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  • 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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• 1998
  • 1998-04-08 FR FR9804389A patent/FR2777291B1/fr not_active Expired - Fee Related
• 1999
  • 1999-04-07 JP JP2000543624A patent/JP2002511271A/ja not_active Withdrawn
  • 1999-04-07 WO PCT/FR1999/000807 patent/WO1999053077A1/fr not_active Application Discontinuation
  • 1999-04-07 CA CA002325599A patent/CA2325599A1/fr not_active Abandoned
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