Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
  • G01N33/9406—Neurotransmitters
  • G01N33/942—Serotonin, i.e. 5-hydroxy-tryptamine

Definitions

• the present invention relates to new polypeptides and the genetic material allowing their expression. More particularly, it relates to new polypeptides having a serotonergic receptor activity.
• Serotonin is a neuromodulator capable of inducing and modulating a wide variety of behaviors such as sleep, appetite, locomotion, sexual activity or even vascular contraction. It is recognized that the activity of serotonin is affected by its interaction with receptors, designated serotonergic receptors or 5-HT receptors (for 5-hydroxytryptamine). Molecular biology studies as well as pharmacological studies have revealed that there are a large number of 5-HT receptor subtypes. The 5-HT receptors which have been described until now belong either to the family of receptors linked to ion channels (5-HT3 receptors), or to the family of receptors which interact with G proteins and which have seven transmembrane domains.
• 5-HT receptors which have been described until now belong either to the family of receptors linked to ion channels (5-HT3 receptors), or to the family of receptors which interact with G proteins and which have seven transmembrane domains.
• the 5-HT1 receptors comprising the mammalian subtypes 5HT1A, 5HT1B and 5HT1D as well as three 5HT Drosophila receptors; and 5HT2 receptors including the 5HT2 and 5HT1C subtypes.
• 5HT4 receptors are probably not the only 5HT receptors existing, since pharmacological studies have revealed other subtypes such as 5HT4 receptors as well as certain receptors related to the 5HT1 subtype ("5HT1 like" receptors). . In addition, additional molecular biology studies have also revealed heterogeneities within the 5HT1B / 1D subtypes.
• the present invention results from the discovery of new polypeptides having a serotonergic receptor activity. Although belonging to the family of receptors which interact with G proteins, these new polypeptides differ from the serotonergic receptors already described (5HT1, 5HT2, 5HT3 and 5HT4) from the structural point of view as from the pharmacological point of view. More particularly, the invention results from the isolation and characterization of these new polypeptides, designated 5HT5b, as well as genetic material allowing their expression or their identification.
• a first object of the invention therefore resides in polypeptides comprising all or part of the peptide sequence SEQ ID No. 2 or a derivative thereof.
• the term derivative designates any molecule obtained by modification of genetic and / or chemical nature of the peptide sequence SEQ ID No. 2.
• modification of genetic and / or chemical nature one can hear any mutation, substitution , deletion, addition and / or modification of one or more residues.
• Such derivatives can be generated for different purposes, such as in particular that of increasing the affinity of the peptide for its ligand (s), that of improving its production levels, that of increasing its resistance to proteases, that of increasing and / or modifying its activity, or that of giving it new pharmacokinetic and / or biological properties.
• derivatives resulting from an addition mention may be made, for example, of the chimeric polypeptides comprising an additional heterologous part linked to one end.
• the term derivative also includes polypeptides homologous to polypeptide SEQ ID No. 2, derived from other cellular sources and in particular from cells of human origin, or from other organisms, and having an activity of the same type. Such homologous polypeptides can be obtained by hybridization experiments as described in the examples.
• the polypeptides of the invention are polypeptides having the capacity to bind serotonin. Even more preferably, they are polypeptides having a serotonergic receptor activity. Still according to a preferred mode, the polypeptides of the invention are capable of being recognized by antibodies recognizing the complete SEQ ID No. 2 peptide sequence.
• a particular embodiment of the invention is represented by the polypeptide 5HT5b comprising the entire peptide sequence SEQ ID No. 2. As indicated in the examples, this polypeptide can be expressed in different cell types to form a functional serotonergic receptor.
• polypeptides of the invention can be obtained by expression in a cellular host of a nucleotide sequence as described below, by synthesis chemical, based on the sequence SEQ ID No. 2 using the techniques known to those skilled in the art, or by a combination of these techniques.
• polypeptides of the invention as defined above are designated by polypeptides 5HT5b.
• the present invention also relates to any nucleotide sequence coding for a 5HT5b polypeptide. More preferably, it is a sequence chosen from:
• the different nucleotide sequences of the invention can be of artificial origin or not. They can be genomic sequences, cDNA, RNA, hybrid sequences or synthetic or semi-synthetic sequences. These sequences can be obtained for example by screening DNA libraries (cDNA library, genomic DNA library) by means of probes prepared on the basis of the sequence SEQ ID No. 1. Such libraries can be prepared for starting from cells of different origins by conventional molecular biology techniques known to those skilled in the art.
