Classifications

• • 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
  • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
  • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
  • C12N9/6416—Metalloendopeptidases (3.4.24)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• New endopeptidase Its activity of cleavage and inactivation of peptide substrates.
• the invention relates to a new endopeptidase and its activity of cleavage of peptide substrates and, where appropriate, the inactivation of these substrates.
• NEP neutral endopeptidase
• ACE angiotensin converting enzyme
• peptidases act by cleavage of peptide bonds within determined substrates, with a narrow selectivity both at the level of the cleaved substrate (s) and at the level of the cleaved peptide bonds. They sometimes act by cleavage of multiple sites within the same peptide substrate.
• BNP Brain Natriuretic peptide
• Bourne et al have described the fact that the membrane preparations make it possible to obtain a hydrolysis of the Ser 25 -Phe 26 bond of the ⁇ -hANP which releases a COOH-terminal Phe-Arg-Tyr fragment. They also note in their article that endopeptidase 24.11 acts at the same time as other endopeptidases which according to them are metalloendopeptidases and cysteine endopeptidases.
• Xaa-Phe, Xaa-Ile, Xaa-Leu type in which Xaa is an amino acid, preferably chosen from Ser, Phe, Tyr, His, Gly, or Ala, when this doublet is present inside a peptide substrate whose sequence includes more than 5 amino acids,
• divalent cation chelating agents such as EDTA, EGTA, o-phenantroline or DTT at concentrations of the order of 1 mM
• NEP neutral endopeptidase inhibitors
• ACE angiotensin converting enzyme
• the endopeptidase type enzyme is further characterized by its ability to cleave, under determined conditions, the peptide bond of amino acid doublets of the Xaa-Tyr or Xaa-Trp type .
• Xaa has the definition given above and the doublet of amino acids is present inside a peptide substrate whose sequence comprises more than 5 amino acids.
• a sequence of more than 5 amino acids corresponding to the preceding definition capable of being cleaved by the endopeptidase of the invention is in particular present in the carboxy-terminal part of hormones such as ANF or of hormones of the family tachykinins, kinins, bombesin, or angiotensin.
• This sequence of more than 5 amino acids advantageously comprises, for example, 6 amino acids; this sequence is involved in the formation of the binding site of the enzyme.
• the conditions for cleavage of the amino acid doublets were determined for different substrates in the examples provided on the following pages.
• these conditions for determining the cleavage capacity of an enzyme according to the invention are as follows: 2 nmoles of ANF (5-28) are incubated in a phosphate buffer at pH 7.4 or 7, 5 for 2 hours in the presence of an amount of enzyme protein equivalent to 0.1 / mole of the pure enzyme (final volume of 20 ⁇ l). At the end of the incubation, the entire sample (20 ⁇ l) is injected onto an HPLC column (C 18 ).
• the ANF fragments (5-25) and (26-28) are identified by their retention time by compared to standards and by their amino acid composition.
• the reaction between the enzyme and the substrate can be stopped by heating for 10 min at 100oC.
• the enzyme can be characterized by its capacity to hydrolyze bonds in the N-terminal position of hydrophobic amino acids.
• the enzyme when the enzyme is in contact with a substrate capable of being cleaved, and when this substrate comprises a doublet of hydrophobic amino acids, the cleavage takes place preferably between the two hydrophobic amino acids.
• the substrate of an enzyme thus defined can be characterized by the fact that it is a peptide in which the hydrophobic amino acid in the C-terminal position in the doublet capable of being cleaved, also has a carboxyl group. - ⁇ (CH 2 ⁇ ) free.
• the enzyme binding site is hydrophobic in nature.
• the enzyme according to the invention is capable of cleaving the peptide bonds of the doublets according to Gly 33 -Leu 34 , Phe 19 -Phe 20 , Lys 16 -Leu 17 , His 13 -His 14 and His 6 -Asp 7 of the human amyloid peptide whereas the corresponding fragment (25-35) containing the same doublet Gly 33 -Leu 34 constituted only an inhibitor.
• the enzyme also cleaved the Gly-Leu doublet of neurokinin A, while it did not cleave the Ser-Phe doublet of this neurokinin A.
• the Ala-Leu doublet of the PGLa enzyme remained intact while the Lys-Ile doublet was cleaved.
