EP1991573A2 - Peptide mit den eigenschaften eines für den alpha-1a-adrenergen rezeptor selektiven allosteren antagonisten und anwendungen davon - Google Patents

Peptide mit den eigenschaften eines für den alpha-1a-adrenergen rezeptor selektiven allosteren antagonisten und anwendungen davon

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Publication number
EP1991573A2
EP1991573A2 EP07731030A EP07731030A EP1991573A2 EP 1991573 A2 EP1991573 A2 EP 1991573A2 EP 07731030 A EP07731030 A EP 07731030A EP 07731030 A EP07731030 A EP 07731030A EP 1991573 A2 EP1991573 A2 EP 1991573A2
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EP
European Patent Office
Prior art keywords
peptide
adrenergic receptor
alphala
receptor
cysteine
Prior art date
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Withdrawn
Application number
EP07731030A
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English (en)
French (fr)
Inventor
Nicolas Gilles
André Menez
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Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
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Commissariat a lEnergie Atomique CEA
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Application filed by Commissariat a lEnergie Atomique CEA filed Critical Commissariat a lEnergie Atomique CEA
Publication of EP1991573A2 publication Critical patent/EP1991573A2/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • 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
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • 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
    • A61P9/12—Antihypertensives
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors

Definitions

  • PEPTIDES WITH ALPHAIA ADRENERGIC RECEPTOR SELECTIVE AJLOSTERIC ANTAGONIST PROPERTIES AND THEIR APPLICATIONS.
  • the present invention relates to peptides having alphala adrenergic receptor selective allosteric antagonist activity and their therapeutic and pharmacological applications.
  • the alphal receptors are mainly expressed in the postsynaptic position of the sympathetic nervous system and their activation leads to a contraction of the smooth muscles under their control. These receptors play a major role in the cardiovascular and genitourinary system, in the normal or pathological state (For a review see: Piascik et al, Pharmacol Ther, 1996, 72, 215-241, Michelotti et al, Pharmacology & Therapeutics, 2000, 88, 281-309).
  • blockade of alphala receptors results in smooth muscle relaxation of the urogenital tract that can be used in the treatment of urinary dysfunction and erectile dysfunction (Moreland et al, The Journal of Pharmacology & Experimental Therapeutics, 2004, 308, 797-804, Guiliano et al, Progress in Urology, 1997, 7, 24-33).
  • the main pathologies that can be treated are: - Functional obstructions of the urinary system in women or in men.
  • Prostate adenoma, or benign prostatic hyperplasia (BPH) is the normal progression of prostate size that increases from 40 years old. This hypertrophy of the prostate whose frequency increases with age (80% of men over 70) may be accompanied by a more or less significant obstruction of the urethra, causing urinary disorders.
  • Incontinence may be due to a pressure of the bladder greater than the retention force of the urethra. Blocking alpha-adrenoceptors would reduce bladder pressure.
  • Erectile disorders Some erectile problems can be treated by inhibiting the activity of alphala adrenoceptors, in order to promote the irrigation of cavernous bodies.
  • alpha-adrenoceptor blockade would have a preventive and curative effect on prostate tumors (European Patents EP 0 799 618 and EP 0 799 619, Thebault et al, The J. Clin Invest, 2003, 111, 1691-1701).
  • KMD3213 silodosin
  • KMD3213 is the first competitive antagonist with selectivity for the alphala subtype (Shibata et al., Mol., Pharmacol., 1995, 48-250). 258). This molecule is in clinical phase for the treatment of prostatic hypertrophy (Drugs, RD, 2004, 5, 50-51).
  • the p-TIA peptide is a natural peptide of 19 residues crosslinked by two disulfide bridges (FNWRCCLIPACRRNHKKFC, SEQ ID NO: 1), extracted from Conus tulipa marine cone venom. This peptide has an affinity of the order of 100 nM for ⁇ 1 adrenoceptors, and a low selectivity (affinity of 10 nM) for the ⁇ 1b subtype which controls the tonicity of the vessels.
  • European Patent EP 1,117,681 envisages the use of the p-TIA peptide for the prevention and treatment of cardiovascular (hypertension), urinary (prostatic hypertrophy), pain and inflammation pathologies.
