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WO2012007777A1 - Nouvelles protéines: procédés de préparation et utilisation correspondants - Google Patents

Nouvelles protéines: procédés de préparation et utilisation correspondants Download PDF

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Publication number
WO2012007777A1
WO2012007777A1 PCT/HU2011/000066 HU2011000066W WO2012007777A1 WO 2012007777 A1 WO2012007777 A1 WO 2012007777A1 HU 2011000066 W HU2011000066 W HU 2011000066W WO 2012007777 A1 WO2012007777 A1 WO 2012007777A1
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WO
WIPO (PCT)
Prior art keywords
masp
seq
protein
proteins
enzyme
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.)
Ceased
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PCT/HU2011/000066
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English (en)
Inventor
Péter GÁL
Gábor PÁL
Dávid HÉJA
Péter ZÁVODSKY
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.)
Magyar Tudomanyos Akademia Szegedi Biologiai Kozpont Enzimologiai Intezete
Eotvos Lorand University
Original Assignee
Magyar Tudomanyos Akademia Szegedi Biologiai Kozpont Enzimologiai Intezete
Eotvos Lorand University
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Publication of WO2012007777A1 publication Critical patent/WO2012007777A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the invention relates to novel proteins, and it also relates to a process for the production of such proteins and to the use of such proteins in the production of medicines .
  • the complement system is one of the most important
  • the complement system as the immune system in general, is able to recognise, label and remove intruding pathogens and altered host structures (e.g. apoptotic cells) .
  • the complement system as a part of the innate immune system, forms one of the first defence lines of the organism against pathogenic microorganisms, but it also links to the adaptive (acquired) immune system at several points forming a bridge, as it were, between innate and adaptive immune mechanism (Walport 2001a; Walport 2001b; Morgan 2005).
  • the complement system is a network consisting of about 30 protein components, which components can be found in the blood plasma in soluble form, and also in the form of receptors and modulators (e.g. inhibitors) attached to the surface of cells.
  • the main components of the system are serine protease zymogens, which activate each other in a cascade-like manner in strictly determined order. Certain substrates of the activated proteases are proteins
  • cytotoxic activity which is triggered by the membrane attack complex (MAC) formed from the terminal components of the complement system.
  • MAC membrane attack complex
  • complement system Another important function of the complement system is opsonisation, when the active complement components (e.g. Clq, MBL, C4b, C3b) settling on the surface of the cells promote the phagocytosis by leukocytes (e.g. macrophages). These leukocytes engulf the cells to be destroyed.
  • active complement components e.g. Clq, MBL, C4b, C3b
  • leukocytes e.g. macrophages
  • the components of the complement system are present in blood plasma in an inactive (zymogenic) form until the activation of the complement cascade is triggered by an appropriate signal (e.g. intrusion of a foreign cell, pathogen) .
  • an appropriate signal e.g. intrusion of a foreign cell, pathogen.
  • the complement system can be activated via three different pathways: the classical pathway, the lectin pathway and the alternative pathway.
  • the classical pathway the classical pathway
  • the lectin pathway the alternative pathway.
  • the CI complex binds to the surface of the classical pathway
  • the CI complex is a supramolecular complex
  • C4bC2a precursors of the C3-convertase enzyme complex
  • C4bC2a precursors of the C3-convertase enzyme complex
  • the C3-convertase splits C3 components and transforms into C5-convertase (C4bC2aC3b) .
  • the C5-convertase cleaves C5, after which the activation of the complement system
  • MBL mannose-binding lectin
  • H, L and M types ficolins
  • MASP-2 MASP-2
  • the activated MASP-2 cleaves the C4 and C2 components, which results in the formation of the C3-convertase enzyme complex already described in the course of the classical pathway, and from this point the process continues as described above.
  • C3bB serine protease
  • the alternative pathway may also be triggered spontaneously, independently, by the slow hydrolysis of the C3 component (C3w) , but if either the classical or the lectin pathway gets to the point of C3 cleavage, the alternative pathway significantly amplifies their effect.
  • C3w C3 component
  • MASP-2 even in itself is able to initiate the complement cascade (Ambrus 2003; Gal 2005) , but this latter enzyme is present in a smaller amount (0.5 ⁇ g/ml) than MASP-1.
  • the physiological function of the MASP-1 protease present in a higher amount (7 g/ml) has not been completely explored yet.
  • MASP-1 on its own is not able to initiate the complement cascade (it can only cleave C2 but not C4), its activity may supplement the activity of MASP-2 at several points, therefore active MASP-1 may be necessary for amplifying and consummating the effect of the lectin pathway.
  • MASP-1 is a protease similar to thrombin, forming a bridge between the two major proteolytic cascade systems - the complement system and the blood coagulation system - in the blood (Hajela 2002; Krarup 2008) .
  • the gene of both MASP-1 and MASP-2 has an alternative splicing product.
  • the MApl9 (sMAP) protein is produced from the MASP-2 gene, containing the first two domains of MASP-2 (CUB1-EGF) .
  • the Map44 and the MASP-3 mRNA are transcribed from the MASP-1 gene.
  • the MAp44 is also a truncated protein: it contains only the first four non-catalytic domains CUB1-EGF-CUB2-CCP1 ) , so it does not have proteolytic activity (Degn 2009) . Its function is unknown, it probably plays a role in regulation.
  • MASP-3 The first five domains of MASP-3 are the same as the domains of MASP- 1, but they differ in their serine protease domain.