• the invention also relates to any fragment of the enzyme defined above, in particular any fragment of this enzyme verifying the properties stated above, containing in particular the active site of the enzyme.
• enzyme and “endopeptidase” will apply to both the enzyme and its fragments.
• amino acid doublets capable of being cleaved by the enzyme of the invention have been given above by way of example.
• Other doublets can be cleaved by endopeptidase, as long as the amino acid designated by Xaa has a structural relationship with one of the amino acids of the group Ser, Phe, Tyr, Lys or Gly, within the bond with an amino acid chosen from, Phe, Ile, or Leu.
• amino acid Phe, Ile or Leu is substituted by another amino acid since the doublet formed, capable of being cleaved retains the structural and conformational properties of one of the doublets given above. .
• amino acid doublets capable of giving rise to cleavage by the action of the endopeptidase of the invention are present inside a peptide substrate, that is to say that they neither form the doublet of the carboxy-terminal end (COOH-terminal) nor the doublet of the amino-terminal end (NH 2 -terminal) of the peptide substrate. Furthermore, the potential cleavage does not take place by the action of the enzyme of the invention, only when the substrate contains more than five amino acids.
• the doublet capable of being cleaved is present in the carboxy-terminal half (part) of the substrate.
• the endopeptidase enzyme of the invention has an insensitivity relative to NEP or ACE inhibitors such as phosphoramidon or captopril.
• relative insensitivity is meant the fact that the cleavage activity of the endopeptidase of the invention is not inhibited by more than 50% by concentrations of the order of 1 mM or 1 ⁇ M of these usually active inhibitors on the cleavage activity of NEP or ACE respectively on enkephalinase and on angiotensin.
• phosphoramidon or captopril partially inhibits the endopeptidase of the invention only at concentrations 100 to 1000 times greater than those which make it possible to obtain the inhibition of the cleavage activity of NEP. or ACE.
• the endopeptidase of the invention can cleave a given substrate at the level of several amino acid doublets; in this case, one of the cleavages carried out remains the majority in proportion to the cleavage product obtained.
• the cleavage can be carried out at a single site of the substrate.
• This cleavage can also be selective and therefore only relate to a particular amino acid doublet, even if other doublets capable of being cleaved according to the preceding definition, are present in the substrate.
• peptide substrates capable of being cleaved by endopeptidase are substance P, the bradykinin, somatostatin especially somatostatin 14, neuromedins B and C, litorin, angiotensin II, or related substrates from the point of view of their amino acid sequence.
• the endopeptidase of the invention is also characterized in that it does not cleave minigastrin I, enkephalin and its carboxamide derivative, oxytocin, vasopressin.
• Cleavage by the enzyme can also, by respecting the conditions previously given, cause cleavage of polypeptides or peptides contained in the peptide substrates described above.
• the enzyme corresponding to the preceding definitions is capable of cleaving, by a single and selective cleavage, the Ser 25 -Phe 26 bond of human ANF and in particular of the peptide comprising amino acids 1 to 28 of ANF (ANF ( 1-28)) or of the peptide comprising amino acids 5 to 28 of ANF (ANF (5-28)).
• This cleavage is advantageously an inactivating cleavage of ANF, abolishing its hormonal functions, in the regulation of blood pressure and water-sodium metabolism: in particular thanks to its capacity to increase the flow of ions and water in renal tubules (diuresis and natriuresis) by intervention of surface receptors whose recognition by ANF results in a hypotensive effect.
• ANF decreases the secretion of 2 hormones (aldosterone and vasopressin) and renin.
• the endopeptidase of the invention leads to an inactivation completely controllable by the fact of this cleavage unique and selective, at the ANF level. Also within the framework of the family of enzymes, the definition of which has been given above, an enzyme capable of cleaving the Gly 33 -Leu 34 bond of the ⁇ 1-40 peptide of the protein / 3-amyloid.
• This cleavage can be accompanied by a second cleavage at the level of the Lys 16 -Leu 17 bond of the ⁇ 1-40 peptide.
• the ⁇ -amyloid protein has for example been described in a publication by Yankner et al (Science Vol. 250: 279-282, October 12, 1990).
