EP1068336A2 - Membranemetalloprotease nepii - Google Patents

Membranemetalloprotease nepii

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Publication number
EP1068336A2
EP1068336A2 EP99911898A EP99911898A EP1068336A2 EP 1068336 A2 EP1068336 A2 EP 1068336A2 EP 99911898 A EP99911898 A EP 99911898A EP 99911898 A EP99911898 A EP 99911898A EP 1068336 A2 EP1068336 A2 EP 1068336A2
Authority
EP
European Patent Office
Prior art keywords
nep
polypeptide
seq
tissue
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99911898A
Other languages
German (de)
French (fr)
Inventor
Tanja Ouimet
Claude Gros
Christiane Rose
Marie-Chantal Bonhomme
Patricia Facchinetti
Jean-Charles Schwartz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut National de la Sante et de la Recherche Medicale INSERM
Original Assignee
Institut National de la Sante et de la Recherche Medicale INSERM
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Publication date
Application filed by Institut National de la Sante et de la Recherche Medicale INSERM filed Critical Institut National de la Sante et de la Recherche Medicale INSERM
Publication of EP1068336A2 publication Critical patent/EP1068336A2/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • 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

Definitions

  • the subject of the present invention is a new membrane metalloprotease called NEP II and its use in particular for the screening of inhibitors useful in therapy
  • Membrane metalloproteases such as nepysiysin
  • the present invention therefore relates to an isolated polypeptide comprising an amino acid sequence chosen from the sequence SEQ ID No. 2 or SEQ ID No. 4, a sequence derived from or homologous to said sequence SEQ ID No. 2 or SEQ ID n ° 4, or a biologically active fragment of said sequence SEQ ID No. 2 or SEQ ID No. 4, said isolated polypeptide being designated by "NEP II"
  • sequence SEQ ID No. 2 is the amino acid sequence of NEP II identified in the rat.
  • sequence SEQ ID No. 4 is a (partial) amino acid sequence of NEP II identified in humans
  • derivative polypeptide any polypeptide resulting from a genetic and / or chemical modification of the sequence SEQ ID No. 2 or SEQ ID No. 4, that is to say by mutation, deletion, addition , substitution and / or chemical modification of at least one amino acid, or any isoform having a sequence identical to the sequence SEQ ID No. 2 or SEQ ID No. 4 but containing at least one amino acid in the D form
  • substitutions are preferably conservative substitutions, that is to say substitutions of amino acids of the same class, such as substitutions of amino acids with uncharged side chains (such as asparagine, glutamine, serine, threonme, and tyrosine), amino acids with basic side chains (such as lysine, argmine, and histidine), amino acids with acid side chains (such as aspartic acid and glutamic acid), amino acids with non-polar side chains (such as glycine, alanine, valine, leucine, isoleucine, pro ne, phenylalanine, methionine, tryptophan, and cysteine ).
  • amino acids with uncharged side chains such as asparagine, glutamine, serine, threonme, and tyrosine
  • amino acids with basic side chains such as lysine, argmine, and histidine
  • amino acids with acid side chains such as aspartic acid and glutamic acid
  • amino acids with non-polar side chains
  • homologous polypeptide is meant more particularly any polypeptide which can be isolated from other mammalian species than the rat or man.
  • Said homologous polypeptides preferably have a sequence homology greater than 70%, more preferably still greater than 75%, with the complete sequence SEQ ID No. 2 or SEQ ID No. 4, the homology being particularly high in the portion of said polypeptide, containing the active site.
  • sequence analysis software e.g. Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705
  • Amino acid sequences similar are aligned to obtain the maximum degree of homology (ie identity)
  • it may be necessary to artificially introduce spaces (“gaps") in the sequence Once the optimal alignment achieved, the degree homology (ie, identity) is established by Recorded i strat all positions for which the amino acid - t »
  • Said polypeptides derived, homologous or the polypeptide fragments of the polypeptide of sequence SEQ ID No. 2 or SEQ ID ⁇ ° 4 are biologically active, that is to say exhibit biological properties identical or similar to the biological properties of the NEP II polypeptide of sequence SEQ ID No. 2 or SEQ ID ⁇ ° 4, namely a metalloprotease activity
  • Preferred polypeptide fragments include the sequence of the active site responsible for the binding of the zinc atom essential for catalysis.
  • This active site has been identified as including the residues HEX 1 X 2 H, Xi and X 2 representing variable amino acids II this is in particular the sequence HEITH (amino acids 608 to 612 of the sequence SEQ ID No. 2) in the NEP II polypeptide in rats and humans
  • HEITH amino acids 608 to 612 of the sequence SEQ ID No. 2
  • sequence SEQ ID No. 1 is the cDNA sequence comprising the coding phase for NEP II identified in the rat
  • sequence SEQ ID No. 3 is the cDNA sequence comprising (partially) the coding phase for NEP II identified in humans
  • derived nucleotide sequence means any nucleotide sequence coding for a polypeptide derived from NEP II as defined above, that is to say a sequence resulting from a modification of the sequence SEQ ID No. 1 or SEQ ID No. 3, in particular by mutation, deletion, addition or substitution of at least one nucleotide
  • sequences derived from the sequence SEQ ID No. 1 or SEQ ID No. 3 are included by degeneration of the genetic code
  • homologous sequence more particularly means any nucleotide sequence coding for a homologous NEP II polypeptide NEP II polypeptide of sequence SEQ ID No. 2 or SEQ ID No. 4 in other mammalian species than rats or humans
  • Such a homologous sequence preferably has a homology greater than 70%, more preferably greater than 75%, with the sequence SEQ ID No. 1 or SEQ ID No. 3, the homology being particularly high in the central part of the coding sequence.
  • such a homologous nucleotide sequence hybrid specifically to the sequences complementary to the sequence SEQ ID No. 1 or No. 3, under stnngent conditions
  • the parameters defining the st ⁇ ngence conditions depend on the temperature at which 50% of the paired strands separate (Tm)
  • Tm 4 (G + C) + 2 (A + T)
  • Tm 4 (G + C) + 2 (A + T)
  • Tm more preferably 5 to 10 ° C below Tm (strong st ⁇ gence)
  • the hybridization buffers used are preferably solutions of high ionic strength such as a 6xSSC solution for example
  • nucleotide sequences according to the invention can be used for the production of a recombinant NEP II protein according to the invention, according to techniques for the production of recombinant products known to those skilled in the art.
  • An efficient system for producing a recombinant protein requires a vector, for example of plasmid or viral origin, and a compatible host cell.
  • the cellular host can be chosen from prokaryotic systems, such as bacteria, or eukaryotic systems, such as, for example, yeasts, insect or mammalian cells, such as CHO cells (Chinese hamster ovary cells) or any other advantageously available system
  • the vector must include a promoter, translation initiation and termination signals, as well as the appropriate regions of transcription regulation II must be able to be integrated into the cell and may possibly have specific signals specifying the secretion of the protein.
  • nucleotide sequences according to the invention can be inserted into vectors with autonomous repation within the chosen host, or integrative vectors of the host chosen Such vectors will be prepared according to the methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as for example electroporation
  • vectors of interest are the plasmids pcDNA 3 1, PCR2 1 (Invitrogen), or pMbac (Stratage ⁇ e)
  • the invention relates to the cloning and / or expression vectors containing a nucleotide sequence according to the invention, and further relates to the host cells transfected with these vectors. These cells can be obtained by the introduction into host cells of a nucleotide sequence inserted into a vector as defined above, then culturing said cells under conditions allowing rephcation and / or expression of the transfected nucleotide sequence
  • the method for producing a polypeptide of the invention in recombinant form is itself included in the present invention, and is characterized in that the transfected cells are cultured under conditions allowing the expression of a recombinant polypeptide according to the invention, and that said recombinant polypeptide is recovered
  • the purification methods used are known to those skilled in the art.
  • the recombinant polypeptide can be purified from lysates and cell extracts, from the culture medium supernatant, by methods used separately or in combination, such as fractionation, methods chromatography, immunoaffinity techniques using monoclonal antibodies or polyclonal serum, etc.
  • the present invention also relates to nucleotide probes, capable of hybridizing strongly and specifically with a nucleic acid sequence, of a genomic DNA or of a messenger RNA, coding for a polypeptide according to the invention.