• the nucleotide sequences of the invention can also be prepared by chemical synthesis, in particular according to the phosphoramidite method, or also by mixed methods including chemical or enzymatic modification of sequences obtained by screening of libraries.
• the nucleotide sequences of the invention can be used for the production of the 5HT5b polypeptides as defined above.
• the part coding for said polypeptide is generally placed under the control of signals allowing its expression in a cellular host.
• the choice of these signals can vary depending on the cell host used.
• the nucleotide sequences of the invention can be part of a vector, which can be autonomous or integrative replication. More particularly, autonomously replicating vectors can be prepared using autonomously replicating sequences in the chosen host.
• integrative vectors s these can be prepared for example by using sequences homologous to certain regions of the host genome, allowing, by homologous recombination, the integration of the vector.
• the cellular hosts which can be used for the production of the 5HT5b polypeptides of the invention by the recombinant route are both eukaryotic and prokaryotic hosts.
• suitable eukaryotic hosts there may be mentioned animal cells, yeasts, or fungi.
• yeasts mention may be made of yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansen la.
• nucleotide sequences of the present invention can also be used in the pharmaceutical field, either for the production of antisense sequences which can be used in the context of gene therapy, or else for the production of probes allowing detection, by hybridization experiments, the expression of serotonergic receptors in biological samples and the detection of genetic anomalies (polymorphism, mutations) or outliers.
• Antisense oligonucleotides are small oliogonucleotides, complementary to an mRNA, and therefore capable of specifically hybridizing with it, inhibiting its translation into protein.
• the subject of the invention is therefore the antisense oligonucleotides capable of at least partially inhibiting the production of 5HT5b polypeptides as defined above.
• the invention also relates to the antisense sequences capable of at least partially inhibiting the production of 5HT5b polypeptides.
• Antisense sequences produce transcripts in the target cell that are complementary to cellular mRNAs.
• oligonucleotides or sequences can consist of all or part of the nucleotide sequences defined above. They are generally sequences or fragments of sequences complementary to sequences coding for peptides of the invention. Such oligonucleotides can be obtained from the sequence SEQ ID No. 1, by fragmentation or by chemical synthesis, etc. As indicated above, the invention also allows the production of nucleotide probes, synthetic or not, capable of hydriding with the nucleotide sequences defined above which code for polypeptides 5HT5b of the invention, or with the corresponding mRNAs .
• probes can be used in vitro as a diagnostic tool, for the detection of the expression of a 5HT5b serotoninergic receptor, or even for the detection of genetic anomalies (poor splicing, polymorphism, point mutations, etc.). Given the multiple activities of serotonin, the probes of the invention can thus make it possible to identify neurological, cardiovascular or psychiatric conditions as being linked to the 5HT5b receptors. These probes can also be used for the detection and isolation of homologous nucleic acid sequences coding for 5HT5b polypeptides as defined above, from other cellular sources and preferably from cells of human origin.
• the probes of the invention generally contain at least 10 bases, and they can contain up to the entire sequence SEQ ID No.
• these probes are, prior to their use, marked.
• different techniques known to those skilled in the art can be used (radioactive, enzymatic labeling, etc.).
• the hybridization conditions under which these probes can be used are indicated in the general cloning techniques below as well as in the examples.
• Another subject of the invention relates to recombinant cells capable of expressing on their surface a 5HT5b polypeptide as defined above. These cells can be obtained by introducing a nucleotide sequence as defined above coding for a polypeptide of the invention, then culturing said cells under conditions of expression of said sequence.
• the recombinant cells according to the invention can be both eukaryotic and prokaryotic cells.
• eukaryotic cells which are suitable, mention may be made of animal cells, yeasts, or fungi.
• yeasts mention may be made of yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula.
• animal cells mention may be made of COS, CHO, C127, NIH-3T3 cells, etc.
• the mushrooms there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp.
• prokaryotic cells it is preferred to use the following bacteria E.coli, Bacillus, or Streptomyces.
• the cells thus obtained can be used to measure the capacity of different molecules to behave as a ligand or as modulator of the activity of the polypeptides of the invention. More particularly, they can thus be used in a process for the detection and isolation of ligands or of modulator of the activity of the polypeptides of the invention, and, more preferably, of agonists and antagonists of serotonin .