  • the toxins of the family "three-toed toxins” or family of “cholinergic toxins” are peptides from 63 to 66 amino acids having four disulfide bridges (between cysteines 1 and 3, 2 and 4, 5 and 6 and 7 and 8: bridges 1-3, 2-4, 5-6 and 7-8) and a characteristic three-finger structure in which the loops I, II and III form the three central fingers of the hand and the disulfide bridges, the palm from the hand.
  • These toxins are divided into several groups depending on their activity: muscarinic toxins (MTs) that bind to muscarinic acetylcholine receptors, alpha neurotoxins ( ⁇ -neurotoxins) that bind to acetylcholine receptors.
  • MTs muscarinic toxins
  • ⁇ -neurotoxins alpha neurotoxins
  • MTX1 MT1
  • MTX2 MT2
  • MTX3 MT3 or m4-tox
  • MTX4 MT4
  • MTX5 MT5
  • MTX7 MT7, ML-tox
  • MT ⁇ Dendroaspis polylepis
  • these peptides have been used as a tool to determine the physiological role of certain muscular receptor subtypes.
  • AdTxI selectively binds the adrenergic receptor alpha 1a and allosterically decreases the affinity of orthosteric ligands.
  • AdTxI has a sequence of 65 amino acids (SEQ ID NO: 2):
  • AdTx1 comprises 4 disulfide bridges, between cysteines at positions 3 and 24 (cysteines 1 and 3: bridge 1-3), 17 and 42 (cysteines 2 and 4: bridge 2-4), 46 and 57 (cysteines 5 and 6: bridge 5-6), 58 and 63 (cysteines 7 and 8: bridge 7-8), characteristics of the family of three-finger toxins acting on the cholinergic system.
  • the ATx1 sequence has a very strong homology with certain sequences of the group of muscarinic toxins in the family of three-finger toxins: Table I: Homology Between AdTxI and Certain Muscarinic Three-Finger Toxins
  • This new toxin defines a subgroup of three-fingered muscarinic toxin group peptides, characterized by their allosteric alpha-adrenergic receptor antagonist property.
  • this allosteric antagonist property of the alphala adrenergic receptor is novel and does not clearly follow from the properties of the three-finger muscarinic toxins which are described in the state of the art.
  • the present invention relates to peptides for use as a drug, said peptides being characterized by: a) a sequence selected from the group consisting of the sequence SEQ ID NO: 2, and derived variants having at least 70% d identity or 80% similarity to the entire SEQiD sequence NO: 2, b) a three-finger structure including eight cysteine residues
  • cystes 1 to 8 connected by four disulfide bridges, respectively between cysteines 1 and 3, 2 and 4, 5 and 6, 7 and 8 (bridges 1-3, 2-4, 5-6 and 7-8), and c) an allosteric antagonist activity which is selective for the alphala adrenergic receptor ( ⁇ la ).
  • the peptides as defined in the present invention are capable of specifically blocking the adrenergic alphala receptors, because of their selectivity for the alphala adrenergic subtype. .
  • inhibitors competitive antagonists
  • they act as modulators of agonist affinity; such modulators have the advantage of not blocking the functioning of the receptor but only of modulating their responses when they are activated by their agonist natural. This modulation seems easier to control than with competitive antagonists.
  • three-finger structure is understood to mean the characteristic structure of the family of three-finger toxins as defined above, which structure comprises three loops (loops I, II, II ) maintained by four disulfide bridges (bridges 1-3, 2-4, 5-6, 7-8),
  • sequence with respect to the sequence of SEQ ID NO: 2 as a reference sequence is evaluated according to the percentage of amino acid residues which are identical, when the two sequences are aligned, so as to get the maximum of correspondence between them.
  • the percentage of identity can be calculated by those skilled in the art using a computer program for comparing sequences such as, for example, that of the BLAST suite (Altschul et al, NAR, 1997, 25, 3389-3402).
  • the BLAST programs are implemented on the comparison window constituted by the whole of SEQ ID NO: 2, indicated as a reference sequence.
  • a peptide having an amino acid sequence having at least X% identity with a reference sequence is defined in the present invention as a peptide whose sequence may include up to 100-X alterations per 100 amino acids of the sequence reference, while retaining the functional properties of said reference peptide, in this case its selective antagonist activity of the alphala adrenergic subtype.