• the sequence of the ⁇ 1-40 peptide is as follows, according to the letter code for amino acids:
• ⁇ -amyloid protein has neurotrophic and neurotoxic effects on neurons, depending on the stage of maturation of these neurons and the concentration of ⁇ -amyloid protein. These effects of the ⁇ -amyloid protein have been associated with certain observations relating to neuronal degeneration appearing in patients suffering from Alzheimer's disease and in particular to the formation of so-called senile plaques.
• the endopeptidase according to the invention could be used to promote or on the contrary attenuate the effects of the ⁇ -amyloid protein depending on the stage of maturation of the neurons.
• An endopeptidase of the invention can also be defined by the following characteristics: it has a single peak in HPLC chromatography, on a column of C4-silica 300 A balanced with a buffer of 10 mM sodium phosphate and 1.5 sodium chloride M at pH 7.5 and at room temperature, with a flow rate of 0.5 ml / min, and it is eluted with a linear gradient consisting of a 10 mM sodium chloride buffer at pH 7.5 to a buffer 10 mM sodium phosphate to pH 7.5 in 10 minutes and a gradient from 0 to 30% methanol in a 10 mM sodium phosphate buffer at pH 7.5 in 60 minutes, the enzyme being eluted between 10 and 30% methanol.
• Enzymes of the invention are also characterized in that their affinity constant (Km) for a substrate chosen from ANF (5-28), substance P, angiotensin II, bradykinin, somatostatin-14 , neuromedin C, neuromedin B or litorin, is between 18 and 63 ⁇ M.
• Km affinity constant
• the activity of the enzyme under the experimental conditions described below is optimal at pH 7.4 or 7.5.
• the enzymes of the invention are in pure form; in other words they are homogeneous from the point of view of their activity of cleavage of determined substrates and devoid of contaminants.
• a particular group of endopeptidases of the invention is represented by the enzyme Xenopus laevis which meets the definitions given above and which has an apparent molecular mass of 100 kDa.
• Another group of enzymes according to the invention comprises human enzymes.
• the inventors have isolated and characterized an enzyme related to that of the xenope, present in particular in the nervous system. This enzyme was found and characterized in cells of the gliobastoma clone U-373 HG, human astrocytoma. This clone is identified by its reference in a cell bank.
• the human enzyme is indistinguishable from the enzyme X. laevis by its selectivity with regard to the substrates and its inhibition profile by the agents described in Table II.
• This enzyme can be isolated and purified according to a technique analogous to that described for the xenopus enzyme.
• an endopeptidase of the invention can be obtained by implementing the following steps:
• this enzyme is characterized in that molecular filtration takes place on Sephadex G50, chromatography on ion exchanger takes place on DEAE Sephadex, hydrophobic absorption chromatography takes place on aminoethylagarose.
• this enzyme is further characterized in that the elution is carried out for 10 minutes with a 10 mM sodium phosphate buffer, 1.5 M sodium chloride at pH 7.5 with a flow rate of 0.5 ml / min. then with a linear gradient with a 10 mM sodium phosphate buffer and 1.5 M sodium chloride at pH 7.5 to a 10 mM sodium phosphate buffer, devoid of sodium chloride at pH 7.5.
• the invention also relates to polyclonal or monoclonal antibodies characterized in that they recognize the endopeptidase described above or any part of this endopeptidase and in particular any site involved in the functional cleavage properties of the enzyme given above.
• Antibodies can be obtained by conventional methods and in particular from hybridomas resulting from the fusion of spleen cells from an animal previously immunized with the enzyme, with myeloma cells.
• Human monoclonal antibodies directed against this enzyme can also be prepared in vitro, for example according to the following technique:
• the fusion of immortalized B lymphocytes is carried out with human B lymphocytes previously stimulated in vitro with the endopeptidase according to the invention against which it is sought to form monoclonal antibodies, under culture conditions. allowing the stimulation of lymphocytes.
• the antibodies of the invention selectively recognize an enzyme meeting the criteria given above and in other words are devoid of reaction with respect to other endopeptidases such as NEP or ACE.
• the invention further relates to the use of endopeptidase for the screening of known molecules or for the development of new molecules capable of acting as an inhibitor or selective modulator of this endopeptidase.