  • MASP-3 has low proteolytic activity on synthetic substrates, and its natural substrate is not known. Unlike other early proteases, it does not form a complex with the Cl-inhibitor molecule. Probably the presence of MApl9, Map44 and MASP-3 acts against the activation of the lectin pathway, as these proteolytically inactive proteins compete with the active MASP-2 and MASP-1 enzymes for the binding sites on the recognition molecules.
  • ischemia-reperfusion (hereinafter: IR) injury, which occurs, when the oxygen supply of a tissue is temporarily restricted or interrupted (ischemia) for any reason (e.g. vascular obstruction) , and after the restoration of blood circulation (reperfusion) cellular destruction starts.
  • IR ischemia-reperfusion
  • ischemic cells as altered self cells and starts an
  • the lectin pathway probably plays a role in the development of IR injury. For this reason the deliberate suppression of the lectin pathway may reduce the extent and the consequences of IR injury.
  • the lectin pathway may also become activated in the case of rheumatoid arthritis (hereinafter: RA) as MBL binds to the antibody form IgG-GO having altered glycosylation accumulated in the joints during RA.
  • RA rheumatoid arthritis
  • the uncontrolled activity of the complement system also plays a role in the development and maintenance of different neurodegenerative diseases (e.g. Alzheimer's, Huntington's and Parkinson's diseases, Sclerosis Multiplex) , and it is one of the main factors in the pathogenesis of age-related macular
  • the complement system can also be associated with one of the forms of autoimmune nephritis (glomerulonephritis) and with another autoimmune disease, namely SLE (systemic lupus erythematosus) .
  • SLE systemic lupus erythematosus
  • liposomes may generate allergic type reactions independent from antibodies in patients. This pseudo-allergic reaction is due to the activation of the complement system
  • CARPA complement activation-related pseudo-allergy
  • the efficient and selective inhibition of certain activation pathways becomes possible without triggering general immunosuppression.
  • the lectin pathway can be blocked selectively (e.g. in the case of the diseases mentioned above) , and by this the classical pathway responsible for the elimination of immunocomplexes is left untouched, that is functioning.
  • the Clr, Cls, MASP-1, MASP-2 and MASP-3 enzymes form an enzyme family having the same domain structure (Gal 2007) .
  • the trypsin-like serine protease (SP) domain responsible for proteolytic activity is preceded by five non-catalytic domains.
  • Growth Factor are responsible for the dimerization of the molecules (both in the case of MASP-1 and MASP-2) and for interacting with other molecules, e.g. for binding to the recognition molecules.
  • CCP1-CCP2-SP fragment Complement Control Protein
  • the SP domain contains the active centre characteristic of serine proteases, the substrate binding pocket and the oxyanion hole. Eight surface loop regions, the conformation of which is quite different in the different proteases, play a decisive role in determining subsite specificity.
  • the CCP modules stabilise the structure of the catalytic region, and on the other part they contain binding sites for large protein substrates.
  • the small-molecule compounds generally used for inhibiting trypsin-like serine proteases e.g. benzamidine, NPGB, FUT- 175
  • the activity of complement proteases too
  • proteases in the blood plasma e.g. blood coagulation enzymes, kallikreins.
  • the only known natural inhibitor of the complement system, CI inhibitor protein circulating in blood and belonging to the serpin family is also characterised by relatively wide specificity .
  • the first lectin pathway selective inhibitors according to the prior art, developed by the inventors of the present invention were described in Hungarian patent application no. P0900319.
  • the reversibly binding peptide inhibitors according to this earlier invention inhibit the activation of the lectin pathway by inactivating the MASP-1 and MASP-2 enzymes, while the classical and the alternative pathways remain intact.
  • These earlier inhibitors are based on the SFTI (Sunflower Trypsin Inhibitor) , and in a functional sense two types have been developed: the inhibitor
  • SFTI Sunflower Trypsin Inhibitor
  • the inhibition of the complement system may be an efficient tool in fighting against human and animal diseases occurring as a result of the abnormal activity of the complement system.
  • inhibitors having the SFTI structure can either inhibit both proteases (MASP-1 and MASP-2) or they are selective MASP-2 inhibitors.
  • the proteins according to the present invention show a greater affinity to the target enzymes and are more
  • One of the main aims of increasing specificity is to throw light upon the specific role of the individual MASP enzymes in the lectin pathway and to open up more selective
  • Xi is M, F, V, A, I, and
  • X 2 is R, K, and
  • X 3 is K, R, L, A and
  • X 4 is L, G, M, A, , Y, and
  • X 5 is W, Y, L, M, N, E, G.
  • the invention relates to proteins containing sequences according to general formula (I), their salts, esters and pharmaceutically acceptable prodrugs .
  • the proteins according to the invention are modified proteins within the pacifastin family, especially preferably modified SGCI protein, where the modification lies in that the protein contains one of the sequences according to formula (I) .
  • the invention also relates to pharmaceutical preparations that contain at least one protein containing sequences according to general formula (I), its salt, ester or prodrug and at least one further additive.
  • This additive is preferably a matrix ensuring controlled active agent release .
  • the invention relates especially to pharmaceutical
  • the invention most preferably relates to pharmaceutical preparations that contain at least one of the proteins containing the following sequences:
  • the invention also relates to kits containing at least one protein containing sequences according to general formula (I), its salt or ester.
  • the invention also relates to the screening procedure of compounds potentially inhibiting MASP enzymes, in the course of which a labelled protein according to the invention is added to a solution containing MASP, then the solution containing one or more compounds to be tested is added to it, and the amount of the released labelled protein is measured.
  • the MASP enzyme is preferably MASP-1 or MASP-2 enzyme.