  • alteration includes deletions, substitutions or consecutive or dispersed insertions of amino acids in the reference sequence. This definition applies by analogy to nucleotide sequences.
  • a peptide having an amino acid sequence having at least X% similarity to a reference sequence is defined in this
  • non-conservative alterations includes deletions, non-conservative substitutions or consecutive or dispersed insertions of amino acids in the reference sequence.
  • the term "selective allosteric antagonist of the adrenergic receptor alphala” is intended to mean a peptide which selectively binds the adrenergic receptor alphala and is capable of allosterically decreasing the affinity of the orthosteric ligands of said receptor.
  • the orthosteric site is the site of binding of the endogenous receptor agonist (adrenaline in the case of the adrenergic receptor alphala).
  • ⁇ This site is also of some ⁇ antagonistic binding site (prazosin, in the case of alphalA adrenergic receptor).
  • Allosteric modulation implies that the receptor is capable of binding two ligands concomitantly through two topographically distinct binding sites; the orthosteric ligand binds to the orthosteric site while the modulator binds to a distinct site (allosteric site).
  • the two binding sites are conformationally linked, so that the binding of a ligand at site 1 disrupts the structure of site 2, thus changing its affinity for its own ligand.
  • the binding of the peptide to the adrenergic receptor alphala decreases the affinity of receptor-specific antagonists, such as prazosin and vice versa.
  • the invention encompasses the use of natural, synthetic or recombinant peptides having allosteric antagonist activity selective for the adrenergic alpha-alphal receptor.
  • the invention notably encompasses the use of variants obtained by mutation (insertion, deletion, substitution) of one or more amino acids in the sequence SEQ ID NO: 2, since said variant retains a selective allosteric antagonist activity of the alphala adrenergic receptor.
  • the invention also encompasses the use of modified peptides derived from the foregoing by introduction of any modification at the level of amino acid residue (s), peptide bond or peptide ends, as long as said modified peptide retains activity.
  • selective allosteric antagonist alphala adrenergic receptor alphala adrenergic receptor.
  • modifications which are introduced into the peptides by conventional methods known to those skilled in the art, include in a nonlimiting manner: the substitution of a natural amino acid by a nonproteinogenic amino acid (amino acid D or the like of amino acid) ; the addition of a chemical group (lipid, oligo or polysaccharide) at a reactive function, in particular the side chain R; modification of the peptide bond (-CO-NH-), in particular by a retro-type or retro-inverso bond (-NH-CO-) or a bond different from the peptide bond; cyclization; the fusion of the sequence of said peptide with that of a peptide or a protein of interest (epitope of interest for immunodetection; labels (biotin, peptides, flag, in particular) useful for the purification of the peptide, in particular under a protease cleavable form, a fluorescent protein, the coupling to a suitable molecule, in particular a marker, for example a fluor
  • the peptide is advantageously modified by means well known to the skilled person, to change its physiological properties, and in particular to improve his time of 1 A life in the body (glycosylation: HAUBNER R. et al., J. Nucl Med., 2001, 42, 326-36, conjugation with PEG: KIM TH et al., Biomaterials, 2002, 23, 2311-7), its solubility (hybridization with albumin: KOEHLER MF et al., Bioorg Med Med Chem Lett, 2002, 12, 2883-6), its resistance to proteases (non-natural amino acids (L conformation, for example), and / or its intestinal absorption (Lien et al. , TIB, 2003, 21, 556-).
  • natural or synthetic amino acid is meant the naturally occurring ⁇ -amino acids commonly found in the proteins (A, R, N, D, C, Q, E, G, H, I, L, K, M, F). P, S, T, W, Y and V), some amino acids rarely encountered in proteins (hydroxyproline, hydroxylysine, methyllysine, dimethyllysine ..), amino acids that do not exist in proteins such as ⁇ -alanine , ⁇ -aminobutyric acid, homocysteine, ornithine, citrulline, canavanine, norleucine, cyclohexylalanine ..., as well as the enantiomers and diastereoisomers of the preceding amino acids.
