easily possible by measuring the resulting cAMP via antibody binding. There are various commercially available assay kits such as the cAMP [ ³ H-] or [ ^{1 5} -l] Biotrak ^® cAMP SPA assays from Amersham ^® or the AlphaScreen ^® cAMP assay from PerkinElmer ^® : They are all based on the principle that In the AC reaction, unlabeled cAMP arises from ATP. This competes with exogenously added 3H-, 1251-, or biotin-labeled cAMP for binding to a cAMP-specific antibody. The more unmarked cAMP is bound, the weaker is the signal triggered by the marked cAMP. The signal strengths can be assigned to the corresponding cAMP concentrations using a standard curve. Analogous to the High-Efficiency Fluorescence Polarization (HEFP ™) PDA assay from Molecular Devices, which is based on IM AP technology, fluorescence-labeled substrates can also be used instead of radioactive. The HEFP-PDE assay uses fluorescein-labeled cAMP (FI-cAMP), which the PDE converts to fluorescein-labeled 5'AMP (FI-AMP). The FI-AMP binds selectively to special beads and the fluorescence is strongly polarized. FI-cAMP does not bind to the beads, so an increase in polarization is proportional to the amount of FI-AMP produced. For a corresponding AC test, fluorescence-labeled ATP can be used instead of FI-cAMP and beads which bind selectively to FI-cAMP instead of FI-cAMP (e.g. beads which are loaded with cAMP antibodies).

If the assay is carried out with cGMP or cAMP as the substances to be tested, the dose-response curve according to FIG. 5 results. The PDE5 / CyaB1 chimera is activated approximately 7.8 times by 100 μM cGMP. This corresponds to a% basal value of 7800 and shows that cGMP is a PDE5-GAF agonist. cAMP has no activating effect and has a% basal value of approximately 100, i.e. it is neither a GAF agonist nor an antagonist.

The present invention further relates to:

A. A polypeptide comprising (a) the GAF _A and GAF _B domains from human PDE5 and (b) the catalytic domain of CyaBI.

B. The polypeptide according to A, characterized in that it (a) the N-terminus of human PDE5A1 up to the amino acid E513 and (b) the C-terminus of CyaBI from the amino acid L386 to K859, L386 of CyaBI being replaced by V386 , includes.

C. The polypeptide according to A, comprising the polypeptide sequence as shown in FIG. 1.

D. A polypeptide as shown in Figure 3.

E. A polynucleotide coding for the polypeptide according to A.

F. A polynucleotide coding for the polypeptide according to B

G. A polynucleotide coding for the polypeptide according to C. H. A polynucleotide coding for the polypeptide according to D.

I. An isolated DNA molecule comprising the nucleotide sequence as shown in Figure 2. J. A recombinant DNA molecule comprising a cDNA sequence encoding a polypeptide according to A, B, C or D.

K. A recombinant DNA molecule comprising a cDNA sequence which codes for a polypeptide with at least 90% sequence identity with a polypeptide according to A, B, C or D.

L. A recombinant plasmid vector comprising a polynucleotide according to E, F, G or H.

M. A recombinant host cell comprising a plasmid vector according to L.

N. A method of identifying an antagonist against PDE5 comprises the steps of (a) contacting a possible antagonist against PDE5 with a polypeptide according to A, B, C or D and (b) determining whether the possible antagonist inhibits the activity of PDE5.

O. The method according to N, wherein in step (a) in addition to the antagonist against PDE5 cGMP is contacted with a polypeptide according to A, B, C or D.

P. The method according to N and O, comprising a further step in which the adenylate cyclase activity of the polypeptide used in step (a) is measured after step (a) and before step (b).

^'■ "^' Q. The method according to P, whereby the reduction in adenylate cyclase activity is measured.