  • the invention also relates to the use of proteins
  • diseases can be selected preferably from the following group: inflammatory and autoimmune diseases, especially preferably ischemia- reperfusion injury, rheumatoid arthritis, neurodegenerative diseases, age-related macular degeneration,
  • the invention also relates to a procedure for isolating MASP enzymes, in the course of which a carrier with one or more immobilised protein with sequences according to general formula (I) are contacted with a solution
  • the MASP enzyme is preferably MASP-1 or MASP-2 enzyme .
  • figure 1 shows a schematic representation of the phage display method
  • figure 2 shows the sequence logo diagrams of the obtained sequences
  • figure 2. a shows the sequence diagram relating to the sequences selected on the MASP-1 enzyme and specific to it; figure 2b shows the sequence diagram relating to the sequences selected on the MASP-2 enzyme and specific to it.
  • the present invention relates to proteins and protein derivatives selectively inhibiting ASP-1 and MASP-2 enzymes .
  • the present invention also relates to proteins and protein derivatives which are sequentially analogous to the
  • the scope of protection of the present invention also includes proteins in which elements ensuring detectability (e.g. fluorescent group, radioactive atom, etc.) are integrated. Furthermore, the scope of protection of the present invention
  • proteins that contain a few further amino acids at their N-terminal, C-terminal, or both ends, if these further amino acids do not have a significant influence on the biological activity of the original sequence.
  • the aim of such further amino acids positioned the ends may be to facilitate immobilisation, ensure the possibility of linking to other reagents, influence
  • the present invention also relates to the pharmaceutically acceptable salts of the proteins containing sequences according to general formula (I) according to the
  • salts which, during contact with human or animal tissues, do not result in an
  • hydrochloride hydrobromide, hydroiodide, lactare, maleate, methane sulphonate, oxalate, propionate, succinate,
  • salts based on the following are mentioned: alkali metals and alkaline earth metals
  • prodrugs are compounds that transform in vivo into a protein according to the present invention. Transformation can take place for example in the blood during enzymatic hydrolysis.
  • the proteins according to the invention can be used in pharmaceutical preparations, where one or more additives are needed to reach the appropriate biological effect. Such preparations may be pharmaceutical preparations combined, for example, with matrices ensuring controlled active agent release, widely known by a person skilled in the art.
  • matrices ensuring controlled active agent release are polymers that, when entering the appropriate tissue (e.g. blood plasma), decompose, for example in the course of enzymatic or acid-base hydrolysis (e.g. polylactide, polyglycolide) .
  • additives known in the state of the art can also be used, such as diluents, fillers, pH regulators, substances promoting dissolution, colouring additives, antioxidants, preservatives, isotonic agents, etc. These additives are known in the state of the art.
  • the pharmaceutical preparations according to the invention can be entered in the organism via parenteral (intravenous, intramuscular, subcutaneous, etc.)
  • compositions may be aqueous or non-aqueous solutions, dispersions, suspensions, emulsions, or solid (e.g. powdered) preparations, which can be transformed into one of the above fluids directly before use.
  • suitable vehicles, carriers, diluents or solvents may be for example water, ethanol, different polyols (e.g.
  • glycerol propylene glycol, polyethylene glycols and similar substances
  • carboxymethyl cellulose carboxymethyl cellulose
  • different (vegetable) oils organic esters, and mixtures of all these substances .
  • preparations according to the invention include among others infusions, tablets, powders, granules,
  • the administered dose depends on the type of the given disease, the patient's sex, age, weight, and on the
  • the preferable daily dose may vary for example between 0.01 mg and 1 g
  • parenteral administration e.g. a preparation administered intravenously
  • the preferable daily dose may vary for example between 0.001 mg and 100 mg in respect of the active agent.
  • the dose to be selected depends very much on the molecular weight of the given protein used.
  • the pharmaceutical preparations can also be used in liposomes or microcapsules known in the state of the art.
  • the proteins according to the invention can also be entered in the target organism by state-of-the-art means of gene therapy.
  • selective inhibitory proteins should be preferably
  • inventions selectively inhibiting the MASP-2 enzyme may be the peptide with the sequence VCTRLYCN (SEQ ID NO 7), while the sequence according to the invention selectively
  • inhibiting the MASP-1 enzyme may be the peptide with the sequence FCTRKLCY (SEQ ID NO 3) .
  • FCTRKLCY SEQ ID NO 3
  • the rest of the protein does not influence selectivity.
  • a given protein may have parts beyond the sequence part according to the invention, which may influence the inhibitory activity shown towards the MASP target enzymes. Practically such undesired effects should be filtered out by performing experiments, and planning these in advance is quite
  • proteins are not preferred, which, apart from the sequence part according to the invention, also contain parts showing any interaction with MASP enzymes having a negative influence on inhibition.
  • the proteins according to the invention can be preferably used in different kits, which can be used for measuring or localising different MASP enzymes (either in a way specific to any MASP enzyme, or both to the MASP-1 and MASP-2 enzymes at the same time) . Such use may extend to
  • kits are especially preferable, which are suitable for the examination of the potential inhibitors of MASP enzymes, e.g. in competitive binding assays.
  • a potential inhibitor' s ability of how much it can displace the protein according to the invention from a MASP enzyme can be measured.
  • the protein according to the invention needs to be labelled in some way (e.g.
  • kits according to the invention may also contain other solutions, tools and starting substances needed for
  • invention according to general formula (I) can also be used for screening compounds potentially inhibiting MASP
  • the compounds binding to the MASP enzyme (if the tested compound binds to the surface of the enzyme partly or completely at the same site as the sequence of the protein according to the invention, or somewhere else, but its binding alters the conformation of the MASP enzyme in such a way that it loses its ability to bind the protein) displace the labelled protein from the MASP molecule to the extent of their inhibiting ability.