  • said peptide is the MT-beta toxin (SWISSPROT P80495, SEQ ID NO: 3), a natural peptide extracted from the venom of the snake Dendroaspis polylepis (black mamba); the MT-beta and AdTxI toxin sequences differ only at the residues at positions 38 and 43 which are respectively I 38 and V 43 (MT-beta), and S 38 and A 43 (AdTxI).
  • cysteine 1 is the first or the second amino acid residue and / or cysteine 8 is the penultimate or the last amino acid residue of the sequence of said peptide.
  • This peptide represents a truncated peptide derived from the preceding peptides by deletion of at least one of the N residues and / or or C terminal located upstream of cysteine 1 or downstream of cysteine 8.
  • the subject of the present invention is also an expression vector for use as a medicament, said vector comprising a polynucleotide coding for a peptide as defined above, under the control of appropriate regulatory sequences, the transcription and optionally the translation.
  • the sequence of said polynucleotide is that of the cDNA coding for said peptide; it is in particular the sequence SEQ ID NO: 4 coding for AdTxI. Said sequence may advantageously be modified in such a way that codon usage is optimal in the host in which it is expressed.
  • said polynucleotide may be linked to at least one heterologous sequence.
  • Heterologous sequence refers to a nucleic acid sequence encoding a peptide as defined in the present invention, any nucleic acid sequence other than those which, in nature, are immediately adjacent to said nucleic acid sequence encoding said peptide.
  • said recombinant vector comprises an expression cassette including at least one polynucleotide as defined above, under the control of transcriptional regulatory sequences and optionally the appropriate translation (promoter, activator, intron, initiation codon (ATG), stop codon, polyadenylation signal),
  • viral vectors such as adenoviruses, retroviruses, lentiviruses, AAVs and baculoviruses, in which the sequence of interest has been inserted beforehand; said sequence (isolated or inserted in a plasmid vector) may also be associated with a substance enabling it to cross the membrane of the host cells, such as a transporter such as a nanotransporter or a preparation of liposomes, or of cationic polymers, or the to introduce into said host cell using physical methods such as electroporation or microinjection.
  • these methods can be advantageously combined, for example by using electroporation associated with liposomes.
  • the present invention also relates to a pharmaceutical composition, characterized in that it comprises at least one peptide, a polynucleotide encoding said peptide, or a vector as defined above, and a pharmaceutically acceptable vehicle.
  • composition according to the invention is in a dosage form suitable for parenteral (subcutaneous, intramuscular, intravenous), enteral (oral, sublingual), or local (nasal, rectal, vaginal) administration.
  • the pharmaceutically acceptable vehicles are those conventionally used.
  • the present invention also relates to the use of at least one peptide and / or a vector as defined above for the preparation of a medicament having an allosteric antagonist activity selective for the adrenergic receptor alphala ( ⁇ la ), for the treatment of a urogenital or cardiovascular pathology, or cancer.
  • the peptides as defined in the present invention may be labeled directly or indirectly by a radioactive or non-radioactive compound, by covalent or non-covalent coupling, in order to obtain a delectable and / or quantifiable signal.
  • the marking is in particular a radioactive, magnetic, fluorescent marking, carried out according to the methods well known to those skilled in the art.
  • the markers detectable directly are in particular radioactive isotopes such as as tritium (H) and iodine (125 I) or luminescent compounds such as radioluminescent agents, chémoluminescents, bioluminescent, fluorescent or phosphorescent.
  • Indirectly detectable markers include, in particular, biotin and B epitopes.
  • Such labeled peptides are especially used to localize alphala adrenergic receptors, in vitro and in vivo, so as to determine their tissue expression profile under physiological, pathological conditions or in response to an endogenous or exogenous stimulus.
  • the present invention also relates to a process for detecting adrenergic receptor (s) alphala, in vitro and in vivo, comprising at least the following steps:
  • the detection of receptors, in vivo, in the body of a mammal comprises a prior step of administering said peptide to said mammal (parenteral injection, oral administration).
  • the labeling of the cells is in particular fluorescent labeling or magnetic labeling, detectable by any technique known to those skilled in the art (fluorescence microscopy, flow cytometry, magnetic resonance imaging).
  • the labeled peptides are used to screen libraries of molecules for the purpose of identifying other allosteric alphala adrenergic receptor ligands.