• selective modulator is understood to mean any molecule capable of either inhibiting or activating the enzyme, depending on the conditions of concentration of the enzyme and of the modulator respectively and according to the conditions relating to the reaction medium.
• the invention also relates to a method for detecting the capacity of a molecule to behave as a substrate for the enzyme of the invention, characterized in that it comprises the following steps:
• the fragments possibly obtained by cleavage of the molecule tested can be identified by their amino acid composition, for example using the Pico Tag Station kit from Waters. If necessary, the amino-terminal part of these possible cleavage products is sequenced.
• the invention also relates to a method for the detection or screening of molecules capable of behaving as an inhibitor, activator or modulator of the enzyme of the invention, characterized by the following steps:
• the invention also relates to a medicament characterized in that it contains, as active principle, an enzyme of endopeptidase type corresponding to the definitions given in the preceding pages, or a fragment of this enzyme, in particular a fragment having retained the capacity of cleavage of this enzyme.
• Another medicament according to the invention contains, as active principle, an activator, an inhibitor or a modulator of the enzymatic functions of the endopeptidase of the invention.
• the invention relates to the preparation of an active drug as a hypotensive agent, capable of acting on a substrate such as ANF.
• modulator a substance capable of acting on the intensity of the activity of endopeptidase either by activation or by inhibition.
• Figure 2 Elution profile of fragments of different hormonal peptide substrates resulting from the action of the endopeptidase of Xenopus Laevis
• ANF the fragments (5-25) and (26-28) of ANF were both identified by reference to standards and by their amino acid composition;
• Substance P the fragments generated (a, b, c and d) have been identified by their amino acid composition.
• Bradykinin the four fragments generated (a, b, c and d) were identified by their amino acid composition.
• Somatostatin-14 the only reaction product generated by the cleavage at the Phe 6 -Phe 7 link was identified by the amino acid composition and by NH2-terminal sequencing.
• Neuromedin C the four reaction products (a, b, c and d) were determined by their amino acid composition.
• FIG. 3 Amino acid sequences of peptide hormones used as a substrate for the endopeptidase inactivation of Xenopua Laevis peptide hormone
• the solid arrows indicate the site of the main cleavage observed.
• the dotted arrows correspond to the minor cleavage sites. When no arrow is indicated no cleavage has been obtained.
• the sequences were aligned so as to demonstrate the Xaa-Phe, Xaa-Leu and Xaa-Ile bonds. All the corresponding fragments were unambiguously identified by their amino acid composition. In some cases the sequencing of the amino-terminal part has also been carried out (Chang et al (1983) Methods Enzymol. 91: 455-466). Km values were measured using five different substrate concentrations under conditions where a maximum of 5% cleavage was measured.
• FIG. 4 pH and phosphoramidon inhibition profiles of endopeptidase activity A): The activity was measured for each pH value on a standard test (2 nmol of substrate, for example [R 8 ] -Kermit or (5-28) -ANF in the presence of 10 ng of pure enzyme in 0.1 M phosphate buffer, in a final volume of 20 ⁇ l for 1 hour at 37oC). The buffer was adjusted to cover a pH range of 5.8 to 8.0.
• endopeptidase was carried out according to a technique essentially analogous to that used in the publication of Kuks et al (J. Biol. Chem. 264: 14609-14612, 1989) to isolate the endopeptidase RXVRG.
• Exudates were obtained from three X. laevis, subjected to molecular filtration on Sephadex G50, followed by ion exchange chromatography on DEAE Sephadex.
• the active fractions were then subjected to hydrophobic absorption chromatography on aminoethylagarose, according to the description given in the reference Kuks et al previous.
• fractions with endopeptidase activity recovered after the absorption step on aminoethylagarose (in total 60 ml) in 10 mM phosphate buffer at pH 7.4, 0.1% NP-40, were concentrated by ultrafiltration (Unisep TM cartridges with Ultracent-30 membrane, Bio-Rad) to obtain an approximate final volume of 0.6 ml .
• This sample was then placed on an HPLC column (SFCC, France) (Nucleosil TM C4 300 ⁇ (8 ⁇ 75mm)), and eluted with a buffer containing 10 mM sodium phosphate and 1.5 M sodium chloride at pH 7.4, with a flow rate of 0.5 ml / min.