  • concentration of the displaced proteins can be determined using any method suitable for detecting the (e.g. fluorescent or
  • radioactive labelling used on the protein molecules.
  • the incubation periods, washing conditions, detection methods and other parameters can be optimised in a way known by th person skilled in the art.
  • the screening procedure
  • HTS high- throughput screening
  • the proteins according to the invention can be used first of all in the medical treatment of diseases, in the case o which the inhibition of the operation of the complement system has preferable effects. Consequently the present invention also relates to the use of proteins in the production of medicaments for the treatment of such diseases. As it has been explained above in detail, such diseases are first of all certain inflammatory and
  • autoimmune diseases especially the following diseases: ischemia-reperfusion injury, rheumatoid arthritis,
  • neurodegenerative diseases e.g. Alzheimer's, Huntington's and Parkinson's disease, Sclerosis Multiplex
  • age-related macular degeneration e.g. glomerulonephritis, systemic lupus erythematosus, complement activation-related pseudo- allergy .
  • the proteins according to the invention can also be used for isolating MASP proteins, by immobilising proteins and making the preparation made in this way come into contact with the solution presumably containing MASP enzyme. If this solution really contains MASP enzyme, it will be anchored via the immobilised protein. This procedure can b suitable both for analytical and preparative purposes. If the geometry of the binding of the given protein on the MASP enzyme is not known, during this procedure a peptide anchored from several directions or even several proteins should be used to ensure appropriate linking.
  • the solution containing the MASP enzyme can be a pure protein solution, an extract purified to different extents, tissue preparation, etc.
  • proteins containing the sequences according to the invention we mean the following.
  • such protein we mean any amino acid sequence, which consists of the sequence according to general formula (I) at least.
  • this sequence is a part of a larger protein to make sure that the two extreme members of the sequence of general formula (I) according to the invention (that is amino acids marked Xi and X 5 in general formula (I)) are situated at an appropriate
  • the sequence part according to the invention can assume the appropriate optimal geometry for inhibiting the MASP enzymes. For this reason the distance measured between the alpha carbon atoms of the two extreme amino acids is preferably 20 ⁇ 4 A.
  • the appropriate distance between the two extreme amino acids of the sequence according to the invention can be ensured by inserting it in a larger protein, and also by adding a suitable shorter sequence part, even a few amino acids, and by creating a covalent or ionic bond between them.
  • cysteine side chains can be inserted a few positions before and after the two extreme amino acids mentioned above, and by creating appropriate conditions between these cysteine side chains, a covalent disulfide bridge can be created. It follows from the above that according to the present invention shorter peptides and modified peptides are also regarded as
  • the protein according to the invention is preferably a protein within the pacifastin family, carrying the sequence according to the invention in the given position.
  • the protein is an SGPI-2 protein within the pacifastin family, which has the sequence according to the invention at the given position.
  • the SGPI-2 protein has the following sequence (SEQ ID NO 13) :
  • underlined part indicates the sequence part to be replaced with the sequence part according to the present invention .
  • the protein according to the invention is preferably an SGPI-2 protein which preferably contains the FCTRKLCY (SEQ ID NO 3) sequence, then the protein according to the invention has the following amino acid sequence (SEQ ID NO 14) :
  • pacifastin and SGCI which has the sequence with general formula (I) according to the
  • Such proteins can be referred to as modified proteins within the pacifastin family, or as modified SGCI proteins.
  • the proteins according to the invention were developed using the phage display method.
  • the phage display is suitable for the realisation of directed in vitro evolution, the main steps of the state- of-the-art procedure (Smith 1985) can be seen in figure 1.
  • the gene of the protein involved in evolution is linked to a bacteriophage envelope protein gene.
  • the phage particle carries the gene of the foreign protein inside, while on its surface it displays the foreign protein.
  • the protein and its gene are physically linked via the phage.
  • phage protein library is created. Each phage displays only one type of protein variant and carries only the gene of this variant.
  • the individual variants can be separated from each other using analogue methods to affinity chromatography, on the basis of their ability to bind to a given target molecule chosen by the researcher (and generally linked to the surface) .
  • phage protein variants selected in this way have two important characteristic features. On the one part they are able to multiply, on the other part they carry the coding gene wrapped in the phage particle.
  • Binding variants are multiplied, and after several cycles of selection-multiplication a population rich in functional variants is obtained. From this
  • the marked inhibitor structure should be: canonic, highly efficient on human trypsin and small.
  • a further expectation in connection with the new inhibitor sceleton was that it should have a protease binding loop with the least possible structural constraints.
  • the structure of the member named SGPI-2 in the pacifastin inhibitor family fulfils these requirements: it is canonic, it can inhibit several different types of human serine protease, and with its length of 35 amino acids it can still be regarded small.
  • SGPI-2 Schistocerca Gregaria Peptidase Inhibitor-2
  • SGCI S. Gregaria Chymotrypsin Inhibitor
  • the inhibitors within the family have a triple antiparallel stranded beta sheet structure consisting of about 35 amino acids. They can be characterised with the following consensus sequence:
  • the six cysteines form bridges between the three beta strands suiting the abcacb pattern.
  • the inhibition loop contacting the protease is situated near the C-terminus (Malik 1999) .
  • the initial SGCI inhibitor loop's amino acid sequence ACTLKACP (SEQ ID NO 9) .
  • amino acid sequence of the inhibition loop characteristic of the library is XCXXXXCX,
  • X can be any one of the 20 amino acids.
