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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • 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
• • 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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

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 mediated 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 5HT2 receptors have been very little studied.
• the 5HT2 receptors act through phospholipase C, and are responsible for many physiological activities of serotonin at the central and peripheral levels.
• They are involved in the contraction of blood vessels and in changes in the morphology of platelets; at the level of the central nervous system, they act on the neurons sensitization to tactile stimuli and to mediate the hallucinogenic effects of lysergic diethylamide and related phenylisopropylamines.
• 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 5HT2C, as well as from the genetic material allowing their expression or their identification. The pharmacological study of these receptors shows that they could account for the atypical effects of serotonin observed previously.
• 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 conferring on it new pharmacokinetic and / or biological properties.
• derivatives resulting from an addition mention may, for example, be made of chimeric polypeptides comprising an additional heterologous part linked at one end.
• the term derivative also includes polypeptides homologous to the polypeptide SEQ ID n ° 2, 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.
• Example 5 describes a derivative according to the invention (truncated form).
• 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 5HT2C polypeptide 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.
• This polypeptide comprises 479 amino acids, and has a calculated molecular weight of 56,508 Daltons.
• polypeptides of the invention can be obtained by expression in a cellular host of a nucleotide sequence as described below, by chemical synthesis, on the basis of the sequence SEQ ID No. 2 using the techniques known in the art. skilled in the art, or by a combination of these techniques.
• polypeptides of the invention as defined above are designated by 5HT2C polypeptides.
• the present invention also relates to any nucleotide sequence coding for a 5HT2C polypeptide. More preferably, it is a sequence chosen from:
• sequences derived from sequences (a) and (b) due to the degeneration of the genetic code 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 5HT2C 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. As regards integrative vectors, 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 5HT2C 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 Hansenula.
• 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 hosts it is preferred to use the following bacteria E.coli, Bacillus, or Streptomyces.
• nucleotide sequences of the present invention are also usable in the pharmaceutical field, either for the production of antisense sequences usable in the context of gene therapy, or also for the production of probes allowing the detection, by hybridization experiments, of the expression of serotonergic receptors in biological samples and the detection of genetic anomalies (polymorphism, mutations) or outliers. Inhibition of the expression of certain genes by antisense sequences has proven to be a promising strategy in controlling the activity of a gene.
• Antisense sequences are DNA sequences coding for complementary RNAs and, therefore, capable of specifically hybridizing with a given mRNA, inhibiting its translation into protein.
• the subject of the invention is therefore the antisense sequences capable of at least partially inhibiting the production of 5HT2C polypeptides as defined above.
• 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 sequences can be obtained from the sequence SEQ ID No. 1, by fragmentation, etc. or by chemical synthesis.
• the invention also allows the production of nucleotide probes, synthetic or not, capable of hybridizing with the nucleotide sequences defined above which code for 5HT2C polypeptides 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 5HT2C serotoninergic receptor, or also 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 5HT2C receptors.
• probes can also be used for the detection and isolation of homologous nucleic acid sequences coding for 5HT2C polypeptides as defined above, from other cellular sources and preferably from cells of human origin, as well as illustrated in the examples.
• the probes of the invention generally contain at least 10 bases, and they can contain up to the entire sequence SEQ ID No. 1 or its complementary strand. Preferably, these probes are, prior to their use, marked. For this, different techniques known to those skilled in the art can be used (radioactive, enzymatic labeling, etc.). Conditions hybridization in 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 5HT2C 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.
• the cells thus obtained can be used to measure the capacity of different molecules to behave as a ligand or as a 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 5HT2C polypeptides of the invention, according to which the following steps are carried out:
• a molecule or a 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 the interaction between said polypeptide of the invention and said molecule in the event that it has an affinity for said polypeptide, and, - the molecules linked to said polypeptide of the invention are detected and / or isolated.
• this method of the invention is suitable for the detection and / or isolation of agonists and antagonists of serotonin for the 5HT2C polypeptides.
• Another object of the invention relates to a method for detecting and / or isolating modulators of the 5HT2C 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, in the presence of 5HT, under conditions allowing interaction between said polypeptide of the invention and 5HT, and,
• 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 5HT2C receptors.