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

example 1

Production of the recombinant DNA encoding a PDE5 / CyaB1 chimera

The cloning was carried out according to standard methods. The original clone with the gene for human PDE5A1 (Genbank Accession No. AF043731) was provided by Prof. A. Friebe in pcDNA-Zeocin-Vector. PCR was carried out analogously to the cloning described in Kanadier et al., EMBO J. 2002 the PDE2-GAF chimera. With specific primers, a gene fragment hPDEδi- _M β was amplified, which codes for the PDE5-N terminus with the GAF-A domain and contains a BglII N-terminal and a Xbal C-terminal interface Fragment hPDE5 _349J ) ₅ o, which codes for the GAF-B domain and contains an X-terminal interface at the N-terminal and a Sall interface at the C-terminal. The two fragments were subcloned in the cloning vector pBluescriptil SK (-) via the XbaI interface to hPDE5 _{1- 50} together. A gene fragment CyaBI _{386 59} generated by PCR with the catalytic domain of the adenylate cyclase CyaBI (Genbank Accession No. D89623) was attached to the gene fragment hPDE5 _1-45 o via the Sall interface C-terminal. The N-terminal Sall interface of hPDE5ι-45o was cloned onto the C-terminal Xhol interface of CyaBI ₃₈₆ * ₅₉ and L386 was mutated to V by CyaBI. All cloning steps were done in E. coliXUblueMRF.

The gene for the PDE5-GAF chimera was cloned into the expression vector pQE30 (from Quiagen).

Example 2

Expression and purification of the polypeptide

For expression, the DNA construct obtained from Example 1, coding a PDE5 / CyaB1 chimera, was inserted into the PQE30 expression vector from Quiagen via the BamHI and Sall restriction enzyme interface of the MCS. The pQE30 vector with the gene for the PDE5-GAF chimera was retransformed in E.coli BL21 cells. The expression and purification of the protein was analogous to The QIAexpressionist ^® ", Fifth Edition, June 2003. In this case, the optimal protein yield in the expression conditions of induction with 25 uM IPTG, 16h incubation at 16 ° C and subsequent French Press Treatment of E. coli , achieved

Example 3

Carrying out the assay

The adenylate cyclase activity of the PDE5 / CyaB1 chimera is measured with and without the substance to be investigated. The adenylate cyclase activity is converted by converting a defined amount of ATP into cAMP and separating it over 2 column steps according to Salomon et al. (Salomon Y., Londos C, and Rondbell M .: A highly sensitive adenylate cyclase assay. 1974, Anal. Biochem., 58, 541-548). To detect the conversion, [α- ³² P] -ATP is used as the radioactive tracer and the resulting amount of [α- ³² P] -AMP is measured. ³ H-cAMP serves as an internal standard for the recovery rate. The incubation time should be between 1 and 120 min, the incubation temperature between 20 and 45 ° C, the Mg ^ cofactor concentration between 1 and 20 mM (corresponding amounts of Mn ²⁺ can also be used as a cofactor) and the ATP concentration between 0 , 5 µM and 5 mM. An increase in sales with substance compared to that without substance indicates a GAF agonistic effect. If the turnover is inhibited by the addition of substance, this indicates a GAF-antagonistic effect of the substance. GAF antagonism can also be measured by blocking the activation of the PDE5 / CyaB1 chimera by the native GAF ligand cGMP. For this purpose, the turnover is measured with increasing or fixed cGMP concentrations with and without substance. If the sales with substance are below those without substance, this indicates a GAF antagonism of the substance. One reaction batch contains: • 50 μl AC test cocktail (glycerol 43.5% (V / V), 0.1 M Tris / HCl pH 7.5, 20 mM MgCl ₂ ) • 40-xy μl enzyme dilution ( Depending on the activity, contains 0.1-0.3 μg PDE5 / CyaB1 chimera in 0.1% (W / V) aqueous BSA solution) • x μl substance • y μl cGMP • 10 μl 750 μM ATP start solution 16-30 kBq [α- ³² P] -ATP included. •

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

Each test approach is carried out twice. A test batch with water instead of enzyme was used as blank. The enzyme basal activity is determined with a test mixture without substance and cGMP. To separate ATP and cAMP, each sample is placed on glass columns with 1.2 g of Dowex-50WX4-400 and, after infiltration, washed with 3-4 ml of water. The mixture was then eluted with 5 ml of water on aluminum oxide columns (9 × 1 cm glass columns with 1.0 g of AL ₂ 0 ₃ 90 active, neutral) and these with 4 ml of 0.1 M TRIS / HCl, pH 7.5 in distillation vessels eluted with 4 ml of Ultima XR Gold scintillator. After thorough mixing, the liquid scintillation count was counted. The amounts of radioactively labeled cAMP and ATP used are counted as ³ H and ³² P totals directly in 5 ml of elution buffer and 4 ml of scintillator.