  • the underlined part indicates the so-called Pi group, which generally bears outstanding significance from the aspect of specificity, as it reaches into deep binding pocket of the enzyme responsible for primary selectivity.
  • the binding molecule displayed should be presented in a low copy number per phage, ideally in one single copy (monovalent phage display) .
  • the phage-SGCI library was created through a glycine-serine linker as the N-terminal fusion of the p8 main envelope protein. In connection with the SGCI molecule described in this way we have pointed out earlier that it appears on the phage surface in one single copy (Szenthe 2007) .
  • Example 1 creating the phage library 1.1.
  • Preparation of a Kunkel-template In the first step, from pGP8-Tag-SGCI phagemid a single- stranded Kunkel-template is prepared, in which stop codons were inserted using Kunkel's method (Kunkel 1985) in the positions to be randomised at a later point. The role of the stop codons is to eliminate possible wild-type SGCI backgrounds while creating the DNA library. The mutagenesis used when creating the library is never 100% efficient, some of the created population is of the same sequence as the template. In our case this population does not appear as a displayed peptide, as it contains numerous stop codons.
  • the DNA library was also created using Kunkel's method. For this we used degenerate oligonucleotides.
  • the DNA library created in this way was introduced into supercompetent cells by electroporation .
  • the phage protein library was created by the helper phage infection of the cell culture. 1.2. Creating the DNA library
  • the DNA solution was cooled on ice.
  • 1.2.1.2 Production and isolation of uracil-containing phage From a separate colony cells were inoculated in 2 ml 2YT/ampicillin (100 ⁇ g/ml) , chloramphenicol (5 ⁇ g/ml) medium and grown overnight, shaken at 37 °C. On the following day 30 ⁇ culture was inoculated in 30 ml medium of the same composition. As soon as the light dispersion of the cell suspension measured at 600 nm (OD600) reached 0.4, it was infected with M13-K07 helper phage allowing 10 phages per coli cell on average. After shaking it for 30 minutes at 37 °C kanamycin was added to the solution in a final concentration of 25 ⁇ g/ml.
  • the cells were shaken for 16 more hours at 37 °C. Then the cells were isolated from the culture by centrifugation (10,000 rpm, 10 minutes, 4 °C) , and from the supernatant containing the phages the phages were precipitated in a clean centrifuge tube: adding 1/5 volume PEG/NaCl solution (20% PEG 8000, 2.5 M NaCl). After thoroughly mixing in the precipitation agent, the sample was left alone for 20 minutes at room temperature. Then the phage particles were settled by centrifuging (12,000 rpm, 10 minutes, 4 °C) .
  • the liquid stuck to the wall of the tube was collected by centrifuging it for a while (1,000 rpm, 1 minute, 4 °C) and then it was removed with a pipette.
  • the phages were suspended in 800 ⁇ PBS, and the remaining cell fragments were removed from the sample by centrifuging it in a microcentrifuge (12,000 rpm, 10 minutes, 4 °C) .
  • the supernatant obtained in this way contained pure phages.
  • ssDNA Single-stranded DNA
  • Stop mutations were introduced with the following oligonucleotide (SEQ ID NO 10) : 5' -
  • the library mutagenesis was realised in a similar way as described above in point 1.2., but using ten times the amounts determined therein.
  • the library oligo is analogous with the stop mutation oligo, but here there are degenerate triplets on the place of the TAA stop codons .
  • Oligo phosphorylation was performed as described above.
  • the template for the mutagenesis was the uracil-containing ssDNA carrying the stop codons, which was created from the pGP8-Tag-SGCI-STOP phagemid obtained as a result of the procedure described above in detail, in CJ236 cells, by M13K07 helper phage infection.
  • the amount of the template was used: 20 ⁇ g, and the annulation volume was also increased by ten times to 250 ⁇ .
  • the incubation periods were extended: 90°C 2 minutes, 50°C 5 minutes. The amounts were multiplied by ten for polymerisation too, the reaction took place overnight at 16 °C.
  • the library was introduced to the supercompetent cells via electroporation. Our aim was to introduce the plasmid to as many cells as possible, so that our library contains 10 8 -10 9 pieces.
  • the DNA library which is situated in USP distilled water so it is salt-free, was added to 2 x 350 ml supercompetent cells.
  • the operation was performed in a cuvette with a diameter of 0.2 cm, according to the following protocol: 2.5 kV, 200 ohm, 25 F.
  • the cells were carefully transferred into 2 x 25 ml of SOC medium, incubated for 30 minutes at 100 rpm, at 37 °C, then a sample was taken, a sequence was diluted from it and dripped onto [LB] , [LB; 100 ⁇ g/ml ampicillin] and [LB; 10 g/ml tetracycline] plates, and it was grown overnight at 37 °C. The same procedure was followed in the case of non-electroporated control products and control products electroporated with water. After taking a sample, the 2 x 25 ml culture was infected with 2 x 250 ⁇ M13K07 helper phage, shaken at 37 °C for 30 minutes at 220 rpm, and then the whole product was
  • Human MASP-targets consist of a serine-protease (SP) domain and two complement control protein domains (CCP-1,-2) (Gal 2007). These are recombinant fragment products, which carry the catalytic activity of the entire molecule.
  • SP serine-protease
  • CCP-1,-2 complement control protein domains
  • the proteins were produced in the form of inclusion bodies, from which the conformation with biological activity was obtained by renaturation . Purification was performed by anion and cation exchange separation. The activity of the proteins was tested in a solution and also in a form linked to the ELISA plate. (For the precise details of production see Ambrus 2003) .