  • 'appropriate hybridization correspond to the conditions of temperature and ionic strength usually used by those skilled in the art (Sambrook et al, 1989), preferably to conditions of high st ⁇ ngence, that is to say conditions of temperature between (T m minus 5 ° C) and (T m minus 15 ° C) and preferably still, at temperature conditions between T m and (T m minus 10 ° C) (strong emergency)
  • the preferred probes are in particular the oligonucleotide probes chosen from sequences SEQ ID No. 5 to SEQ ID No. 27. Such probes are useful for specific sequencing or amplification reactions according to the so-called PCR technique (polymerase chain reaction ) or any other variant thereof
  • Such probes are also useful in a method of detecting expression of the NEP II polypeptide in a cell or tissue sample or in cells or tissue by in situ hybridization, comprising the steps of - prepare the RNA of said sample or of said cells or of said tissue,
  • the subject of the invention is also the mono- or polyclonal antibodies or their fragments, chimeric or immu ⁇ oconjugate antibodies, characterized in that they are obtained from a polypeptide according to the invention administered to an animal, and are capable of recognizing specifically a polypeptide according to the invention
  • the invention further relates to the use of these antibodies for the purification or detection of a NEP II polypeptide in a biological sample
  • polyclonal antibodies can be obtained from the serum of an animal immunized against the NEP II protein, produced for example by genetic recombination according to the method described above, according to the usual procedures.
  • the monoclonal antibodies can be obtained according to the conventional method of culturing hybridomas described by Kohler and Milstem (Nature, 1975, vol 256, pp 495-497)
  • the antibodies can be chimeric antibodies, humanized antibodies, Fab and F fragments (ab ') 2 They can also be in the form of immu ⁇ oconjugates or labeled antibodies
  • the antibodies according to the invention are particularly useful for detecting the presence of NEP II
  • the present invention therefore relates to a method of detecting i mmuniques CIP II in a cell or tissue sample or in cells or tissue comprising the steps of - bringing said cell or tissue sample into contact with said cells or said tissue with a detectable antibody according to the invention
  • detectable antibody is meant either an antibody labeled with a detectable group, such as a radioactive, enzymatic, fluorogenic or fluorescent group, etc., or an antibody to which another detectably labeled antibody binds
  • the antibodies according to the invention can thus make it possible to assess an overexpression of polypeptide II, which can be indicative of neuroendocrine tumor cells in particular
  • the subject of the invention is also a method of identifying compounds which are substrates of the NEP II polypeptide as defined above, in which said compounds, optionally labeled, are brought into contact with the NEP II polypeptide, and the cleavage of said compounds is evaluated by NEP II, indicative of the metalloprotease activity of NEP II towards said substrate compounds
  • Such specific NEP II substrates can in particular be used in a method for detecting the metalloprotease activity of NEP II in a cell or tissue sample or in cells or tissue comprising the steps consisting in
  • the cells capable of being thus tested are in particular the cells transfected with a polynucleotide coding for the NEP II polypeptide as defined above.
  • the tissue extracts which can be tested are in particular the testicle membranes, which are particularly rich in 9
  • the subject of the invention is moreover a method of screening for compounds capable of inhibiting the metalloprotease activity of the NEP II polypeptide according to the invention, in which said compounds are brought into contact with said NEP II polypeptide and the level of d is evaluated.
  • the compounds capable of inhibiting the metalloprotease activity of NEP II are preferably short peptides of 2 or 3 natural or modified amino acids
  • the synthetic peptides identified as inhibitors of the metalloprotease activity of NEP II by this screening method can be coupled to a zinc chelating group such as the thiol, phosphate or hydroxamic acid groups, according to the conventional techniques known to those skilled in the art.
  • the inhibitor compound obtained is a good candidate as an active principle of a medicament, in combination with a pharmaceutically acceptable vehicle.
  • Said chelating group can optionally be protected in a transient manner, for example with a thiol ester, to improve the bioavailability of said principle.
  • active The NEP II polypeptide according to the invention is particularly useful for the screening of compounds inhibiting the metalloprotease activity of NEP II useful for the manufacture of a medicament intended for treating disorders involving peptidergic transmissions in which NEP II participates
  • NEP II substrate compounds or inhibitors of NEP II metalloprotease activity obtained according to the methods described above can also be useful for detecting the NEP II protein.
  • the present invention therefore also relates to a method for detecting NEP II in a cell or tissue sample or in cells or tissue comprising the steps consisting in
  • labeled substrate compound or “labeled inhibitor” means a substrate compound or an inhibitor compound detectably labeled, for example by a radioactive, enzymatic, fluorogenic or fluorescent group, etc.
  • oligonucleotides were obtained from the alignment of the peptide sequences of the enzymes ECE, NEP I and Kell and from the delimitation of the zones of strong homology
  • RNA from different rat tissues was reverse transcribed (RT) and amplified by polymerase chain reaction (PCR), using a pair of o degenerate gonucleotides on the N-terminal region rich in cysteine residues
  • PCR polymerase chain reaction
  • the new isolated gene codes for a protein of 774 amino acids (SEQ ID No. 2) which, in addition to strong homologies with the enzymes NEP I,
  • ECE and Kell (52%, 40% and 28% amino acid identity, respectively) has the consensus sequence of the active site HEXXH, a transmembrane region (amino acids 24 to 40 on the sequence SEQ ID No. 2) followed by four cysteine residues characteristic of this family, and seven potential glycosylation sites
  • Three alternative splices have been identified by RACE sequencing and by RT-PCR One of these alternative splices eliminates a potential glycosylation site and could affect the transit of the protein to the cell surface or its activity
  • Each splicing also corresponds to an exon of NEP I, which suggests a similar gene structure
  • oligonucleotides were designed, based on the protein sequence of rat NEP II.
  • the sequences were chosen on the one hand for their low degeneration (such as for example a tryptophan, represented by a only codo ⁇ in the genetic code) and secondly for their degree of conservation (such as the zinc binding site)
  • HNII-2 5'- CTG ACT GCT CCC GGA AGT GCT GGG TG - 3 '(SEQ ID n ° 25)
  • ectoprotease responsible for the metabolism of neuronal and / or hormonal messenger peptides
  • the native NEP II polypeptide is expressed heterogeneously in the nervous system, the glands (pituitary gland, testis), the digestive system (especially the small intestine), the cardiovascular system (especially the heart)
  • In situ hybridization techniques also indicate a high expression of the NEP II protein in neurons and adenohypophysial cells expressing the POMC (propiomelanocortin) gene, precursor of ACTH.
  • POMC propiomelanocortin

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Abstract

The invention concerns an isolated polypeptide comprising an amino acid sequence selected among the sequence SEQ ID n° 2 or n° 4, a sequence derived from or homologous with said sequence SEQ ID n° 2 or n° 4, or a biologically active fragment of said sequence SEQ ID n° 2 or n° 4, said isolated polypeptide being designated as 'NEP II', and the use of said NEP II polypeptide for screening inhibitors useful in therapy.

Description

- 1 - " Nouvelle métalloprotéase membranaire NEP II et son utilisation pour le criblage d ' inhibiteurs utiles en thérapie" . - 1 - "New NEP II membrane metalloprotease and its use for the screening of inhibitors useful in therapy".
La présente invention a pour objet une nouvelle métalloprotéase membranaire appelée NEP II et son utilisation notamment pour le criblage d'inhibiteurs utiles en thérapieThe subject of the present invention is a new membrane metalloprotease called NEP II and its use in particular for the screening of inhibitors useful in therapy
Les métalloprotéases membraπaires telles que la népπiysineMembrane metalloproteases such as nepysiysin
(NEP I, EC 3 4 24 1 1 ) jouent un rôle important dans l'activation ou l'inactivation des messagers peptidiques neuronaux ou hormonaux Leur inhibition sélective par des composés synthétiques a déjà conduit à des médicaments couramment utilisés en thérapeutique ou en cours de développement clinique, notamment dans les domaines gastroenterologique (Baumer et coll , Gut, 1992, 33 753-758) et cardiovasculaire (Gros et coll , Proc Natl Acad Sci USA, 1991 , 88 4210-4214) L'isolement des ADNc de gènes de nouvelles métalloprotéases apparentées est de nature à permettre le développement de nouvelles classes d'inhibiteurs spécifiques à applications thérapeutiques prometteuses C'est ainsi que le clonage et l'expression du gène de l'enzyme de conversion de l'endothéhne (ECE) (Xu et coll , Cell, 1994, 78 473-485) a permis la mise au point d'inhibiteurs potentiellement utiles dans certaines affections cardiovasculaires(NEP I, EC 3 4 24 1 1) play an important role in the activation or inactivation of neuronal or hormonal peptide messengers Their selective inhibition by synthetic compounds has already led to drugs commonly used in therapy or in the process of clinical development, particularly in the gastroenterological (Baumer et al, Gut, 1992, 33 753-758) and cardiovascular (Gros et al, Proc Natl Acad Sci USA, 1991, 88 4210-4214) fields Isolation of cDNAs from genes new related metalloproteases is likely to allow the development of new classes of specific inhibitors with promising therapeutic applications. This is how the cloning and expression of the gene for the endothéhne converting enzyme (ECE) (Xu and coll, Cell, 1994, 78 473-485) has made it possible to develop inhibitors which are potentially useful in certain cardiovascular diseases
Les auteurs de la présente invention ont mis en évidence une nouvelle métalloprotéase membranaire appartenant à la famille ECE/NEP/Kell (Lee S et coll , 1991 , PNAS 88(14) 6353-57), qu'ils ont appelée NEP IIThe authors of the present invention have revealed a new membrane metalloprotease belonging to the ECE / NEP / Kell family (Lee S et al, 1991, PNAS 88 (14) 6353-57), which they have called NEP II
La présente invention a donc pour objet un polypeptide isolé comprenant une séquence d'acides aminés choisie parmi la séquence SEQ ID n°2 ou SEQ ID n° 4, une séquence dérivée ou homologue de ladite séquence SEQ ID n°2 ou SEQ ID n° 4, ou un fragment biologiquement actif de ladite séquence SEQ ID n°2 ou SEQ ID n° 4, ledit polypeptide isolé étant désigne par «NEP II »The present invention therefore relates to an isolated polypeptide comprising an amino acid sequence chosen from the sequence SEQ ID No. 2 or SEQ ID No. 4, a sequence derived from or homologous to said sequence SEQ ID No. 2 or SEQ ID n ° 4, or a biologically active fragment of said sequence SEQ ID No. 2 or SEQ ID No. 4, said isolated polypeptide being designated by "NEP II"
La séquence SEQ ID n° 2 est la séquence d'acides aminés de NEP II identifiée chez le rat.The sequence SEQ ID No. 2 is the amino acid sequence of NEP II identified in the rat.