The conversion is calculated as enzyme activity using the following formula:

pmol [cAMP] substrate | uM] 10 ⁵ mg [protein] x min time [min] amount of protein [μg]

sample

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

If the% basal value for 100 μM substance is less than 50, this indicates a PDE5-GAF antagonist, while the% basal value greater than 200 indicates GAF agonists. In a test approach with cGMP, a PDE5-GAF antagonist is present if the% basal value is less than 90 when 100 μM of the possible PDE5-GAF antagonist is used.

After use, the columns were regenerated as follows: Dowex columns: 5 ml of 2N HCl, 2x 5 ml of water

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

Example 4

Carrying out the assay with test substances

Analogously to Example 3, the adenylate cyanase activity of the PDE5 / CyaB1 chimera was measured in the presence of cGMP and the known PDE5 inhibitors Sildenafii, Tadalafil and Vardenafil. The results are shown graphically in FIG. 6. None of the measured PDE5 inhibitors showed a PDE5-anatgonistic effect which works via binding to the GAF domain of PDE5.

Example 5

Perform the assay with a subsequent LANCE ^® cAMP assay

The adenylate cyclase activity of the PDE5 / CyaB1 chimera is measured with and without the substance to be investigated. The adenylate cyclase activity is determined by converting a defined amount of ATP into cAMP. To detect the cAMP formed by the PDE5 / CyaB1 chimera, the actual enzymatic reaction in the reaction mixture is followed by an antibody-based homogeneous assay (eg Lance cAMP, Perkin Elmer; HitHunter cAMP assay, DisvoverX; cAMP AlphaScreen, Perkin Elmer). The incubation time of the enzymatic Assays should be between 1 and 120 min, the incubation temperature between 20 and 45 ° C, the Mg ²⁺ cofactor concentration between 1 and 20 mM (appropriate amounts of Mn ²⁺ can also be used as cofactor) and the ATP concentration between 0 , 5 µM and 5 mM. An increase in sales with substance compared to that without substance indicates a GAF agonistic effect. If the turnover is inhibited by the addition of substance, this indicates a GAF-antagonistic effect of the substance. GAF antagonism can also be measured by blocking the activation of the PDE5 / CyaB1 chimera by the native GAF ligand cGMP (as shown below using a reaction approach). For this purpose, the turnover is measured with increasing or fixed cGMP concentrations with and without substance. If the sales with substance are below those without substance, this indicates a GAF antagonism of the substance.

For the assays with a subsequent LANCE cAMP assay, the following pipetting steps and incubations must be carried out in 384 well plates • 1 μl of the compound to be tested (-3 to -12 log M in DMSO) 1 μl cGMP (2 to 0.2 mM; activator of the GAF domain) 3 μl reaction buffer (22% Gylcerin, 50mM Tris-HCl pH 8.5, 10 mM MgCl2.1 mg / ml bovine serum albumin) 10 μl PDE5 / CyaB1 - Enzyme in reaction buffer (contains between 0.001 and 100 ng enzyme) 5 min. Incubation at 37 ° C 5 μl ATP (300 μM) 15 min Incubation at 37 ° C Add 10 μl AlexaFluor647-anti-cAMP antibody solution (Perkin Elmer, LANCE cAMP Assay) 30-60 min incubation Add 10 μl LANCE Eu- W8044 labbeled streptavidin solution mixed with biotin cAMP ((Perkin Elmer, LANCE cAMP assay) 30 min - 24 h incubation measurement at an excitation wavelength of 340 +/- 10 nm and an emission wavelength of 665 +/- 10 nm and 615 +/- 10 nm (in LANCE or HTRF mode)

Each test approach is carried out twice. A test batch without adding ATP as a substrate was used as blank. The enzyme basal activity is determined using a test mixture without substance and cGMP.