  • MASP-1 (hereinafter MASP-1) .
  • phages were produced in 2 x 250 ml of culture for 18 hours. In the first step of the selection they were isolated to be able to use the library immediately for display.
  • the cell culture was centrifuged at 8,000 rpm for 10
  • the white phage precipitate was solubilised in 25 ml [PBS; 5 mg/ml BSA; 0.05% Tween-20] buffer. In order to remove possible cell fragments it was centrifuged again, the supernatant was transferred into clean tubes.
  • the first selection cycle a) Immobilisation: The target molecules were immobilised on a 96-well Nunc Maxisorp ELISA plate (cat#442404 ) . During immobilisation the concentration of MASP-1 and
  • MASP-1 was incubated while mixing at 110 rev/min. at room temperature for 60 minutes, the antibody was incubated for 30 minutes, and MASP-2 was incubated overnight at
  • E. coli XL1 Blue culture During the term of the selection, XLI Blue cells were inoculated from a plate freshly picked in advance using an inoculating loop, into 2 x 30 ml of medium [2YT; 10 ⁇ g/ml tetracycline] .
  • the phages produced for 18 hours were isolated as described above, but at the end they were solubilised in 10 ml of sterile PBS buffer.
  • the concentration of the phage solutions was measured at 268 nm, and then they were diluted with [PBS; 2 mg/ml casein; 0.05% Tween-20] buffer so that each of them has a uniform OD268 value of 0.5, and this is how they were used in the step of display.
  • 2.7 ml of fresh exponentially growing XL1 Blue cells was infected with 300 ⁇ of eluted phage.
  • casein was also kept in the buffers. After isolation the phages were solubilised in 2.8 ml of sterile PBS, and for display they were diluted to OD268 -0.5.
  • the antibody can be much more preferably immobilised on the surface of the ELISA plate, and so much more phages were eluted. Due to the high phage concentration there is the risk of one cell being infected by several phages, which results in a mixed, incomprehensible sequences.
  • MASP-1 and MASP-2 proteins were immobilised in a concentration of 0.01 ⁇ g/ ⁇ l, while the anti-Flag-tag antibody in a concentration of 1 g/ml, in a volume of 100 ⁇ /well, as described above in connection with selection, on Nunc ELISA Maxisorp plates. Each clone was tested on its own target protein, on the background and on anti-Flag-tag antibody .
  • Phage application The phages produced and isolated as described above were diluted by 2 times using [PBS; 2 mg/ml casein; 0.05% Tween-20] buffer, and 100 ⁇ was placed in the wells. From the same clone samples were pipetted into a total of 3 wells. Incubation was performed at room temperature, for 1 hour, while mixing at 110 rev/min.
  • Anti-M13 antibody 100 ⁇ of monoclonal anti-M13 HRP conjugated antibody (Amersham, cat#27-9421-01 ) diluted in [PBS; 2 mg/ml casein; 0,05% Tween-20] buffer 10,000 times was placed in the wells, and then it was incubated for 30 minutes at room temperature, while mixing it at 110 rev/min.
  • the NNK codon pattern used when constructing the DNA library does not ensure the same initial frequency for the individual amino acids.
  • an amino acid may have one, two or three codons .
  • the simplest way of correction is to use codon normalisation, and divide all amino acid frequencies by the number of codons the given amino acid is represented by in the NNK set.
  • the other, more realistic approach is normalisation with the data of the sequences selected from the antibody. Not all theoretically possible sequence types can be displayed on the surface of the phages, as some of them do not result in a realisable construction, or they significantly deteriorate peptide production or the efficiency of leaving the cell.
  • the sequence of the clones selected with anti-Flag-tag antibody reveals this non- random nature relating to producibility, so it can be used for normalisation.
  • the amino acid frequency values of the MASP-selected population are divided by the corresponding frequency values of the population selected on the Flag-tag antibody.
  • sequence logo diagrams are shown in figure 2, where the figure numbers (that is 2. a and 2.b) relate to the sequence logo diagram of the above two groups marked a) and b) , in the same order.
  • the sequence logo is the graphic display of the information content and amino acid distribution per position in a set of multiple aligned sequences, using the single-letter abbreviations of the amino acids.
  • the column height of the logo indicates how even the occurrence of the elements (20 different types of amino acids in our case) is. The less even this occurrence is, the higher the column. In the case of completely even distribution (all 20 amino acids occur in a proportion of 5%) the height is zero. The maximum value belongs to the case, when only one type of element (amino acid) occurs. Within the column the individual amino acids are arranged on the basis of the frequency of occurrence, the most frequent one is at the top.
  • the height of the letter indicating the amino acid is in proportion with its relative frequency of occurrence in the given position (for example, in the case of 50% frequency of occurrence, it is half the height of the column) .
  • generally amino acids with similar chemical characteristics are shown in the same or in a similar colour, for which we used different shades of grey in the figure belonging to the present patent description.
  • site PI corresponds to position 4.
  • the information content of the positions is determined in bits.
  • the logo diagrams illustrate the selection taking place in the individual positions.
  • the necessity to bind to the MASP enzymes resulted in intensive selection, which especially affected positions 3-6 (P2-P2' ) .
  • P2-P2' affected positions 3-6
  • the significant reduction of the combination range is basically due to two reasons. One of these is that the spatial structure needed for efficient inhibition dictates forced conditions for the sequence. The other restriction is due to the quite narrow substrate-specificity of the MASP enzymes.
  • the former one is illustrated by the only dominant threonine side change in position P2, which dominates this position independently from the enzyme.