La séquence SEQ ID n° 4 est une séquence (partielle) d'acides aminés de NEP II identifiée chez l'homme Par polypeptide "dérivé", on entend tout polypeptide résultant d'une modification de nature génétique et/ou chimique de la séquence SEQ ID n° 2 ou SEQ ID n° 4, c'est-à-dire par mutation, délétion, addition, substitution et/ou modification chimique d'au moins un acide aminé, ou toute isoforme ayant une séquence identique à la séquence SEQ ID n° 2 ou SEQ ID n° 4 mais contenant au moins un acide aminé sous la forme DThe sequence SEQ ID No. 4 is a (partial) amino acid sequence of NEP II identified in humans By "derivative" polypeptide is meant any polypeptide resulting from a genetic and / or chemical modification of the sequence SEQ ID No. 2 or SEQ ID No. 4, that is to say by mutation, deletion, addition , substitution and / or chemical modification of at least one amino acid, or any isoform having a sequence identical to the sequence SEQ ID No. 2 or SEQ ID No. 4 but containing at least one amino acid in the D form
Lesdites substitutions sont de préférence des substitutions conservatives, c'est-à-dire des substitutions d'acides aminés de même classe, tels que des substitutions d'acides aminés aux chaînes latérales non chargées (tels que l'asparagine, la glutamine, la serine, la thréonme, et la tyrosine), d'acides aminés aux chaînes latérales basiques (tels que la lysine, l'argmine, et l'histidine), d'acides aminés aux chaînes latérales acides (tels que l'acide aspartique et l'acide glutamique) , d'acides aminés aux chaînes latérales apolaires (tels que la glycine, l'alanine, la valine, la leucine, l'isoleucine, la pro ne, la phénylalanine, la méthionine, le tryptophane, et la cystéine).Said substitutions are preferably conservative substitutions, that is to say substitutions of amino acids of the same class, such as substitutions of amino acids with uncharged side chains (such as asparagine, glutamine, serine, threonme, and tyrosine), amino acids with basic side chains (such as lysine, argmine, and histidine), amino acids with acid side chains (such as aspartic acid and glutamic acid), amino acids with non-polar side chains (such as glycine, alanine, valine, leucine, isoleucine, pro ne, phenylalanine, methionine, tryptophan, and cysteine ).
Par polypeptide "homologue", on entend plus particulièrement tout polypeptide isolable chez d'autres espèces de mammifères que le rat ou l'hommeBy "homologous" polypeptide is meant more particularly any polypeptide which can be isolated from other mammalian species than the rat or man.
Lesdits polypeptides homologues présentent préférentiellement une homologie de séquence supérieure à 70 %, de préférence encore supérieure à 75 %, avec la séquence SEQ ID n° 2 ou SEQ ID n° 4 complète, l'homologie étant particulièrement élevée dans la partie dudit polypeptide, contenant le site actif.Said homologous polypeptides preferably have a sequence homology greater than 70%, more preferably still greater than 75%, with the complete sequence SEQ ID No. 2 or SEQ ID No. 4, the homology being particularly high in the portion of said polypeptide, containing the active site.
L'homologie est généralement déterminée en utilisant un logiciel d'analyse de séquence (par exemple, Séquence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705) Des séquences d'acides aminés similaires sont alignées pour obtenir le maximum de degré d'homologie (i e identité) A cette fin, il peut être nécessaire d'introduire de manière artificielle des espaces (« gaps ») dans la séquence Une fois l'alignement optimal réalisé, le degré d'homologie (i e identité) est établi par enregistrement de toutes les positions pour lesquelles les acides aminés des - t»Homology is usually determined using sequence analysis software (e.g. Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705) Amino acid sequences similar are aligned to obtain the maximum degree of homology (ie identity) For this purpose, it may be necessary to artificially introduce spaces ("gaps") in the sequence Once the optimal alignment achieved, the degree homology (ie, identity) is established by Recorded i strat all positions for which the amino acid - t »
deux séquences comparées sont identiques, par rapport au nombre total de positionstwo compared sequences are identical, compared to the total number of positions
Lesdits polypeptides dérivés, homologues ou les fragments polypeptidiques du polypeptide de séquence SEQ ID n° 2 ou SEQ ID π° 4 sont biologiquement actifs, c'est-à-dire présentent des propriétés biologiques identiques ou similaires des propriétés biologiques du polypeptide NEP II de séquence SEQ ID n° 2 ou SEQ ID π° 4, à savoir une activité métalloprotéasiqueSaid polypeptides derived, homologous or the polypeptide fragments of the polypeptide of sequence SEQ ID No. 2 or SEQ ID π ° 4 are biologically active, that is to say exhibit biological properties identical or similar to the biological properties of the NEP II polypeptide of sequence SEQ ID No. 2 or SEQ ID π ° 4, namely a metalloprotease activity
Les fragments polypeptidiques préférés comprennent la séquence du site actif responsable de la liaison de l'atome de zinc indispensable à la catalyse Ce site actif a été identifié comme englobant les résidus HEX1X2H, Xi et X2 représentant des acides aminés variables II s'agit en particulier de la séquence HEITH (acides aminés 608 à 612 de la séquence SEQ ID n° 2) dans le polypeptide NEP II chez le rat et l'homme La présente invention a également pour objet un acide nucléique isolé comprenant une séquence nucléotidique choisie parmi la séquence SEQPreferred polypeptide fragments include the sequence of the active site responsible for the binding of the zinc atom essential for catalysis. This active site has been identified as including the residues HEX 1 X 2 H, Xi and X 2 representing variable amino acids II this is in particular the sequence HEITH (amino acids 608 to 612 of the sequence SEQ ID No. 2) in the NEP II polypeptide in rats and humans The present invention also relates to an isolated nucleic acid comprising a sequence nucleotide chosen from the sequence SEQ
ID n°1 ou SEQ ID n° 3, une séquence dérivée ou homologue de ladite séquence SEQ ID n°1 ou SEQ ID n° 3, ou leurs séquences complémentairesID No. 1 or SEQ ID No. 3, a sequence derived from or homologous to said sequence SEQ ID No. 1 or SEQ ID No. 3, or their complementary sequences
La séquence SEQ ID n° 1 est la séquence d'ADNc comprenant la phase codante pour NEP II identifiée chez le ratThe sequence SEQ ID No. 1 is the cDNA sequence comprising the coding phase for NEP II identified in the rat
La séquence SEQ ID n° 3 est la séquence d'ADNc comprenant (partiellement) la phase codante pour NEP II identifiée chez l'hommeThe sequence SEQ ID No. 3 is the cDNA sequence comprising (partially) the coding phase for NEP II identified in humans
Par séquence nucléotidique "dérivée", on entend toute séquence nucléotidique codant pour un polypeptide dérivé de NEP II tel que défini précédemment, c'est-à-dire une séquence résultant d'une modification de la séquence SEQ ID n° 1 ou SEQ ID n° 3, notamment par mutation, délétion, addition ou substitution d'au moins un nucléotide Sont en particulier comprises les séquences dérivées de la séquence SEQ ID n° 1 ou SEQ ID n° 3 par dégénérescence du code génétique Par séquence "homologue", on entend plus particulièrement toute séquence nucléotidique codant pour un polypeptide NEP II homologue du polypeptide NEP II de séquence SEQ ID n° 2 ou SEQ ID n° 4 chez d'autres espèces de mammifères que le rat ou l'hommeThe term “derived” nucleotide sequence means any nucleotide sequence coding for a polypeptide derived from NEP II as defined above, that is to say a sequence resulting from a modification of the sequence SEQ ID No. 1 or SEQ ID No. 3, in particular by mutation, deletion, addition or substitution of at least one nucleotide In particular, the sequences derived from the sequence SEQ ID No. 1 or SEQ ID No. 3 are included by degeneration of the genetic code By "homologous" sequence , more particularly means any nucleotide sequence coding for a homologous NEP II polypeptide NEP II polypeptide of sequence SEQ ID No. 2 or SEQ ID No. 4 in other mammalian species than rats or humans