An exemplary measurement using different amounts of enzyme is shown in FIG. 7.

figure description

Figure 1: Amino acid sequence of the PDE5 / CyaB1 chimeric

Figure 2: PDE5 / CyaB1 chimeric cDNA sequence

3: Protein sequence of the PDE5 / CYAB1 chimera after purification, italics = purification day from the expression vector (PQE30 from Quiagen); bold = N-terminus with PDE-5 GAF domains; bold and underlinedπ = GAFA domain and GAFB domain; V386 was mutated to insert the cloning interface from L386; underlined = C-terminus of CyaBI with catalytic domain.

Fig. 4: Schematic representation of the chimeric PDE5 / CYAB1 polypeptide

FIG. 5 Activation of the PDE5 / CyaB1 chimera by cydic nucleotides. If the assay is carried out with cGMP or cAMP as the substances to be tested, the dose-response curve according to FIG. 5 results. The PDE5 / CyaB1 chimera is approximately 7.8-fold by 100 μM cGMP activated. This corresponds to a% basal value of 780 and shows that cGMP is a PDE5-GAF agonist. cAMP has no activating effect and has a% basal value of approximately 100, i.e. it is neither a GAF agonist nor an antagonist.

Fig. 6 Carrying out the assay with Sildenafii, Tadalafil and Vardenafil

Carrying out the assay by measuring the adenylate cyanase activity of the PDE5 / CyaB1 chimera in the presence of the known PDE5 inhibitors Sildenafii, Tadalafil and Vardenafil. The results are shown graphically in FIG. 6. None of the measured PDE5 inhibitors showed a PDE5-anatgonistic effect which acts via a binding to the GAF domain of PDE5.

Fig. 7 is an exemplary measurement with the Lance® assay as a read out when using different amounts of enzyme.

Claims

claims

1. The polypeptide is functionally linked (a) the GAF _A domain and GAF _B domain of a human phosphodiesterase 5 (PDE5) or their functionally equivalent variants and (b) the catalytic domain of an adenylate cyanase or their functionally equivalent variants.

2. Polypeptide according to claim 1, characterized in that the phosphodiesterase 5 (PDE5) is selected from the group PDE5A1, PDE5A2, PDE5A3, PDE5A4 or their respective functionally equivalent variants.

3. Polypeptide according to claim 1 or 2, characterized in that the phosphodiesterase 5 (PDE5) has the isoform PDE5A1.

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

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

6. A polypeptide according to any one of claims 1 to 5, characterized in that the GAF _ß - domain having an amino acid sequence comprising the amino acid sequence SEQ. ID. NO. Domain comprises - _ß 8 and the characteristic of a GAF: a group derived from this sequence by substitution, insertion or deletion of amino acid sequence or 8 which has an identity of at least 90% at the amino acid level with the sequence SEQ ID NO.

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

8. Polypeptide according to one of claims 1 to 7, characterized in that the functionally linked GAF _A domain and GAF _B domain of a human phosphodiesterase 5 (PDE5) or its functionally equivalent variants has an amino acid sequence containing the amino acid sequence SEQ. ID. NO. 10 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which has an identity of at least 70% at the amino acid level with the sequence SEQ ID NO: 10 and the regulatory property of the GAF domain of a human phosphodiesterase 5 (PDE5), where the contained amino acid sequences of the GAF _A domain, SEQ. ID. NO. 6 and the GAF _B domain, SEQ. ID. NO. 8 vary by substitution, insertion or deletion of amino acids by a maximum of 10%.