  • the reason for this is that the group is an internal structural element, which ensures the rigidity of the inhibition loop, and as a result of this it is one of the basic pillars of fulfilling the inhibitor function.
  • positions (4-6) it can be seen how strict selection by the enzyme is asserted.
  • the strong commitment in different directions in the same positions of the two logos was a promising sign concerning the selectivity of the inhibitors .
  • PSS means periplasmic signal sequence
  • MBP Maltose Binding Protein, E. coli's own periplasmic protein, as a fusion partner;
  • poliN linker means an asparagin distance keeping member consisting of ten members in the manufacturer's vector, between the fusion members;
  • TEV cleavage site means the specific recognition and cleavage site created for the TEV protease (Tobacco Etch Virus protease) ;
  • Stop means TAATAA translation stop codons.
  • the individual SGCI clones were removed from the phagemid vector used for selection, using PCR reaction.
  • universal 5' primer with which we inserted an EcoRI cleavage site, which is underlined in the sequence; a TEV protease recognition site, which is marked in the sequence in bold.
  • the protease enzyme cleaves after the TEV protease recognition site.
  • GlySerGly linker which is marked in italics.
  • the underlined part and the part in italics is on the N-terminal of the SGCI (SEQ ID NO 12) : 5' -ACTGGAATTCGAAAACCTGTATTTTCAGGGATCCGGCGAGGrGACCrGCGAACCG- 3'
  • the 3' primers were already clone-specific, they had to be planned separately for each variant, as the randomised part was near the C-terminal.
  • At the common part of the 3' PCR primers we inserted a Hindlll cleavage site and two stop codons in each product.
  • the recipient vector (pMal-p2G) was opened under the same conditions as the PCR product, with EcoRI and Hindlll enzymes, 1 ug plasmid was used. The digested product was run in 0.8% agarose gel, then it was cut out from there and isolated with a QIAgen Gel Extraction kit and eluted with 50 ul 0.1 x EB.
  • the reaction was incubated for 2 hours at room temperature.
  • the cell suspension was divided into two 50 ml falcon tubes and frozen overnight at -20 °C. On the following day the cells were defrosted at room temperature, and immediately after this they were centrifuged (10 minutes, 10, 000 rpm, 4°C) and the supernatant was kept. This is the periplasm containing fusion protein. During freezing the cytoplasm of the cells is protected by the cell wall, but a smaller part of the cells is unavoidably extruded and genomic DNA gets into the periplasm. The DNA content was removed with general nuclease treatment and then with salting out. 20U Benzonase nuclease (Novagen) was added to the periplasm per ml and incubated overnight at room temperature.
  • the fusion protein was salted out with 90% saturated ammonium sulphate and the precipitated proteins were centrifuged (10 minutes, 10,000 rpm, 4°C) .
  • the precipitate was suspended in 70 ml 2.5 mM HC1 solution, and it was dialysed for two times one hour in 2 litres of 2.5 mM HC1 solution.
  • the majority of the contaminating protein is precipitated, and the precipitation was removed by centrifuging (10 minutes, 15000 rpm, 4 °C) .
  • Mass spectrometry analysis was realised with HP1100 type HPLC-ESI-MS system, with flow-injection method, using 10 mM ammonium formate, pH 3.5 solution.
  • the settings of the device were the following. Both the drying and the pulverising gas was nitrogen, the flow rate of the drying gas was 10 1/minute, its temperature was 300 Celsius degrees.
  • the pressure of the pulverising gas was 210 kPa, the capillary voltage was 3500 V.
  • the total ion current (TIC) chromatogram was recorded in positive ion setting within the range of 100-1500 mass/charge.
  • the mass data were evaluated with Agilent ChemStation software.
  • the inhibition constant of all eight inhibitor variants produced was measured on both enzymes.
  • catalytic enzyme fragments also used for selection (CCP1-CCP2-SP) .
  • the synthetic substrate used in the measurements was Z-L-Lys-SBzl hydrochloride (Sigma, C3647), from which a 10 mM stock solution was prepared.
  • the reactions were performed in a volume of 0.5 ml, at room temperature, in a buffer consisting of [20 mM HEPES; 145 mM NaCl; 5 mM CaCl 2 ; 0.05% Triton-X100 ] .
  • the substrate cleaved by the enzyme entered into a reaction with the dithiodipyridine auxiliary substrate (Aldrithiol-4 , Sigma, cat#143057) present in the solution in 2x excess.
  • the release of the chromophore group created in this way was monitored in a spectrophotometer at 324 nm.
  • a dilution sequence was prepared from the individual inhibitors, the enzyme was added to it, and it was incubated for 1 hour at room temperature.
  • the concentration of the substrate and the length of the measuring period were chosen so that under the given conditions the enzyme should consume less than 10% of the substrate.
  • a measuring method developed for the characterisation of tight-binding inhibitors was used (Empie, 1982) .
  • the MASP-1 and MASP- 2 concentration was determined by titration with CI inhibitor. The results were calculated as the average of parallel measurements. The results are summarised in table 3.
  • Table 3 Summarising table of the enzyme inhibition of the individual inhibitors. In the sequences shown the bold letters have the same meaning as in table 2.
  • the complement system can be activated through three pathways and it leads to the same single end-point.
  • Three activation pathways include the classical, the lectin and the alternative pathway.
  • MASP-s are the enzymes of the initial phase of the lectin pathway, so it is important to know what effect the MASP inhibitors developed by us have on the lectin pathway, on the other two activation pathways and on the joint phase following the meeting of the three pathways .
  • the so-called IELISA kit Euro- Diagnostica AB, COMPL300 developed for the selective measuring of the complement pathways, on the basis of the instructions for use attached to the kit.