  • the present invention also relates to a method for screening allosteric alpha-alphal adrenergic receptor ligands, comprising at least the following steps:
  • the complexes between the peptide as defined in the present invention and the alphala adrenergic receptor can be advantageously used to obtain crystals; such crystals make it possible to determine the three-dimensional structure of the alphala adrenergic receptor, by ray diffraction
  • the subject of the present invention is also a method for preparing alphal adrenergic receptor crystals, comprising at least the following steps: a) contacting the alpha 1 adrenergic receptor with a peptide as defined above, so as to form receptor / ligand complexes, and b) incubation of the complexes obtained in a) under conditions and for a time sufficient to obtain the formation of crystals.
  • the present invention also relates to a receptor / ligand complex in which the receptor is the alphala adrenergic receptor and the peptide is a peptide as defined above, optionally coupled to a suitable marker.
  • the subject of the present invention is also the peptide of sequence SEQ ID NO: 2 and the variant peptides of SEQ ID NO: 2 comprising the substitution of lysine at position 34 of the sequence SEQ ID NO: 2 by another amino acid, especially arginine or homoarginine. According to an advantageous embodiment of said peptide, it is coupled to a suitable marker.
  • the present invention further relates to a polynucleotide, an expression cassette, a recombinant vector, and a modified prokaryotic or eukaryotic host cell derived from the foregoing peptide.
  • said polynucleotide has the sequence SEQ ID NO: 4 coding for AdTxI.
  • the invention encompasses in particular: a) expression cassettes comprising at least one polynucleotide as defined above, under the control of appropriate transcriptional regulatory and optionally translational sequences (promoter, activator, intron, initiation codon (ATG), stop codon, polyadenylation signal), and b) recombinant vectors comprising a polynucleotide according to the invention.
  • these vectors are expression vectors comprising at least one expression cassette as defined above.
  • Polynucleotides, recombinant vectors and transformed cells such as ⁇ defined above are useful in particular for the production of peptides as defined in the present invention.
  • polynucleotides according to the invention are obtained by conventional methods, known per se, following the standard protocols such as those described in Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and his Inc, Library of Congress , USA) and Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Spring Harbor CoId, New York: CoId Spring Harbor Laboratory Press). For example, they can be obtained by amplification of a nucleic sequence by PCR or RT-PCR, by screening genomic DNA libraries by hybridization with a homologous probe, or by total or partial chemical synthesis. Recombinant vectors are constructed and introduced into host cells by conventional methods of recombinant DNA and genetic engineering, which are known per se.
  • peptides and their derivatives are prepared by standard techniques known to the human being. in particular by solid or liquid phase synthesis or by expression of a recombinant DNA in a suitable cellular system (eukaryotic or prokaryotic). More precisely,
  • the peptides and their derivatives can be synthesized in solid phase, according to the Fmoc technique, originally described by Merrifield et al. (J. Am.
  • FIG. 1 illustrates the displacement of the binding of the radiolabeled AdTxI peptide ( 125 I-AdTxI) to the adrenergic receptor alphala, in the presence of prazosin (•) or of AdTx1 (O).
  • the IC 50 values are 2.2 ⁇ 10 -10 M and 5.10 -10 M, respectively for prasozine and AdTxI peptide.
  • FIG. 2 illustrates the displacement of the binding of the 3 H-prazosin, 3 H-rauwolscine and 3 H-CPG1 2177 binding to the various adrenergic receptor subtypes by prazosin or the AdTxI peptide.
  • IC 50 displacement by AdTxI of 3 H-prazosin binding to the receptor alphala
  • AdTxI of the 3 H-prazosin binding to the alp.99 receptor
  • FIG. 3 represents the hot saturation of 125 I-AdTxI on yeast membranes expressing the alphala adrenergic receptor. Different concentrations of 125 I-AdTxI are incubated for 20 hours in the presence of 20 ⁇ g of yeast membranes. Nonspecific binding is measured in the presence of 1 ⁇ M AdTxI. ( ⁇ ) Non-specific binding. (•) Specific binding. (O) Total binding. Specific binding is saturable and high affinity, equal to 0.8 ⁇ 0.2 nM.