Une telle séquence homologue présente préférentiellement une homologie supérieure à 70 %, de préférence encore supérieure à 75 %, avec la séquence SEQ ID n° 1 ou SEQ ID n° 3, l'homologie étant particulièrement élevée dans la partie centrale de la séquence codant pour le polypeptide NEPSuch a homologous sequence preferably has a homology greater than 70%, more preferably greater than 75%, with the sequence SEQ ID No. 1 or SEQ ID No. 3, the homology being particularly high in the central part of the coding sequence. for the NEP polypeptide
IIII
De manière préférentielle, une telle séquence nucléotidique homologue hybride spécifiquement aux séquences complémentaires de la séquence SEQ ID n° 1 ou n° 3, dans des conditions stnngentes Les paramètres définissant les conditions de stπngence dépendent de la température à laquelle 50% des brins appariés se séparent (Tm)Preferably, such a homologous nucleotide sequence hybrid specifically to the sequences complementary to the sequence SEQ ID No. 1 or No. 3, under stnngent conditions The parameters defining the stπngence conditions depend on the temperature at which 50% of the paired strands separate (Tm)
Pour les séquences comprenant plus de 30 bases, Tm est définie par la relation Tm=81 , 5+0,41 (%G+C)+16, 6Log(concentratιon en cations) - 0,63(%formamιde) -(600/nombre de bases) (Sambrook et al, MolecularFor sequences comprising more than 30 bases, Tm is defined by the relation Tm = 81.5 + 0.41 (% G + C) +16.6Log (concentration of cations) - 0.63 (% formamide) - (600 / number of bases) (Sambrook et al, Molecular
Clonmg, A laboratory manual, Cold Spring Harbor laboratory Press, 1989, pages 9 54-9 62)Clonmg, A laboratory manual, Cold Spring Harbor laboratory Press, 1989, pages 9 54-9 62)
Pour les séquences de longueur inférieure à 30 bases, Tm est définie par la relation Tm= 4(G+C) + 2 (A+T) Dans des conditions de stπngence appropriées, auxquelles les séquences aspécifiques n'hybπdent pas, la température d'hybridation est approximativement de 5 à 30°C, de préférence de 5 à 15°C en dessous deFor sequences of length less than 30 bases, Tm is defined by the relation Tm = 4 (G + C) + 2 (A + T) Under appropriate conditions of urgency, to which the aspecific sequences do not hybridize, the temperature d hybridization is approximately 5 to 30 ° C, preferably 5 to 15 ° C below
Tm, de préférence encore de 5 à 10°C en dessous de Tm (forte stππgence), et les tampons d'hybridation utilisés sont de préférence des solutions de force ionique élevée telle qu'une solution 6xSSC par exempleTm, more preferably 5 to 10 ° C below Tm (strong stππgence), and the hybridization buffers used are preferably solutions of high ionic strength such as a 6xSSC solution for example
Les séquences nucléotidiques selon l'invention peuvent être utilisées pour la production d'une protéine recombinante NEP II selon l'invention, selon des techniques de production de produits recombinants connues de l'homme du métier Un système efficace de production d'une protéine recombinante nécessite de disposer d'un vecteur, par exemple d'origine plasmidique ou virale, et d'une cellule hôte compatibleThe nucleotide sequences according to the invention can be used for the production of a recombinant NEP II protein according to the invention, according to techniques for the production of recombinant products known to those skilled in the art. An efficient system for producing a recombinant protein requires a vector, for example of plasmid or viral origin, and a compatible host cell.
L'hôte cellulaire peut être choisi parmi des systèmes procaryotes, comme les bactéries, ou eucaryotes, comme par exemple des levures, des cellules d'insectes, de mammifères, telles que les cellules CHO (cellules d'ovaires de hamster chinois) ou tout autre système avantageusement disponibleThe cellular host can be chosen from prokaryotic systems, such as bacteria, or eukaryotic systems, such as, for example, yeasts, insect or mammalian cells, such as CHO cells (Chinese hamster ovary cells) or any other advantageously available system
Le vecteur doit comporter un promoteur, des signaux d'initiation et de terminaison de la traduction, ainsi que les régions appropriées de régulation de la transcription II doit pouvoir être intégré dans la cellule et peut éventuellement posséder des signaux particuliers spécifiant la sécrétion de la protéine traduiteThe vector must include a promoter, translation initiation and termination signals, as well as the appropriate regions of transcription regulation II must be able to be integrated into the cell and may possibly have specific signals specifying the secretion of the protein. translated
Ces différents signaux de contrôle sont choisis en fonction de l'hôte cellulaire utilisé A cet effet, les séquences nucleotidiques selon l'invention peuvent être insérées dans des vecteurs à rép cation autonome au sein de l'hôte choisi, ou des vecteurs intégratifs de l'hôte choisi De tels vecteurs seront préparés selon les méthodes couramment utilisées par l'homme du métier, et les clones en résultant peuvent être introduits dans un hôte approprié par des méthodes standard, telles que par exemple l'électroporationThese different control signals are chosen as a function of the cellular host used. For this purpose, the nucleotide sequences according to the invention can be inserted into vectors with autonomous repation within the chosen host, or integrative vectors of the host chosen Such vectors will be prepared according to the methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as for example electroporation
Des exemples de vecteurs d'intérêt sont les plasmides pcDNA 3 1 , PCR2 1 (Invitrogen), ou pMbac (Stratageπe)Examples of vectors of interest are the plasmids pcDNA 3 1, PCR2 1 (Invitrogen), or pMbac (Stratageπe)
L'invention vise les vecteurs de clonage et/ou d'expression contenant une séquence nucléotidique selon l'invention, et vise en outre les cellules hôtes transfectées par ces vecteurs Ces cellules peuvent être obtenues par l'introduction dans des cellules hôtes d'une séquence nucléotidique insérée dans un vecteur tel que défini ci-dessus, puis la mise en culture desdites cellules dans des conditions permettant la réphcation et/ou l'expression de la séquence nucléotidique transfectéeThe invention relates to the cloning and / or expression vectors containing a nucleotide sequence according to the invention, and further relates to the host cells transfected with these vectors. These cells can be obtained by the introduction into host cells of a nucleotide sequence inserted into a vector as defined above, then culturing said cells under conditions allowing rephcation and / or expression of the transfected nucleotide sequence
Ces cellules sont utilisables dans une méthode de production d'un polypeptide recombinant selon l'invention 6These cells can be used in a method for producing a recombinant polypeptide according to the invention 6
La méthode de production d'un polypeptide de l'invention sous forme recombinante est elle-même comprise dans la présente invention, et se caractérise en ce que l'on cultive les cellules transfectées dans des conditions permettant l'expression d'un polypeptide recombinant selon l'invention, et que l'on récupère ledit polypeptide recombinantThe method for producing a polypeptide of the invention in recombinant form is itself included in the present invention, and is characterized in that the transfected cells are cultured under conditions allowing the expression of a recombinant polypeptide according to the invention, and that said recombinant polypeptide is recovered
Les procédés de purification utilisés sont connus de l'homme du métier Le polypeptide recombinant peut être purifié à partir de lysats et extraits cellulaires, du surnageant du milieu de culture, par des méthodes utilisées séparément ou en combinaison, telles que le fractionnement, les méthodes de chromatographie, les techniques d'immunoaffinité à l'aide d'anticorps monoclonaux ou de sérum polyclonal, etcThe purification methods used are known to those skilled in the art. The recombinant polypeptide can be purified from lysates and cell extracts, from the culture medium supernatant, by methods used separately or in combination, such as fractionation, methods chromatography, immunoaffinity techniques using monoclonal antibodies or polyclonal serum, etc.