  • the guiding principle of measuring is that according to the three activation pathways it uses three measuring conditions, in which the currently examined complement activation pathway can operate, while the other two pathways are inactive.
  • the product detected during measuring is not a pathway-selective component, but the last element of the joint section of the activation pathways, the C5-9 complex.
  • the blood sample was incubated for 1 hour at room temperature, then it was centrifuged and the serum was stored in small batches at -80 °C.
  • the serum was diluted according to the prescriptions with the buffer belonging to the given complement pathway, it was incubated for 20 minutes at room temperature, the dilution sequence prepared from inhibitors was added to it, it was incubated for 20 minutes at room temperature, then it was pipetted into the appropriate wells of a special ELISA plate. In the following, washing, incubation and antibody addition was performed according to the protocol. It was incubated for 20 minutes with the substrate, and then the data was read at 450 nm in a spectrophotometer. 100% activity was represented by the serum without an inhibitor.
  • the measurements were performed at the same time and on the same plate, from one single melted serum sample.
  • the measurements lead to the result that the inhibitors selected on the MASP-2 enzyme are efficient and specific inhibitors of the lectin pathway of the complement system. This result is in compliance with the result demonstrated earlier, according to which these inhibitors inhibit the MASP-2 enzyme very efficiently, which enzyme, according to our present knowledge, is responsible for the initiation of the lectin pathway.
  • the inhibitor variants selected on the MASP-1 enzyme produced an unexpected result. These variants, without any exception, also proved to be efficient and specific inhibitors of the lectin pathway.
  • Table 4 The inhibitor concentrations (IC50) needed for reducing the uninhibited lectin pathway activity by half, the Ki values, and the proportion of the inhibitor concentrations (IC50) needed for reducing the uninhibited lectin pathway activity by half and the Ki values.
  • VCTRLWCN 110 35 3.1
  • the presence of the inhibitors did not inhibit the creation of the terminal C5-9 complex, it is for certain that the peptides do not inhibit the proteases of the joint section of the complement system, so the inhibition of the lectin pathway really took place at the beginning of the lectin pathway, at the level of the MASP enzymes.
  • the IC50 data obtained in the course of the WIELISA measuring is about 2- 10 times higher than the Ki values obtained in the course of MASP-2 inhibition measurements based on synthetic substrates. A possible explanation for this is the following.
  • the inhibitor peptides bind to the MASP-2 enzyme directly at the substrate binding site, and this binding successfully competes with the relatively weak interaction of small synthetic substrates with the same enzyme surface.
  • physiological substrates can create bonds via other surfaces too (exosites), and they bind to the enzyme with a higher affinity than small synthetic substrates. It is because of this higher affinity that inhibitor molecules must be used in a higher concentration for the balance to be shifted from the enzyme-substrate complex towards the enzyme-inhibitor complex.
  • Prothrombin time (PT) testing the extrinsic pathway of blood coagulation was measured on Sysmex CA-500 (Sysmex, Japan) automatic system using Innovin Reagent (Dale Behring, Marburg, Germany) .
  • Activated partial thromboplastin time (APTT) testing the intrinsic pathway of blood coagulation and thrombin time (TT) directly testing thrombin operation was measured on a Coag-A-Mate MAX (BioMerieux, France) analyser using TriniClot reagent (Trinity Biotech, Wichlow, Ireland) and Reanal reagent (Reanal Finechemical , Hungary) .
  • the effect of the inhibitors on blood coagulation was measured in a final concentration of 5 uM, which is 40-90 times the IC50 values determined in the case of MASP-2 inhibitors and ⁇ 125 times the IC50 value determined in the case of MASP-1 inhibitors in the WIELISA test.
  • MASP-2 inhibitors that in the measuring concentration they are inefficient with respect to all proteases of the blood coagulation cascade. It complies with our knowledge according to which MASP-2 and blood coagulation proteases have no common physiological substrate. From this aspect MASP-1 is different, as it has several known common substrates with thrombin: fibrinogen, coagulation factor XIII or the PAR-4 receptor (Krarup 2008, Megyeri 2009) . Despite all this, in a concentration of 5 uM none of the MASP-1 inhibitor variants inhibited the activity of thrombin, which is clearly indicated by the unchanged nature of the thrombin times.
  • MASP-1 as a thrombin-like enzyme, may contribute to the blood coagulation process.
  • This effect of MASP-1 has been recently demonstrated in MASP-1 knockout transgenic mice (Takahashi 2010) . It is important to point out that this effect on the intrinsic activation pathway is only a slight effect, which can be neglected from a physiological aspect.
  • Table 5 gives a summary of the effect of inhibitors on blood coagulation times.
  • Table 5 The effect of the MASP-1 and MASP2 inhibitors on blood coagulation times.
  • MBL associated serine protease 1 (MASPl) on factor XIII and fibrinogen. Biochim. Biophys . Acta. 1784, 1294-1300.

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Abstract

L'invention concerne des nouveaux peptides représentés par la formule générale (I), X1CTX2X3X4CX5 (I), dans laquelle X1 représente: M, F, V, A, I; X2 représente: R, K; X3 représente: K, R, L, A; X4 représente: L, G, M, A, W, Y; et X5 représente: W, Y, L, M, N, E, G; ainsi que leurs sels, esters et promédicaments de qualité pharmaceutique. Par ailleurs, l'invention concerne des préparations pharmaceutiques et des kits contenant ces peptides, des protocoles de criblage et d'isolement intégrant ces peptides, ainsi que l'utilisation de ces peptides dans la production de préparations pharmaceutiques.
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