9. Polypeptide according to one of claims 1 to 8, characterized in that the functionally linked GAF _A domain and GAF _B domain of a human phosphodiesterase 5 (PDE5) or its functionally equivalent variants has an amino acid sequence selected from the group

(a) N-terminus from human PDE5A1 to amino acid E513 or (b) SEQ. ID. NO. 10

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

11. Polypeptide according to one of claims 1 to 10, characterized in that the adenylate cyclase is an adenylate cyclase selected from group (a) adenylate cyclase from Anabaena sp. PCC 7120 or its functionally equivalent variant, (b) adenylate cyclase from Anabaena variabili ATTC 29413 or its functionally equivalent variant, (c) adenylate cyclase from Nostoc punctiform PCC 73102 or its functionally equivalent variant, (d) adenylate cyclase from TrichodesellMS10 equivalent or the trichodesmium equivalent erytum Variant (e) adenylate cyclase from Bdellovibrio bacteriovorus HD100 or its functionally equivalent variant, (f) adenylate cyclase from Magnetococcus sp. MC-1 or its functionally equivalent variant

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

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

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

(a) C-terminus of CyaBI from amino acid L386 to K859, where L386 of CyaBI is replaced by V386 or (b) SEQ. ID. NO. 12

15. Polypeptide according to one of claims 1 to 14, containing the amino acid sequence SEQ. ID. NO. 1 or SEQ. ID. NO.4 or a sequence derived from these sequences by substitution, insertion or deletion of amino acids, which has an identity of at least 70% at the amino acid level with the sequence SEQ ID NO: 1 or 4 and the regulatory properties of the GAF domain of a human phosphodiesterase 5 (PDE5) and has the catalytic properties of an adenylate cyclase, the amino acid sequences contained in the GAF _A domain, SEQ. ID. NO. 6, the GAF _ß domain, SEQ. ID. NO. 8 and the catalytic domain of adenylate cyanase, SEQ. ID. NO. 12 vary by substitution, insertion or deletion of amino acids by a maximum of 10%.

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

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

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

19. Polynucleotide according to claim 18, comprising as partial sequences (a) SEQ. ID. NO. 5 or a nucleic acid sequence with the nucleic acid sequence SEQ. ID. NO. 5 hybridized under continuous conditions and (b) SEQ. ID. NO. 7 or a nucleic acid sequence with the nucleic acid sequence SEQ. ID. NO. 7 hybridized under constant conditions and (c) SEQ. ID. NO. 11 or a nucleic acid sequence with the nucleic acid sequence SEQ. ID. NO. 11 hybridized under constant conditions.

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

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

22. Recombinant plasmid vector comprising a polynucleotide according to one of claims 18 to 22.

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

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

25. A method for identifying a modulator of a human phosphodiesterase 5 (PDE5) comprising the steps (a) contacting a possible modulator of a human phosphodiesterase 5 (PDE5) with a polypeptide according to one of claims 1 to 17 and (b) determining whether the possible Modulator changes the adenylate cyclase activity of the polypeptide according to any one of claims 1 to 17 compared to the absence of the possible modulator.

26. The method according to claim 25, wherein in step (a) in addition to the possible modulator of a human phosphodiesterase 5 (PDE5) cGMP is contacted with a polypeptide according to one of claims 1 to 17.

27. The method according to claim 25 or 26, characterized in that the determination of the adenylate cyanase activity is carried out by measuring the conversion of radioactive or fluorescence-labeled ATP.

28. The method according to claim 25 to 27, characterized in that in the presence of the modulator a decrease in the adenylate cyanase activity is measured in comparison to the absence of the modulator and the modulator is a PDE5 antagonist.

29. The method according to claim 25 to 28, characterized in that in the presence of the modulator an increase in the adenylate cyanase activity is measured in comparison to the absence of the modulator and the modulator is a PDE5 agonist.

30. The method according to any one of claims 25 to 29, characterized in that to exclude direct modulators of the catalytic domain of adenylate cyclase, a method according to one of claims 25 to 27 is carried out using a polypeptide which has the catalytic domain of an adenylate cyclase and is not functional Has GAF domain of a human phosphodiesterase 5 (PDE5).

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

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