La présente invention a également pour objet les sondes nucleotidiques, capables de s'hybrider fortement et spécifiquement avec une séquence d'acide nucléique, d'un ADN genomique ou d'un ARN messager, codant pour un polypeptide selon l'invention Les conditions d'hybridation appropriées correspondent aux conditions de température et de force ionique usuellement utilisées par l'homme du métier (Sambrook et al , 1989), de préférence à des conditions de forte stπngence, c'est-à-dire des conditions de température comprises entre (Tm moins 5° C) et (Tm moins 15° C) et de préférence encore, à des conditions de température comprises entre Tm et (Tm moins 10° C) (forte stnngence)The present invention also relates to nucleotide probes, capable of hybridizing strongly and specifically with a nucleic acid sequence, of a genomic DNA or of a messenger RNA, coding for a polypeptide according to the invention. 'appropriate hybridization correspond to the conditions of temperature and ionic strength usually used by those skilled in the art (Sambrook et al, 1989), preferably to conditions of high stπngence, that is to say conditions of temperature between (T m minus 5 ° C) and (T m minus 15 ° C) and preferably still, at temperature conditions between T m and (T m minus 10 ° C) (strong emergency)
Les sondes préférées sont notamment les sondes oligonucléotidiques choisies parmi les séquences SEQ ID n°5 à SEQ ID n°27 De telles sondes sont utiles pour des réactions de séquençage ou d'amplification spécifique selon la technique dite de PCR (réaction en chaîne par polymérase) ou toute autre variante de celle-ciThe preferred probes are in particular the oligonucleotide probes chosen from sequences SEQ ID No. 5 to SEQ ID No. 27. Such probes are useful for specific sequencing or amplification reactions according to the so-called PCR technique (polymerase chain reaction ) or any other variant thereof
De telles sondes sont également utiles dans un procédé de détection de l'expression du polypeptide NEP II dans un échantillon cellulaire ou tissulaire ou dans des cellules ou un tissu par hybridation in situ, comprenant les étapes consistant à - préparer l'ARN dudit échantillon ou desdites cellules ou dudit tissu,Such probes are also useful in a method of detecting expression of the NEP II polypeptide in a cell or tissue sample or in cells or tissue by in situ hybridization, comprising the steps of - prepare the RNA of said sample or of said cells or of said tissue,
- mettre en contact ledit ARN obtenu avec au moins une sonde ayant une séquence nucléotidique capable de s'hybnder spécifiquement avec une séquence nucléotidique selon l'invention, ladite sonde pouvant être notamment une sonde oligonucléotidique de séquence SEQ ID n° 5 à SEQ ID n° 27 ,- bringing said RNA obtained into contact with at least one probe having a nucleotide sequence capable of hybridizing specifically with a nucleotide sequence according to the invention, said probe possibly being in particular an oligonucleotide probe of sequence SEQ ID n ° 5 to SEQ ID n ° 27,
- détecter la présence d'ARNm hybndant avec ladite sonde indicatrice de l'expression du polypeptide NEP II- detect the presence of hybridizing mRNA with said probe indicative of the expression of the NEP II polypeptide
L'invention a également pour objet les anticorps mono- ou polyclonaux ou leurs fragments, anticorps chimériques ou immuπoconjugués, caractérisés en ce qu'ils sont obtenus à partir d'un polypeptide selon l'invention administré à un animal, et sont capables de reconnaître spécifiquement un polypeptide selon l'invention L'invention a en outre pour objet l'utilisation de ces anticorps pour la purification ou la détection d'un polypeptide NEP II dans un échantillon biologiqueThe subject of the invention is also the mono- or polyclonal antibodies or their fragments, chimeric or immuπoconjugate antibodies, characterized in that they are obtained from a polypeptide according to the invention administered to an animal, and are capable of recognizing specifically a polypeptide according to the invention The invention further relates to the use of these antibodies for the purification or detection of a NEP II polypeptide in a biological sample
Les anticorps polyclonaux peuvent être obtenus à partir du sérum d'un animal immunisé contre la protéine NEP II, produite par exemple par recombinaison génétique suivant la méthode décrite ci-dessus, selon les modes opératoires usuelsThe polyclonal antibodies can be obtained from the serum of an animal immunized against the NEP II protein, produced for example by genetic recombination according to the method described above, according to the usual procedures.
Les anticorps monoclonaux peuvent être obtenus selon la méthode classique de culture d'hybndomes décrite par Kohler et Milstem (Nature, 1975, vol 256, pp 495-497) Les anticorps peuvent être des anticorps chimériques, des anticorps humanisés, des fragments Fab et F(ab')2 Ils peuvent également se présenter sous forme d'immuπoconjugués ou d'anticorps marquésThe monoclonal antibodies can be obtained according to the conventional method of culturing hybridomas described by Kohler and Milstem (Nature, 1975, vol 256, pp 495-497) The antibodies can be chimeric antibodies, humanized antibodies, Fab and F fragments (ab ') 2 They can also be in the form of immuπoconjugates or labeled antibodies
Les anticorps selon l'invention sont particulièrement utiles pour détecter la présence de NEP II La présente invention a donc pour objet un procédé de détection immunologique de NEP II dans un échantillon cellulaire ou tissulaire ou dans des cellules ou un tissu comprenant les étapes consistant à - mettre en contact ledit échantillon cellulaire ou tissulaire lesdites cellules ou ledit tissu avec un anticorps détectable selon l'inventionThe antibodies according to the invention are particularly useful for detecting the presence of NEP II The present invention therefore relates to a method of detecting i mmunologique CIP II in a cell or tissue sample or in cells or tissue comprising the steps of - bringing said cell or tissue sample into contact with said cells or said tissue with a detectable antibody according to the invention
- détecter la présence dudit anticorps, indicatrice de la présence du polypeptide NEP II Par "anticorps détectable", on entend soit un anticorps marqué par un groupement détectable, tel qu'un groupement radioactif, enzymatique, fluorogène ou fluorescent, etc , soit un anticorps auquel se lie un autre anticorps lui-même marqué de manière détectable- detecting the presence of said antibody, indicative of the presence of the NEP II polypeptide By "detectable antibody" is meant either an antibody labeled with a detectable group, such as a radioactive, enzymatic, fluorogenic or fluorescent group, etc., or an antibody to which another detectably labeled antibody binds
Les anticorps selon l'invention peuvent ainsi permettre d'évaluer une surexpression du polypeptide II, qui peut être indicatrice de cellules tumorales neuroendocriniennes notammentThe antibodies according to the invention can thus make it possible to assess an overexpression of polypeptide II, which can be indicative of neuroendocrine tumor cells in particular
L'invention a également pour objet un procédé d'identification de composés substrats du polypeptide NEP II tel que défini précédemment, dans lequel on met en contact lesdits composés, éventuellement marqués, avec le polypeptide NEP II, et on évalue la coupure desdits composés par NEP II, indicatrice de l'activité métalloprotéasique de NEP II envers lesdits composes substratsThe subject of the invention is also a method of identifying compounds which are substrates of the NEP II polypeptide as defined above, in which said compounds, optionally labeled, are brought into contact with the NEP II polypeptide, and the cleavage of said compounds is evaluated by NEP II, indicative of the metalloprotease activity of NEP II towards said substrate compounds
De tels substrats spécifiques de NEP II peuvent être en particulier utilisés dans un procédé de détection de l'activité métalloprotéasique de NEP II dans un échantillon cellulaire ou tissuiaire ou dans des cellules ou un tissu comprenant les étapes consistant àSuch specific NEP II substrates can in particular be used in a method for detecting the metalloprotease activity of NEP II in a cell or tissue sample or in cells or tissue comprising the steps consisting in