• the purified enzyme was resistant to 30% methanol and 1.5 M sodium chloride (90% of the original activity is retained).
• the eluted fractions were analyzed by UV absorption at 220 nm and the enzymatic activity was monitored on each 1 ml fraction. This last purification step made it possible to obtain complete separation of the endopeptidase from the copurified endoprotease RXVRG which was eluted with 12% methanol in 10 mM sodium phosphate buffer. The active fractions were combined, and concentrated to a final volume of 30 ⁇ l.
• This sample containing the endopeptidase activity was analyzed by polyacrylamide gel electrophoresis under non-denaturing conditions or under denaturing conditions using the "Phast-System" system (Pharmacia, Uppsala, Sweden, Kuks et al, J. Biol. Chem. 264: 14609-14612, 1989). Protein content was assessed by the Bradford as described in Anal. Biochem 72: 248-254 (1976).
• the endopeptidase activity was monitored routinely using the technique described in the publication of Kuks et al above and, as substrate the peptide designated by [R 8 ] -Kermit, derived from the peptide designated by Kermit (DVDERDVRGFASFL NH2 ) which cleaved at the S 12 -F 13 link but was not cleaved by the endoprotease RXVRG at the R 8 -G 9 site .
• ANF Atrial Natriuretic Factor
• substance P substance P
• somatostatin substance P
• bradykinin substance P
• angiotensin II neuromeinins B and C
• litorin the conditions were as follows: 1-2 nmol of peptide were incubated for 1 to 5 hours in a 100 mM phosphate buffer at pH 7.4 in the presence of 10 ng of the pure enzyme for a final volume of 20 ⁇ l. The reaction was stopped by heating for 10 min at 100oC and the resulting products were directly placed on an HPLC column (Nucléosil TM 5 ⁇ C18, 146 ⁇ 4.5 mm), and eluted in accordance with the conditions described in Figure 2.
• HPLC column Nucléosil TM 5 ⁇ C18, 146 ⁇ 4.5 mm
• the substrate remaining and the products obtained were followed directly by UV absorption at 220 nm. All the fragments produced by endopeptidolytic cleavage have been identified with certainty by their amino acid composition using the PicoTag apparatus (Waters). In some cases the identification has also been verified by the sequencing of the amino-terminal part according to the description given by Chang (Methods Enzymol. 91: 455-466, 1983). Inhibitors were tested at different concentrations (see Table II) using the routine conditions of the endopeptidase test, described above and using either the peptide ANF- (5-28) or the peptide [R 8 ] -Kermit as a substrate. The ANF- peptide (5-28) comprises amino acids 5 to 28 of ANF. The inhibitions were expressed as a percentage of the reference activity measured in the absence of chemical reagents, under the same experimental conditions.
• the pH profile of the endoprotease activity was obtained using either [R 8 ] -Kermit or ANF- (5-28) as substrate (2 nmol per test) incubated for 1 hour with 10 ng of pure enzyme in 0.1 M phosphate buffer adjusted to respond to a pH range of 5.8 to 8.0 ( Figure 4).
• the peptides [R 8 ] -Kermit, ANF (24-28), [Ala 25 ] ANF (24-28), [His 25 JANF (24-28) were prepared by solid phase synthesis according to the techniques described by Tarn et al and Merrifield respectively in the publications (J. Am. Chem. Soc. 105: 6442-6455 (1983) and J. Am. Chem. Soc. 85: 2149-2154 (1963)), on an NPS 4000 multisynthesizer ( Neosystem, France) and purified by HPLC.
• the enzymatic activity offered great stability for periods of 24 hours and more under the conditions of routine incubation of the enzymatic test.
• the selectivity of the substrate for the isolated enzyme was verified on a variety of peptides ( Figure 2).
• the endopeptidase of X. laevis has in most cases given rise to a single cleavage or to a majority cleavage at the level of the Ser-Phe, Gly-Phe, Phe-Phe, His-Phe, Gly-Leu, His- Leu or Tyr-Ile peptides tested.
• the pure enzyme exhibited a strong affinity for all of the cleaved substrates.
• the Km was of the order of 18 to 63 ⁇ M, measured with respect to the peptide ANF- (5-28) of substance P, of somatostatin-14, of bradykinin, of neuromedin C and of angiotensin II ( Figure 3).