  • AdTxI peptide is synthesized in the solid phase by the Fmoc (Fluorenyl methyloxycarbonyl) technique, using dicyclohexylcarbodiimide / 1-hydroxy-7-azabenzotriazole (HOAT) as coupling agent and N-methylpyrrolidone as solvent (Mourier et al. al, Molecular Pharmacology, 2003, 63, 26-35). Briefly, the synthesis is carried out from the C-terminus to the N-terminus of the peptide using 0.05 mmol of resin.
  • Fmoc Fluorenyl methyloxycarbonyl
  • the resin / peptide is treated with a mixture of 9 ml of trifluoroacetic acid, 0.5 ml of triisopropylsilane and 0.5 ml of distilled water.
  • the peptide is then cleaved from the resin after two hours of incubation.
  • the mixture is filtered through cold ethyl ether and centrifuged twice.
  • the precipitate thus obtained is dissolved in a solution of 10% acetic acid and freeze-dried.
  • the reduced synthetic toxin is purified by reverse phase chromatography (HPLC) on a Discovery® Bio Wide Pore C5 semi-prepative column, 25 cm x 10 mm, 10 ⁇ m (SUPELCO) with a gradient of 40% to 70% solvent B in 150 minutes (A: 0.1% TFA, B: 50% acetonitrile and 0.1% TFA), with a flow rate of 4.5 ml / min.
  • HPLC reverse phase chromatography
  • A 0.1% TFA
  • B 50% acetonitrile and 0.1% TFA
  • the detection is monitored at 220 nm.
  • the synthetic toxin is then refolded in 100 mM Tris buffer, pH 8.0, in the presence of reduced glutathione (GSSG) and oxidized (GSH) with a GSSG / GSH molar ratio of 1/1 and a concentration of 1 mM.
  • GSSG reduced glutathione
  • GSH oxidized
  • the folded synthetic toxin is purified by reverse phase chromatography (HPLC) under the same conditions as previously described.
  • the concentration of synthetic toxin is 5 ⁇ M.
  • the cloning of the nucleotide sequence coding for AdTxI is carried out by homologous recombination according to the technology (Gateway®), Invitrogen).
  • a polynucleotide fragment comprising successively from 5 'to 3': an attB1 recombination sequence, the TEV cleavage site (ENLYFQG), the sequence coding for AdTxI (SEQ ID NO: 3), a pseudo stop, the coding sequence for the Stag peptide, a stop codon, and the attB2 recombination sequence, was amplified by PCR.
  • the PCR product was cloned by homologous recombination into the donor plasmid pDONR221 (Invitrogen). The clone thus obtained is used to generate recombinant expression vectors, suitable for the expression of AdTxI in a suitable cellular system.
  • Example 2 Assay of AdTx1 binding to alpha 1 adrenergic receptors 1
  • Materials and methods a) Iodization of AdTxI toxin The iodination of AdTxI is by a lactoperoxidase catalyzed halogenation reaction. The reaction mixture containing 50 ⁇ l of 0.1 " M phosphate buffer pH 7.3, 10 ⁇ l of 100 ⁇ M toxin, 10 ⁇ l of H 2 O 2 1/50 000 and 1 mCi [I] and 0.7 Lactoperoxidase unit (Sigma) is incubated for 1 minute at 25 ° C.
  • Each subtype receptor, ⁇ the at ,, ⁇ ⁇ , ⁇ 2a, P 1 is expressed in the yeast Pichia pastoris transformed with an expression plasmid comprising cDNA corresponding to said receiver.
  • Each clone is grown in the same way. Clones of Pichia pastoris are inoculated into 10 ml of medium (1% yeast extract, 2% peptone, 100 mM Potassium Phosphate pH 6, 1.3% yeast nitrogen base, 1% Glycerol), overnight at 30 ° C. °, then the cultures are diluted in 100 ml of fresh medium and incubated again for 4 h at 30 ° C.
  • the cultures are then centrifuged (3000 g, 5 min), resuspended in 500 ml of induction medium (1% yeast extract, 2% peptone, 100 mM Potassium Phosphate pH 6, 1.3% yeast nitrogen base, 0.5% methanol), supplemented with 2.5% DMSO and incubated for 18 h at 20 ° C., with stirring (200 rpm).
  • the cultures are harvested (3000 g, 15 min, 4 ° C) and resuspended in 3 ml of buffer (50 mM Potassium Phosphate pH 7.4, 100 mM NaCl, 5% Glycerol, 2 mM EDTA and 1 mM PMSF), cooled in ice.