• Another object of the invention therefore relates to a process for detecting and / or isolating ligands of the 5HT5b polypeptides of the invention, according to which the following steps are carried out:
• a molecule or mixture containing different molecules, possibly unidentified is brought into contact with a recombinant cell as described above expressing on its surface a polypeptide of the invention under conditions allowing interaction between said polypeptide of the invention and said molecule if the latter has an affinity for said polypeptide, and,
• this method of the invention is suitable for the detection and / or isolation of agonists and antagonists of serotonin for the 5HT5b polypeptides.
• Another subject of the invention relates to a process for detecting and / or isolating modulators of the 5HT5b polypeptides of the invention, according to which the following steps are carried out: - a molecule or a mixture containing different is brought into contact molecules, possibly unidentified, with a recombinant cell as described above expressing on its surface a polypeptide of the invention, in the presence of 5HT, under conditions allowing the interaction between said polypeptide of the invention and 5HT , and, - the molecules capable of modulating the activity of 5HT on said polypeptide of the invention are detected and / or isolated.
• Another object of the invention relates to the use of a ligand or of a modulator identified and / or obtained according to the process described above as a medicament.
• ligands or modulators can indeed make it possible to treat certain neurological, cardiovascular or psychiatric affections linked to the 5HT5b receptors.
• the invention also relates to any medicament comprising as active principle at least one molecule acting on a 5HT5b polypeptide of the invention.
• the molecule is a ligand or a modulator identified and / or isolated according to the method described above.
• Table 1 Pharmacological profile of the 5HT5b receptor. The results correspond to competitive experiments for the binding of [ 125 I] -LSD to the membranes of Cos-7 cells expressing the 5HT5b receptor.
• the numbers in parentheses correspond to the number of independent experiments carried out, each point being carried out in triplicate.
• Table 2 Percentages of peptide sequence homology between the 5HT5b receptor (SEQ ID No. 2) and other receptors of the receptor family coupled to G proteins. The homologies were calculated on the conserved sequences: transmembrane domain and its connection loops.
• Fi ure 1 Saturation curve of [ 125 I] -LSD at the membranes of Cos-7 cells expressing the 5HT5b receptor. The membranes were incubated with ligand concentrations ranging from 50 p to 1.25 nM, with or without 10 ⁇ M of 5HT. The specific link is shown. The insert represents the Scatchard analysis of the results.
• the DNA fragments are separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of DNA.
• phage T4 ligase Biolabs
• the filling of the prominent 5 ′ ends is carried out by the Klenow fragment of DNA Polymerase I of E. coli (Biolabs) according to the supplier's specifications.
• the destruction of the protruding 3 ′ ends is carried out in the presence of the DNA polymerase of phage T4 (Biolabs) used according to the manufacturer's recommendations.
• the destruction of the protruding 5 ′ ends is carried out by gentle treatment with the nuclease Si.
• PCR Polymerase-catalyzed Chain Reaction, Saiki R.K. et al., Science 230 (1985) 1350-1354; Mullis K.B. and Faloona F.A., Meth. Enzym. J ⁇ 5 (1987) 335-350] is carried out using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the manufacturer's specifications. Verification of the nucleotide sequences is carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.
• the normal stringency conditions are generally as follows: hybridization: 3 x SCC in the presence of 5 x Denhart's at 65 ° C; washing: 0.5 x SSC at 65 ° C.
• AGAACTAGTGGATCCAA (A / G) AA (A / G / C / T) GG (A / G / C / T) A (A / G) CCA (A / G) CA
• CTTGATATCGAATTCGA (T / C) (A / G) T (A / G / C T) CT (A / G / Cy) TG (C /) TG (C / T) A C
• GGTATCGATAAGCTTAT (C ⁇ , / A) GC (C /) CT (A / G / CT) GA (C , ) (C / A) G (A / G / C / T) TA
• PCR reactions were carried out as follows: 5 ⁇ g of adult mouse brain RNA were subjected to a reverse transcription reaction in the presence of 500 ng of oligonucleotide (i) and 200 units of MMLV reverse transcriptase (BRL). Half of the product of this reaction was then subjected to 30 amplification cycles in the presence of 5 units of Taq polymerase (Cetus) and 1 ⁇ g of oligonucleotide (i) and of oligonucleotide (ii). 1/20 of the product of this reaction was then subjected to 30 additional amplification cycles in the presence of the oligonucleotides (i) and (iii).