• the invention also relates to any medicament comprising as active principle at least one molecule acting on a 5HT2C polypeptide of the invention.
• the molecule is a ligand or a modulator identified and / or isolated according to the method described above.
• Figure 1 Scatchard curve of the results of the saturation binding experiments of [ 125 I] -DOI to the membranes of Cos-7 cells expressing the 5HT2C receptor.
• Figure 2 Demonstration of homologous sequences by PCR on total RNA (10 ⁇ g) of different mouse tissues: line 1: RNA of the LMTK cell line, line 2: RNA of 10-day embryos, line 3: RNA of testes, line 4: RNA of the 3T6 cell line, line 5: RNA of the MBK cell line, line 6: RNA of liver, line 7: RNA of brain, line 8: RNA of heart, line 9: RNA of intestine, line 10: spleen RNA, line 11: kidney RNA, line 12: total brain RNA.
• Table 1 Pharmacological profile of the whole (N) or truncated (T) 5HT2C receptor.
• the results correspond to competitive experiments for the binding of [ 125 I] -DOI to the membranes of Cos-7 cells expressing the 5HT2C receptor.
• the values, expressed in pKD (-log mol / l) are the result of at least three separate experiments, carried out in triplicate.
• RNA polymerase I DNA polymerase I
• T4 polynucleotide kinase T4 DNA ligase
• T3 or T7 RNA polymerases were provided by New England Biolabs (Biolabs), Bethesda Research Laboratories (BRL ), Boehringer-Mannheim or Stratagene and are used according to supplier recommendations.
• 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 according to supplier's recommendations.
• the protruding 5 'ends are filled with the Klenow fragment of E. coli DNA Polymerase I according to the supplier's specifications.
• the destruction of the prominent 3 'ends is carried out in the presence of the DNA polymerase of phage T4 used according to the manufacturer's recommendations.
• the destruction of the protruding 5 ′ ends is carried out by gentle treatment with nuclease S1.
• 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].
• the normal stringency conditions are generally as follows: hybridization: 5 ⁇ SCC in the presence of 5 ⁇ Denhart's at 65 ° C; washing: 0.5 ⁇ SSC at 65 ° C.
• AGAACTAGTGGTACCCA G, A) IGT (G, A) TAIACIA (G, A) IGG (G, A) TT
• AGAACTAGTGGTACCC (G, C) (A, T) (G, A) (G, A) (G, A) CAIAC (G, A) TAICC (G, A, T) ATCCA
• a cloning multisite was added to the 5 'end of the oiligonucleotides, to allow subsequent subcloning of the amplified fragment.
• the PCR reactions were carried out as follows: 1 ⁇ g of mouse genomic DNA was denatured for 1 minute at 94 ° C, maintained for 2 minutes at 55 ° C, then subjected to 20 cycles of amplification at 72 ° C in the presence of 10 units of Taq polymerase (Cetus), 3 mM MgCl 2 , and 1 ⁇ g of each of the oligonucleotides (i) and (ii).
• This clone was designated NP75.
• the insert carried by the clone NP75 was sequenced on the 2 strands using the dideoxynucleotide technique using synthetic oligonucleotides. Analysis of the sequence obtained reveals the presence of an open reading phase coding for a protein of 479 amino acids (SEQ ID No. 1 and 2). 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 potential site of N- glycosylation, and the suspected cytoplasmic domain contains the consensus sites for phosphorylation by protein kinases C and A. In addition, the presence of 19 serine or threonine residues in the 121 C-terminal amino acids seems to indicate that this region is involved in desensitization of the receptor by kinases.
• the sequence of the 5HT2C receptor isolated above was compared with the sequences of the receptors coupled to the following G proteins: 5HT1A, 5HT1B, 5HT1C and 5HT2. These experiments revealed a homology in the transmembrane domains I to VII with the receptors 5HT1C and 5HT2, superior to the homology encountered with the receptors 5HT1A and 5HT1B. These results suggest that the receptors of the invention belong to the 5HT2 subtype. This is also in agreement with the presence of 2 introns in the gene coding for the 5HT2C receptor (not shown in the sequence).