  • the pellet of each preparation is resuspended in a buffer (50 mM Tris pH 8, 120 mM NaCl, 10% Glycerol, 1 mM PMSF) using a homogenizer, aliquoted and stored at -80 ° C until use. , c) Linkage tests.
  • the ⁇ Nonspecific binding was measured in the presence of 1 .mu.M of prazosin (Sigma) for binding with 3H-Prazosin, 1 .mu.M yohimbine (Sigma) for binding with 3 H-rauwolscine, or l ⁇ M propranolol (Sigma) for the bond with 3H-CPG12177.
  • the reaction is stopped by filtration preceded by a dilution of the reaction medium in 2 ml of washing buffer (Tris-HCl pH 7.2, 10 mM), at 4 ° C.
  • the filtration is carried out on glass filters (GFC, Whatman) pretreated in 0.3% PEI buffer (Polyethyleneimine, Sigma). Two successive and rapid washes are carried out.
  • the filters are dried for one hour at 80 ° C and added with 10 ml of Lipoluma Plus (Lumac LMC).
  • the emissions are detected by a Rockbeta 1211 counter (LKB Wallac) giving the value of each test in cpm (counts per minute).
  • the results are analyzed using Kaleidagraph software (Tools for discovery, Synergy Software, PA, USA).
  • Kaleidagraph software Tools for discovery, Synergy Software, PA, USA.
  • the analysis of the displacement of the adrenergic receptor binding alphala by the iodinated AdTxI peptide (0.1 nM) is carried out according to the same protocol than the one used for tritiated ligands, and the radioactivity is measured by a Multigamma 1261 counter (LKB Wallac).
  • AdTxI The analysis of the saturation of the alphala adrenergic receptor by AdTxI is carried out by incubating increasing concentrations of the iodinated peptide in the presence of 20 ⁇ g of membranes containing the adrenergic alphala receptor, for 20 h, and then measuring the radioactivity as above. Non-specific binding is measured in the presence of 1 ⁇ M AdTxI.
  • the AdTxI peptide is an alphahala adrenergic receptor ligand (FIG. 1).
  • the competitive binding assays with orthosteric ligands specific for the different adrenergic receptor subtypes indicate that the AdTxI peptide selectively binds the alphala adrenergic subtype.
  • Its affinities for the subtypes alphala, Ib, Id, 2a and betal, evaluated by the value of ICso, are respectively l, 8.10 ⁇ 9 M, 2.3.10 "6 M, 9.9.10 " 6 M,> 5.10 5M 5 and> 5.10 "5 M.
  • the displacement curve of the prazosin binding to the alphala adrenergic receptor, in the presence of the AdTxI peptide indicates that the displacement is incomplete, which signifies an allosteric modulation.
  • AdTxI variant K34R was synthesized as described in Example 1 for AdTxI. The AdTxI variant K34R was then coupled to the Cy3B- fluorophore. mono-NHS-ester (AMERSHAM) according to the protocol recommended by the manufacturer.
  • AMERSHAM mono-NHS-ester
  • COS cells transfected either with an alphala human adrenergic receptor expression vector or with an alpched human adrenergic receptor expression vector transiently expressing this alphala or alpschreib receptor were incubated in the presence of 2 ⁇ M of AdTxI -Cy 3b for 16 h, then the fluorescence emitted after laser excitation at 543 nm was analyzed using a fluorescence microscope (Leica TCS SP2, LEICA MICROSYSTEMS) at a magnification of 40. 2) Results Figure 4 illustrates the specific labeling of alphala adrenergic receptors by the fluorescent derivative of AdTxI.
  • FIG. 4A shows intense labeling of h-alphala adrenergic receptors by the fluorescent derivative of AdTx1.
  • FIG. 4B shows the absence of labeling of h-alphak adrenergic receptors (FIG. 4B) by the fluorescent derivative of AdTx1.

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EP07731030A 2006-02-23 2007-02-23 Peptide mit den eigenschaften eines für den alpha-1a-adrenergen rezeptor selektiven allosteren antagonisten und anwendungen davon Withdrawn EP1991573A2 (de)

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