• the products thus obtained were digested with the enzymes BamHI and HindIII, inserted at the corresponding sites of the plas ide Bluescript (Stratagene), and sequences.
• ⁇ NS and carried by the plasmid pNS, contained a 2.1 kb insert. This phage was isolated, and its insert was then introduced into the Bluescript plasmid.
• sequence SEQ ID No. 1 The sequence of this fragment was determined on the 2 strands using the dideoxynucleotide technique using synthetic oligonucleotides.
• the sequence thus obtained corresponds to sequence SEQ ID No. 1. It shows that the isolated cDNA carries an open reading phase of 370 amino acids. Furthermore, the hydrophobicity analysis shows that this protein carries seven hydrophobic domains, a peculiarity encountered in members of the family of receptors coupled to G proteins. The N-terminal end also contains 1 N-glycosylation site , and the suspected cytoplasmic domain contains the consensus sites of phosphorylation by protein kinases C and A.
• a genomic clone encoding the 5HT5b receptor was also isolated, by screening a genomic library using the sequence SEQ ID No. 1 as probe.
• the library was obtained by partial digestion with Sau3A of the genomic DNA of mouse embryo stem cells, then insertion into the lambda phage EMBL3.
• the fragments obtained were subcloned into the Bluescript plasmid and partially sequenced. Analysis of these sequences reveals the presence of an intron, located in the middle of the 3rd cytoplasmic loop.
• the sequence of the 5HT5b receptor isolated above was compared with the sequences of the receptors coupled to the following G proteins: 5HT1B, 5HT1D, 5HT5A, 5HT1A, 5HT-dro2A, 5HT-drol, ⁇ 2, D2, ⁇ l, Dl, H2, 5HT1C and 5HT2.
• Example 1 The cDNA fragment isolated in Example 1 was inserted into a eukaryotic expression vector, which was used to transfect Cos-7 cells. The membranes of the transfected cells obtained were then prepared and tested for their capacity to bind certain labeled serotonergic ligands.
• the 2.1 kb cDNA encoding the 5HT5b receptor was isolated from the plasmid pNS in the form of an EcoRI-.XhoI fragment, then inserted at the sites correspondents of vector p513.
• the vector p513 is derived from the vector pSG5 [Green et al., Nucl. Acids Res. 16 (1988) 369] by adding a multisite of cloning.
• the recombinant vector thus obtained, designated p513NS was then used (20 ⁇ g per 10 cm plate) to transfect the Cos-7 cells in the presence of calcium phosphate.
• the recombinant cells are harvested and the membranes are prepared according to the technique described by Amlaiky and Caron [J. Biol. Chem. 260 (1985) 1983]. Saturation binding and competition experiments were then carried out on these membranes in the presence of different radiolabelled ligands (cf. Table 1). For this, the membrane samples (10-20 ⁇ g of proteins) were incubated for 10 minutes at 37 ° C. in the presence of the ligand in a final volume of 250 ⁇ l of 50 mM Tris-HCl buffer (pH 7.4).
• the reaction is then stopped by vacuum filtration on Whatman GF / C glass fiber filters, and rinsing 4 times with 4 ml of 50 mM Tris-HCl buffer (pH 7.4).
• the non-specific binding was determined in the presence of 10 ⁇ M of 5HT. Radioactivity was measured with a ⁇ counter.
• the nucleotide sequence SEQ ID No. 1 was then used to demonstrate homologous sequences from other tissues by in situ hybridization.
• the in situ hybridization experiments were carried out on cryostatic sections of the adult mouse brain (approximately 8 weeks) according to the technique described by Associates et al. [EMBO J. 2 (1983) 617].
• the probe used for these experiments is a single-stranded RNA obtained by transcription in the presence of T3 polymerase, of [ 35 S] -CTP using as matrix the plasmid pNS linearized by Xhol.
• the human 5HT5b receptor was cloned.
• a human genomic DNA library was prepared from the placenta, by partial digestion with the enzyme Mbol, separation on salt gradients, and under cloning in the vector Lamda GEM 12 linearized by BamHI (host bacterium: TAP 90).
• the library thus obtained was then screened using the sequence SEQ ID No. 1.
• the DNA fragments which hybridize with this probe were isolated, subcloned in a Bluescript plasmid, amplified, then sequenced in both directions according to the technique. dideoxynucleotide.

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