- mettre en contact ledit échantillon cellulaire ou tissuiaire, lesdites cellules ou ledit tissu avec un composé substrat du polypeptide NEP II obtenu selon l'invention, ledit composé substrat étant éventuellement marqué ,bringing said cell or tissue sample, said cells or said tissue into contact with a substrate compound of the NEP II polypeptide obtained according to the invention, said substrate compound being optionally labeled,
- évaluer la coupure dudit composé substrat, indicatrice de l'activité métalloprotéasique de NEP II- evaluate the cleavage of said substrate compound, indicative of the metalloprotease activity of NEP II
Les cellules susceptibles d'être ainsi testées sont notamment les cellules transfectées par un polynucléotide codant pour le polypeptide NEP II tel que défini précédemment. Les extraits tissulaires susceptibles d'être testés sont en particulier les membranes de testicule, particulièrement riches en 9The cells capable of being thus tested are in particular the cells transfected with a polynucleotide coding for the NEP II polypeptide as defined above. The tissue extracts which can be tested are in particular the testicle membranes, which are particularly rich in 9
métalloprotéase NEP IImetalloprotease NEP II
L'invention a par ailleurs pour objet un procède de criblage de composes susceptibles d'inhiber l'activité métalloprotéasique du polypeptide NEP II selon l'invention, dans lequel on met en contact lesdits composés avec ledit polypeptide NEP II et on évalue le taux d'inhibition de l'activité métalloprotéasique de NEP IIThe subject of the invention is moreover a method of screening for compounds capable of inhibiting the metalloprotease activity of the NEP II polypeptide according to the invention, in which said compounds are brought into contact with said NEP II polypeptide and the level of d is evaluated. of NEP II metalloprotease activity
Les composés susceptibles d'inhiber l'activité métalloprotéasique de NEP II sont de préférence des peptides courts de 2 ou 3 acides aminés naturels ou modifiésThe compounds capable of inhibiting the metalloprotease activity of NEP II are preferably short peptides of 2 or 3 natural or modified amino acids
Les peptides synthétiques identifiés comme inhibiteurs de l'activité métalloprotéasique de NEP II par ce procédé de criblage peuvent être couplés à un groupe chélateur de zinc tels que les groupes thiol, phosphate ou acide hydroxamique, selon les techniques classiques connues de l'homme du métier Le composé inhibiteur obtenu est un bon candidat en tant que principe actif d'un médicament, en association avec un véhicule pharmaceutiquement acceptable Ledit groupe chélateur peut éventuellement être protégé de manière transitoire, par exemple par un ester de thiol, pour améliorer la biodisponibilité dudit principe actif Le polypeptide NEP II selon l'invention est particulièrement utile pour le criblage de composés inhibiteurs de l'activité métalloprotéasique de NEP II utiles pour la fabrication de médicament destiné à traiter les troubles impliquant les transmissions peptidergiques auxquelles participe NEP IIThe synthetic peptides identified as inhibitors of the metalloprotease activity of NEP II by this screening method can be coupled to a zinc chelating group such as the thiol, phosphate or hydroxamic acid groups, according to the conventional techniques known to those skilled in the art. The inhibitor compound obtained is a good candidate as an active principle of a medicament, in combination with a pharmaceutically acceptable vehicle. Said chelating group can optionally be protected in a transient manner, for example with a thiol ester, to improve the bioavailability of said principle. active The NEP II polypeptide according to the invention is particularly useful for the screening of compounds inhibiting the metalloprotease activity of NEP II useful for the manufacture of a medicament intended for treating disorders involving peptidergic transmissions in which NEP II participates
Parmi les troubles en cause, on peut citer notamment les maladies cardiovasculaires, neurodégénératives, les troubles de la croissance d'origine endocrinienne, les perturbations de l'axe hypothalamo-hypophysaire et les affections endocriniennes Sont plus particulièrement visés les troubles affectant le métabolisme des neurohormones ou facteurs de la sphère corticotrope Les composés substrats de NEP II ou inhibiteurs de l'activité métalloprotéasique de NEP II obtenus selon les procédés décrits précédemment peuvent également être utiles pour détecter la protéine NEP IIAmong the disorders in question, mention may be made in particular of cardiovascular and neurodegenerative diseases, growth disorders of endocrine origin, disturbances of the hypothalamic-pituitary axis and endocrine disorders. More particularly targeted are disorders affecting the metabolism of neurohormones or factors of the corticotropic sphere The NEP II substrate compounds or inhibitors of NEP II metalloprotease activity obtained according to the methods described above can also be useful for detecting the NEP II protein.
La présente invention a donc également pour objet un procédé de détection de NEP II dans un échantillon cellulaire ou tissuiaire ou dans des cellules ou un tissu comprenant les étapes consistant àThe present invention therefore also relates to a method for detecting NEP II in a cell or tissue sample or in cells or tissue comprising the steps consisting in
- mettre en contact ledit échantillon cellulaire ou tissuiaire, lesdites cellules ou ledit tissu avec un composé substrat du polypeptide NEP II obtenu tel que défini précédemment ou avec un composé inhibiteur de l'activité métalloprotéasique de NEP II obtenu selon le procédé de criblage tel que défini précédemment, ledit composé substrat ou ledit composé inhibiteur étant marqué,- bringing said cell or tissue sample, said cells or said tissue into contact with a substrate compound of the NEP II polypeptide obtained as defined above or with a compound inhibiting the metalloprotease activity of NEP II obtained according to the screening process as defined previously, said substrate compound or said inhibitor compound being labeled,
- détecter la présence dudit composé substrat ou dudit composé inhibiteur, indicatrice de la présence du polypeptide NEP II Par "composé substrat marqué" ou "inhibiteur marqué", on entend un composé substrat ou un composé inhibiteur marqué de manière détectable, par exemple par un groupement radioactif, enzymatique, fluorogène ou fluorescent, etc- detecting the presence of said substrate compound or of said inhibitor compound, indicative of the presence of the NEP II polypeptide By "labeled substrate compound" or "labeled inhibitor" means a substrate compound or an inhibitor compound detectably labeled, for example by a radioactive, enzymatic, fluorogenic or fluorescent group, etc.
Les exemples suivants illustrent l'invention sans la limiterThe following examples illustrate the invention without limiting it
EXEMPLE 1 :EXAMPLE 1:
Clonage de l'ADNc codant pour NEP 11 chez le ratCloning of the NEP 11 coding cDNA in rats
Des oligonucléotides dégénérés ont été obtenus à partir de l'alignement des séquences peptidiques des enzymes ECE, NEP I et Kell et de la délimitation des zones de forte homologieDegenerate oligonucleotides were obtained from the alignment of the peptide sequences of the enzymes ECE, NEP I and Kell and from the delimitation of the zones of strong homology
L'ARN total de différents tissus de rat (cerveau, intestin et testicules) a été soumis à une transcription inverse (RT) et amplifié par réaction en chaîne à la polymérase (PCR), à l'aide d'une paire d'o gonucléotides dégénérés sur la région N-terminale riche en résidus cystéine Les séquences de ces oligonucléotides dégénérés sont les suivantesTotal RNA from different rat tissues (brain, gut and testes) was reverse transcribed (RT) and amplified by polymerase chain reaction (PCR), using a pair of o degenerate gonucleotides on the N-terminal region rich in cysteine residues The sequences of these degenerate oligonucleotides are as follows