• the endopeptidase of X. laevis secretions preferably cleaves the NH2-terminal end of the amino acids Phe, Leu or Ile when they are included in a. doublet of Xaa-Phe, Xaa-Leu or Xaa-Ile type.
• the endopeptidase activity is regulated by other structural parameters such as the size of the substrate and probably its conformation.
• the Xaa-Phe, Xaa-Leu or Xaa-Ile motif forms the carboxy-terminal end of the peptide, it is not cleaved. This has been demonstrated in the case of minigastrin I and angiotensin II ( Figure 3).
• the enzyme described here is of particular interest in terms of its mode of action since it has been possible to clearly show that it produces a main selective cleavage at the level of peptide bonds of doublets Xaa-Phe, Xaa-Ile or Xaa -Leu in a large number of peptide messengers which have in common the presence of these doublets in their sequence. Consequently, the capacity of this enzyme to cleave the Xaa-Phe bond of ANF, somatostatin and substance P for example, cleavages which are recognized in all cases as responsible for inactivation, shows that this enzyme is a prototype of the class of distinct inactivation enzymes of NEP and ACE. Indeed, this hypothesis is supported by a certain number of observations.
• the amino acid doublet corresponds to a selective and inactivating cleavage site in cellular systems containing a receptor.
• ANF undergoes selective elimination of the tripeptide (Phe 26 -Tyr 28 ) by cleavage of the Ser-Phe bond in various tissues including the endothelial cells of the smooth vascular muscle (Johnson et al, J. Biol. Chem. 264: 11637 -11642, 1989 and Biochem. Biophys. Res. Commun. 167: 110-116, 1990), as well as in the cells of neuroblastoma NB-OK-1 (Delporte et al, Eur. J. Pharm. 207: 81-88 , 1991) and in the bovine renal cortex (Toll et al, Biochem. Byophys. Res. Commun. 175: 886-893, 1991).
• the ANF can be degraded in vitro by the enkephalinase which performs cleavages, essentially and in a limited way at the level of the Cys 7 -Phe 8 bond (Roques et al, Trends Pharmacol. Sci. 11: 245-249, 1990 and Bourne et al Biochem. J. 271: 381-385, 1990) whereas only a secondary reaction and more weak was observed at the Ser 25 -Phe 26 bond.
• the skin secretion enzyme which does not cleave enkephalin, was only sensitive to phosphoramidon and other classic EC 24.11 inhibitors, at concentrations one hundred to one thousand higher than those acting on the neutral endopeptidase.
• the enzyme X. laevis is a new metalloendopeptidase which hydrolyzes a bond peptide by selective recognition of an amino acid doublet containing a carboxy-terminal residue Phe or Leu or Ile, thus having thermolysin-like activity (Almenoff et al J. Neurochem 42 (1), 151-157, 1984 and Pozsgay et al, Biochem, soc. Trans. 13: 44-50, 1985).
• the enzyme X. laevis for peptide hormone inactivation (PHIE) effectively cleaves only this type of doublets, when it is included in a peptide containing more than five amino acid residues, this constituting an additional difference. and notable with the conventional structure corresponding to the NEP type activity (Pozsgay et al).

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Zoology (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• General Health & Medical Sciences (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Wood Science & Technology (AREA)
• Biochemistry (AREA)
• Medicinal Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biophysics (AREA)
• General Engineering & Computer Science (AREA)
• Microbiology (AREA)
• Immunology (AREA)
• Biotechnology (AREA)
• Biomedical Technology (AREA)
• Cardiology (AREA)
• Endocrinology (AREA)
• Toxicology (AREA)
• Gastroenterology & Hepatology (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Enzymes And Modification Thereof (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=9418372

Family Applications (1)

Country Status (4)

• 1991
  • 1991-10-28 FR FR9113279A patent/FR2682955A1/fr active Granted
• 1992
  • 1992-10-28 PT PT10101792A patent/PT101017A/pt not_active Application Discontinuation
  • 1992-10-28 MX MX9206217A patent/MX9206217A/es unknown
  • 1992-10-28 WO PCT/FR1992/001009 patent/WO1993009226A1/fr not_active Ceased

Non-Patent Citations (6)

Also Published As

Similar Documents

Legal Events