• 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 cDNA coding for the 5HT2C receptor was isolated in the form of an XbaI-EcoRI fragment of 1372 bp, then inserted into the corresponding sites of the 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 p513NP75 was then used (10 ⁇ 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].
• [ 125 I] -DOI (dimethoxyphenyl isopropylamine) was first used alone, to detect the expression of the gene encoding the receptors of the invention in the membranes of the recombinant cells. For this, 100 ⁇ l of membrane suspension (50 ⁇ g / ml of protein) were incubated for 30 minutes at 30 ° C. in the presence of the radioactive ligand (50 ⁇ l) and 50 ⁇ l of 50 mM Tris-HCl buffer (pH 7.4 ), 150 mM NaCl, 10 mM MgCl 2 , final volume: 200 ⁇ l.
• the reaction is then stopped by adding an ice-cold iso-osmotic solution, then by filtration through Whatman GF / B filters, followed by 4 rinses in 5 ml of the above-frozen buffer.
• the filters are then dried and the radioactivity is measured by counting ( ⁇ counter).
• the non-specific binding determined in the presence of 10 ⁇ M of cold DOI, represented approximately 30% of the total mass.
• the results obtained show that the DOI binds specifically to the membranes of the transfected cells, and not to the membranes of the control cells (transfected with the plasmid pSG5). These cells therefore clearly express a functional serotonin receptor.
• the affinity of DOI for transfected cells is high and the binding is saturable ( Figure 1).
• the apparent KD is 25.8 +/- 0.54 nM, and the apparent Bmax varies between 14.8 and 21.0 pmol / mg of protein, depending on the transformation efficiency.
• the nucleotide sequence SEQ ID No. 1 was then used to demonstrate homologous sequences from other tissues by PCR.
• tissues used for the search for homologous sequences are the following of murine origin: intestine, heart, brain, kidney, spleen and liver.
• the probe (iv) corresponds to position 85 on SEQ ID No. 1 and the probe (v) at position 547 (complementary strand).
• RNAs were prepared from the tissues indicated above according to the technique described by Cathala et al. (DNA 2 (4) (1983). 10 ⁇ g of these RNAs were subjected to reverse transcription in the presence of 13 units of AMV reverse transcriptase, 5 units of Taq polymerase and 1 ⁇ g of probes (iv) and (v ), for 15 minutes at 50 ° C., then amplified (20, 25 and 30 cycles) (Maroteaux et al., Proc.Natl.Acad.Sci. USA 89 (1992) 3020). As internal standard, the primers corresponding to ribosomal elongation factor EF1A mRNA was used, under the same conditions as probes (iv) and (v).
• the products thus obtained were then transferred to nitrocellulose filters and hybrids under the following conditions:
• the probe used for the hybridization had the following sequence:
• Hybridization was carried out under high stringency conditions: 50 ° C, in 20 mM sodium phosphate buffer (pH 6.5) containing 5 ⁇ SSC, 1 ⁇ Denhardt's, 0.1% SDS and 100 ⁇ g / ml of tRNA. Washings were carried out at 50 ° C. in a 1 ⁇ SSC, 0.1% SDS buffer. This study made it possible to highlight specific DNA fragments which are homologous in the intestine, the heart, the kidneys and the brain (FIG. 2).
• RNA comes from the use of an alternative splicing donor at the end of exon 2, leading to the elimination of bases 550-568 from the nucleic sequence SEQ ID No. 1. This causes a phase change and introduces a stop codon into the RNA, producing a truncated protein after the third trans-membrane domain.
• This alternative form of mRNA first isolated from brain RNA, is differentially expressed depending on the tissue. The relative rate of expression of truncated RNA compared to complete RNA is maximum in the heart of mice, where it represents more than 50%, whereas it is only 15% in the intestine.
• ATC AAA AAG CCA ATT CAG GCC AAT CAG TGC AAC ACC CGG GCT ACT GCA 531 Ile Lys Lys Pro Ile Gln Ala Asn Gln Cys Asn Thr Arg Ala Thr Ala

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