DCYS2 CCC AAG (G/T)CG (A/G)G(A/G) CTG GTC DCYS3 T(A/T)(C/T) GC(A C T/G) GG(A/T) GG(A/C) TGGDCYS2 CCC AAG (G / T) CG (A / G) G (A / G) CTG GTC DCYS3 T (A / T) (C / T) GC (ACT / G) GG (A / T) GG (A / C) TGG
Ceci a permis d'amplifier un fragment de 420 paires de bases à partir de l'ARNt de testicule codant pour une phase ouverte de lecture qui présente une homologie de 76% avec la protéine NEP I Cette séquence a été complétée par 3' et 5' RACE (rapid amplification of cDNA ends), à partir d'ARNt de cerveau de de testicules Les séquences ont été confirmées par la vérification de cinq clones différents pour chaque tissu et chaque amplification L'ADNc complet (SEQ ID n° 1 ) a alors été clone dans les vecteurs PCR2 1 et pcDNA3 1 (Iπvitrogen)This made it possible to amplify a fragment of 420 base pairs from the testis tRNA coding for an open reading phase which exhibits 76% homology with the NEP I protein. This sequence was completed by 3 ′ and 5 'RACE (rapid amplification of cDNA ends), from brain testis tRNA The sequences were confirmed by the verification of five different clones for each tissue and each amplification The complete cDNA (SEQ ID No. 1) a then was cloned into the vectors PCR2 1 and pcDNA3 1 (Iπvitrogen)
EXEMPLE 2 :EXAMPLE 2:
Caractéristiques du polypeptide NEP II de ratCharacteristics of the rat NEP II polypeptide
Le nouveau gène isolé code pour une protéine de 774 acides aminés (SEQ ID n° 2) qui, outre de fortes homologies avec les enzymes NEP I,The new isolated gene codes for a protein of 774 amino acids (SEQ ID No. 2) which, in addition to strong homologies with the enzymes NEP I,
ECE et Kell (52%, 40% et 28% d'identité en acides aminés, respectivement) possède la séquence consensus du site actif HEXXH, une région transmembranaire (acides aminés 24 à 40 sur la séquence SEQ ID n° 2) suivie de quatre résidus cystéine caractéristiques de cette famille, et sept sites potentiels de glycosylation Trois épissages alternatifs ont été identifiés par séquençage des RACE et par RT-PCR Un de ces épissages alternatifs élimine un site potentiel de glycosylation et pourrait affecter le transit de la protéine à la surface de la cellule ou son activité Chaque épissage correspond par ailleurs à un exon de la NEP I, ce qui suggère une structure de gène similaire Ces données démontrent une appartenance de cette nouvelle enzyme à la famille des métalloprotéases ECE/NEP/Kell Son homologie marquante avec NEP I a conduit à la nommer NEP II EXEMPLE 3 :ECE and Kell (52%, 40% and 28% amino acid identity, respectively) has the consensus sequence of the active site HEXXH, a transmembrane region (amino acids 24 to 40 on the sequence SEQ ID No. 2) followed by four cysteine residues characteristic of this family, and seven potential glycosylation sites Three alternative splices have been identified by RACE sequencing and by RT-PCR One of these alternative splices eliminates a potential glycosylation site and could affect the transit of the protein to the cell surface or its activity Each splicing also corresponds to an exon of NEP I, which suggests a similar gene structure These data demonstrate that this new enzyme belongs to the ECE / NEP / Kell metalloprotease family Its striking homology with NEP I led to the name NEP II EXAMPLE 3:
Clonage de l'ADNc codant pour NEP II chez l'hommeCloning of the cDNA encoding NEP II in humans
Afin de cloner l'homologue humain de NEP II, deux oligonucléotides ont été conçus, basés sur la séquence proteique de NEP II de rat Les séquences ont été choisies d'une part pour leur faible dégénérescence (comme par exemple un tryptophane, représenté par un seul codoπ dans le code génétique) et d'autre part pour leur degré de conservation (comme le site de liaison du zinc)In order to clone the human NEP II homolog, two oligonucleotides were designed, based on the protein sequence of rat NEP II. The sequences were chosen on the one hand for their low degeneration (such as for example a tryptophan, represented by a only codoπ in the genetic code) and secondly for their degree of conservation (such as the zinc binding site)
1 - (H)EITHFD (SEQ ID n°28) ou 5' - CGA GAT CAC ACA TGG CTT TGA TGA - 3' (S) (SEQ ID n°22)1 - (H) EITHFD (SEQ ID n ° 28) or 5 '- CGA GAT CAC ACA TGG CTT TGA TGA - 3' (S) (SEQ ID n ° 22)
2- QVWCGS (SEQ ID n°29) ou 5'- GGA CCC ACA CCA CAC CTG - 3' (AS) (SEQ ID n°23)2- QVWCGS (SEQ ID n ° 29) or 5'- GGA CCC ACA CCA CAC CTG - 3 '(AS) (SEQ ID n ° 23)
Une réaction en chaîne à la polymérase a été effectuée sur de l'ADNc d'hippocampe humain obtenu à partir d'une banque (Stratagene), et une bande de 330 pb a été amplifiée, sous-clonée et sequencée (SEQ ID n°3) La séquence obtenue présente une homologie de séquence de 82 % avec la NEPII de rat, ce qui permet d'affirmer qu'elle code pour l'homologue humainA polymerase chain reaction was carried out on human hippocampus cDNA obtained from a library (Stratagene), and a band of 330 bp was amplified, subcloned and sequenced (SEQ ID no. 3) The sequence obtained has a 82% sequence homology with rat NEPII, which makes it possible to confirm that it codes for the human counterpart
La présence du site de liaison du zinc HEITH a été confirmée parThe presence of the HEITH zinc binding site has been confirmed by
5' RACE à l'aide des oligonucléotides HNII-2 et HNII-3, spécifiques à l'humain De même, les oligonucléotides HNII-1 et HNII-2 permettront l'amplification de la région 3' par la technique de 3' RACE5 ' RACE using the human specific oligonucleotides HNII-2 and HNII-3 Similarly, the oligonucleotides HNII-1 and HNII-2 will allow the amplification of the 3' region by the technique of 3 'RACE
HNII-1 5'- CGG CCT GGA TCT CAC CCA TGA G - 3' (SEQ ID n°24)HNII-1 5'- CGG CCT GGA TCT CAC CCA TGA G - 3 '(SEQ ID n ° 24)
HNII-2 5'- CTG ACT GCT CCC GGA AGT GCT GGG TG - 3' (SEQ ID n°25)HNII-2 5'- CTG ACT GCT CCC GGA AGT GCT GGG TG - 3 '(SEQ ID n ° 25)
HNII-3 5'- GAG CAG CTC TTC TTC ATC - 3' (SEQ ID n°26)HNII-3 5'- GAG CAG CTC TTC TTC ATC - 3 '(SEQ ID n ° 26)
HNII-4 5'- CTC CAC CAA TCC ATC ATG TTG C - 3' (SEQ ID n°27) EXEMPLE 4 :HNII-4 5 ' - CTC CAC CAA TCC ATC ATG TTG C - 3' (SEQ ID n ° 27) EXAMPLE 4:
Expression tissuiaire de NEP II Des études de Northem-blot et de RT-PCR montrent que NEP II est codé par un transcrit de 2,8 Kb très fortement exprimé dans les testicules de rat et, modérément, dans le coeur, le foie, le système digestif et le cerveau Des études de RT-PCR semi-quantitatives montrent un profil d'expression similaire dans ces tissus ainsi qu'une prédominance des formes longues Toutes ces caractéristiques indiquent clairement que la protéine identifiée pour la première fois est une métalloprotéase membranaire (ectoprotéase) responsable du métabolisme de peptides messagers neuronaux et/ou hormonauxNEP II tissue expression Studies by Northem-blot and RT-PCR show that NEP II is encoded by a 2.8 Kb transcript very strongly expressed in rat testes and, moderately, in the heart, liver, digestive system and brain Semi-quantitative RT-PCR studies show a similar expression profile in these tissues as well as a predominance of long forms All these characteristics clearly indicate that the protein identified for the first time is a membrane metalloprotease ( ectoprotease) responsible for the metabolism of neuronal and / or hormonal messenger peptides
Le polypeptide NEP II natif est exprimé de manière hétérogène dans le système nerveux, les glandes (hypophyse, testicule), l'appareil digestif (intestin grêle notamment), l'appareil cardiovasculaire (coeur notamment)The native NEP II polypeptide is expressed heterogeneously in the nervous system, the glands (pituitary gland, testis), the digestive system (especially the small intestine), the cardiovascular system (especially the heart)
Les techniques d'hybridation in situ indiquent en outre une forte expression de la protéine NEP II dans les neurones et les cellules adénohypophysaires exprimant le gène de la POMC (propiomélanocortine), précurseur de l'ACTHIn situ hybridization techniques also indicate a high expression of the NEP II protein in neurons and adenohypophysial cells expressing the POMC (propiomelanocortin) gene, precursor of ACTH.
Ces localisations indiquent la participation de NEP II dans la proteolyse d'hormones et de neurotransmetteurs peptidergiques ou de leurs précurseurs émanant de ou agissant sur ces divers organes II devient dès lors intéressant dans un but thérapeutique d'affecter les transmissions peptidergiques correspondantes en inhibant NEP II These localizations indicate the participation of NEP II in the proteolysis of hormones and peptidergic neurotransmitters or their precursors emanating from or acting on these various organs II therefore becomes interesting for therapeutic purposes to affect the corresponding peptidergic transmissions by inhibiting NEP II

Claims

1414
REVENDICATIONS
Polypeptide isolé comprenant une séquence d'acides aminés choisie parmi la séquence SEQ ID n°2 ou SEQ ID n° 4, une séquence dérivée ou homologue de ladite séquence SEQ ID n°2 ou SEQ ID n° 4 ou un fragment biologiquement actif de ladite séquence SEQ ID n°2 ou SEQ ID n° 4, ledit polypeptide isolé étant désigné par «NEP II » Acide nucléique isolé comprenant une séquence nucléotidique choisie parmi la séquence SEQ ID n°1 ou SEQ ID n° 3, une séquence dérivée ou homologue de ladite séquence SEQ ID n°1 ou n° 3, ou leurs séquences complémentaires Sonde oligonucléotidique hybridant spécifiquement avec une séquence nucléotidique selon la revendication 2, ladite sonde ayant une séquence nucléotidique choisie parmi les séquences SEQ ID n°5 à SEQ ID n°27 Vecteur de clonage et/ou d'expression contenant une séquence nucléotidique selon la revendication 2 Cellule hôte transfectée par un vecteur selon la revendication 4 Anticorps mono- ou polyclonaux ou leurs fragments, anticorps chimériques ou immunoconjugués, caractérisés en ce qu'ils sont obtenus à partir d'un polypeptide selon la revendication 1 administré à un animal, et sont capables le reconnaître spécifiquement un polypeptide selon la revendication 1Isolated polypeptide comprising an amino acid sequence chosen from the sequence SEQ ID No. 2 or SEQ ID No. 4, a sequence derived from or homologous to said sequence SEQ ID No. 2 or SEQ ID No. 4 or a biologically active fragment of said sequence SEQ ID No. 2 or SEQ ID No. 4, said isolated polypeptide being designated by "NEP II" Isolated nucleic acid comprising a nucleotide sequence chosen from the sequence SEQ ID No. 1 or SEQ ID No. 3, a derived sequence or homolog of said sequence SEQ ID No. 1 or No. 3, or their complementary sequences Oligonucleotide probe specifically hybridizing with a nucleotide sequence according to claim 2, said probe having a nucleotide sequence chosen from the sequences SEQ ID No. 5 to SEQ ID n ° 27 Cloning and / or expression vector containing a nucleotide sequence according to claim 2 Host cell transfected by a vector according to claim 4 Mono- or polyclonal antibodies or their fragments, chimeric or immunoconjugate antibodies, characterized in that they are obtained from a polypeptide according to claim 1 administered to an animal, and are capable of specifically recognizing it a polypeptide according to claim 1
7 Procédé de détection immunologique de NEP II dans un échantillon cellulaire ou tissuiaire ou dans des cellules ou un tissu comprenant les étapes consistant à7 A method of immunological detection of NEP II in a cell or tissue sample or in cells or tissue comprising the steps of
- mettre en contact ledit échantillon cellulaire ou tissuiaire, lesdites cellules ou ledit tissu avec un anticorps détectable selon la revendication 6 , - détecter la présence dudit anticorps, indicatrice de la présence du polypeptide NEP II 8 Procédé de détection de l'expression du polypeptide NEP II dans un échantillon cellulaire ou tissuiaire ou dans des cellules ou un tissu par hybridation m situ, comprenant les étapes consistant à- contacting said cell or tissue sample, said cells or said tissue with a detectable antibody according to claim 6, - detecting the presence of said antibody, indicative of the presence of the NEP II polypeptide 8 A method of detecting the expression of the NEP II polypeptide in a cell or tissue sample or in cells or tissue by m situ hybridization, comprising the steps consisting in
- préparer TARN dudit échantillon ou desdites cellules ou dudit tissu,- prepare TARN of said sample or of said cells or of said tissue,
- mettre en contact ledit ARN obtenu avec au moins une sonde ayant une séquence nucléotidique capable de s'hybnder spécifiquement avec une séquence nucléotidique selon la revendication 2, ladite sonde pouvant être notamment une sonde oligonucléotidique selon la revendication 3 , - détecter la présence d'ARNm hybπdant avec ladite sonde, indicatrice de l'expression du polypeptide NEP II- bringing said RNA obtained into contact with at least one probe having a nucleotide sequence capable of specifically hybridizing with a nucleotide sequence according to claim 2, said probe possibly being in particular an oligonucleotide probe according to claim 3, - detecting the presence of MRNA hybridizing with said probe, indicative of the expression of the NEP II polypeptide
9 Procédé d'identification de composés substrats du polypeptide NEP II selon la revendication 1 , dans lequel on met en contact lesdits composés, éventuellement marqués, avec le polypeptide NEP II, et on évalue la coupure desdits composés par NEP II, indicatrice de l'activité métalloprotéasique de NEP II envers lesdits composés substrats9 A method of identifying substrate compounds of the NEP II polypeptide according to claim 1, in which said compounds, optionally labeled, are brought into contact with the NEP II polypeptide, and the cleavage of said compounds is evaluated by NEP II, indicative of the metalloprotease activity of NEP II towards said substrate compounds
10 Procédé de détection de l'activité métalloprotéasique de NEP II dans un échantillon cellulaire ou tissuiaire ou dans des cellules ou un tissu comprenant les étapes consistant à - mettre en contact ledit échantillon cellulaire ou tissuiaire, lesdites cellules ou ledit tissu avec un composé substrat du polypeptide NEP II obtenu selon le procédé de la revendication 9, ledit composé substrat étant éventuellement marqué ,10 A method of detecting the metalloprotease activity of NEP II in a cell or tissue sample or in cells or tissue comprising the steps consisting in - contacting said cell or tissue sample, said cells or said tissue with a substrate compound of the NEP II polypeptide obtained according to the method of claim 9, said substrate compound being optionally labeled,
- évaluer la coupure dudit composé substrat, indicatrice de l'activité métalloprotéasique de NEP II- evaluate the cleavage of said substrate compound, indicative of the metalloprotease activity of NEP II
11 Procédé de criblage de composés susceptibles d'inhiber l'activité métalloprotéasique du polypeptide NEP II selon la revendication 1 , dans lequel on met en contact lesdits composés avec ledit polypeptide NEP II et on évalue le taux d'inhibition de l'activité métalloprotéasique de NEP II 12 Procédé de détection de NEP II dans un échantillon cellulaire ou tissuiaire ou dans des cellules ou un tissu comprenant les étapes consistant à - mettre en contact ledit échantillon cellulaire ou tissuiaire, lesdites cellules ou ledit tissu avec un composé substrat du polypeptide NEP II obtenu selon le procédé de la revendication 9 ou avec un composé inhibiteur de l'activité métalloprotéasique de NEP II obtenu selon le procédé de criblage de la revendication 11 , ledit composé substrat ou ledit composé inhibiteur étant marqué ,11. A method of screening for compounds capable of inhibiting the metalloprotease activity of the NEP II polypeptide according to claim 1, in which said compounds are brought into contact with said NEP II polypeptide and the rate of inhibition of the metalloprotease activity of is evaluated. CIP II 12 A method of detecting CIP II in a cell or tissue sample or in cells or tissue comprising the steps of - bringing said cell or tissue sample, said cells or said tissue into contact with a substrate compound of the NEP II polypeptide obtained according to the process of claim 9 or with a compound inhibiting the metalloprotease activity of NEP II obtained by the screening process of claim 11, said substrate compound or said inhibitor compound being labeled,
- détecter la présence dudit composé substrat ou dudit composé inhibiteur, indicatrice de la présence du polypeptide NEP II.- detecting the presence of said substrate compound or of said inhibitor compound, indicative of the presence of the NEP II polypeptide.
13 Utilisation du polypeptide NEP II selon la revendication 1 pour le criblage de composés inhibiteurs de l'activité métalloprotéasique de NEP II utiles pour la fabrication de médicament destiné à traiter les troubles impliquant les transmissions peptidergiques auxquelles participe NEP II.13 Use of the NEP II polypeptide according to claim 1 for the screening of compounds inhibiting the metalloprotease activity of NEP II useful for the manufacture of a medicament intended for treating disorders involving peptidergic transmissions in which NEP II participates.
14 Utilisation selon la revendication 13 dans laquelle lesdits troubles sont choisis parmi les maladies cardiovasculaires, neurodégénératives, les troubles de la croissance d'origine endocrinienne, les perturbations de l'axe hypothalamo-hypophysaire et les affections endocriniennes. 14 Use according to claim 13 wherein said disorders are chosen from cardiovascular, neurodegenerative diseases, growth disorders of endocrine origin, disturbances of the hypothalamic-pituitary axis and endocrine disorders.
EP99911898A 1998-04-08 1999-04-07 Membranemetalloprotease nepii Withdrawn EP1068336A2 (en)

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FR9804389 1998-04-08
FR9804389A FR2777291B1 (en) 1998-04-08 1998-04-08 NOVEL MEMBRANE METALLOPROTEASE NEP II AND ITS USE FOR THE SCREENING OF INHIBITORS USEFUL IN THERAPY
PCT/FR1999/000807 WO1999053077A1 (en) 1998-04-08 1999-04-07 Novel nep ii membrane metalloprotease and its use for screening inhibitors useful in therapy

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US20020086405A1 (en) * 2000-09-25 2002-07-04 Inmaculada Silos-Santiago 56638, a novel human neprilysin protease and uses thereof
CA2260376A1 (en) * 1999-02-11 2000-08-11 Universite De Montreal New metalloproteases of the neprilysin family
EP1069188A1 (en) * 1999-07-15 2001-01-17 Sanofi-Synthelabo Three neprilysin-like membrane metallopeptidases
EP1234025B1 (en) * 1999-11-19 2008-08-27 Solvay Pharmaceuticals B.V. Human enzymes of the metalloprotease family
JP2001275687A (en) * 2000-04-04 2001-10-09 Masafumi Matsuo Membrane-bound metalloprotease and soluble secretor thereof
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