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WO2003025001A1 - Systeme de peptides antigeniques multiples (map) servant d'antidote contre une intoxication par des neurotoxines de serpent - Google Patents

Systeme de peptides antigeniques multiples (map) servant d'antidote contre une intoxication par des neurotoxines de serpent Download PDF

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Publication number
WO2003025001A1
WO2003025001A1 PCT/IT2002/000580 IT0200580W WO03025001A1 WO 2003025001 A1 WO2003025001 A1 WO 2003025001A1 IT 0200580 W IT0200580 W IT 0200580W WO 03025001 A1 WO03025001 A1 WO 03025001A1
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group
residue selected
tyr
multiple antigen
glu
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PCT/IT2002/000580
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English (en)
Inventor
Luisa Bracci
Luisa Lozzi
Barbara Lelli
Alessandro Pini
Paolo Neri
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Universita' Degli Studi Di Siena
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    • 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
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention concerns Multiple Antigen Peptides (MAP) as antidotes against snake neurotoxin intoxication.
  • MAP Multiple Antigen Peptides
  • the invention refers to synthetic peptides in the form of Multiple Antigen Peptides (MAP) as therapeutic agents able to neutralize snake neurotoxin toxicity in vivo by inhibition of neurotoxin binding to cell receptors.
  • MAP Multiple Antigen Peptides
  • Snakes belonging to the families Elapidae (cobra, kraits, mambas, tiger snakes, coral snakes and others) and Hydrophidae (sea snakes) have typical paralysing venoms with a high content of curaremimetic neurotoxins. As their name implies, these postsynaptic neurotoxins mimic the action of curare and prevent the depolarizing action of acetylcholine by binding to the nicotinic acetylcholine receptor (nAchR).
  • nAchR nicotinic acetylcholine receptor
  • Nicotinic acetylcholine receptors are ligand gated ion channels made by the association of five homologous membrane-spanning subunits.
  • nAchR are located at muscle endplates and in the central nervous system, where acetylcholine-binding ⁇ subunits ( ⁇ 2 to ⁇ 9) can be assembled in a number of combinations with structural ⁇ subunits ( ⁇ 2 to ⁇ 4) giving rise to nicotinic receptors with different functional and pharmacological properties.
  • the muscle nAchR is composed by 2 ⁇ subunits and one each of ⁇ , ⁇ (or ⁇ ) and ⁇ subunits. Gating of the channel is induced by acetylcholine binding to the ligand sites, located on the two receptor ⁇ subunits, at their interfaces with respectively ⁇ and ⁇ subunits (1, 2).
  • the postsynaptic ⁇ -neurotoxins of Elapidae and Hydrophidae snake venom bind with very high affinity to the nicotinic receptor ligand sites and compete with acetylcholine, thus exerting their potent receptor functional blockade at neuromuscular junctions.
  • Snake neurotoxins can be divided into two subfamilies: short (60-62 residues) and long (64-74 residues) neurotoxins, which have a significant sequence homology and share the same three-fingered structure, as determined by X-ray crystallography and NMR. Thanks to their high affinity and specificity, snake neurotoxins, in particular the long neurotoxin ⁇ -bungarotoxin ( ⁇ -bgt) from the snake Bungarus multicinctus, have been extremely useful in the characterization of nicotinic receptor structure and function (3, 4).
  • ⁇ -bgt not only binds with an affinity in the picomolar range to muscle nicotinic receptors, but it also selectively binds with a similar affinity to a subpopulation of central nervous system nicotinic receptors containing the ⁇ 7 subunit (5-7).
  • the three-dimensional structure of nicotinic receptors has not yet been solved. Nonetheless, chemical modification of specific receptor residues and site-directed mutagenesis have allowed to localize some receptor residues involved in binding of agonist and antagonist molecules, including snake ⁇ -neurotoxins (8). From these studies, a sequence of the ⁇ subunit close to cysteine residues 192 and 193 has been determined as containing at least part of the nAchR ligand binding site.
  • nAchR binding site-mimicking peptide which binds ⁇ -bgt and competes with the native receptor for binding, was selected from a random phage-epitope library.
  • the sequence of this randomly selected peptide is very similar to that of peptides reproducing the regions close to cysteines 192 and 193 of muscle and ⁇ 7 neuronal nAchR (13).
  • Peptide mimotopes have a toxin-binding affinity that is considerably higher than peptides reproducing native receptor sequences (14).
  • the best peptide (p6.7) the authors identified in this work has a ⁇ -bgt binding affinity constant (KA) about 20 times higher than the previously described phage library derived peptide (13) and from 20 to more than 1000 times higher than peptides reproducing native receptor sequences from different species.
  • KA ⁇ -bgt binding affinity constant
  • the IC50 of peptide p6.7 is 30 times lower than that of the previously described phage library derived peptide and at least 50 times lower than the IC50 of peptides reproducing native receptor sequences.
  • Some snake toxins including D-bungarotoxin have now been produced in an active form, either in prokaryotic or eukaryotic cells (18, 19).
  • Antidotes that can specifically inhibit snake neurotoxin binding to their receptors can be effective also against snake neurotoxin intoxications not related to snake bites.
  • efficient and specific detection and identification systems could be fundamental for a defence against biological attack. From the above description it is apparent the need to have the availability of new compounds more efficient in binding snake neurotoxins in comparison to known compounds and to be used as effective antidotes against snake neurotoxin intoxication.
  • MAP4 Multiple Antigen Peptide
  • MAP4 peptides when used to coat plastic wells, are much more effective than monomeric peptides for ⁇ -bgt binding.
  • Peptide mimotopes of receptor binding sites when synthesized in the MAP form can thus be advantageously used as efficient synthetic antidotes against snake neurotoxins and for the specific detection of the same neurotoxins.
  • an object of the present invention is a multiple antigen peptide comprising: a) a dendritic core essentially constituted by at least one bifunctional molecule; b) at least two peptide molecules able to bind snake ⁇ - neurotoxins when injected into mammals and neutralize snake neurotoxin toxicity; and wherein the dendritic core and peptide molecules are linked by covalent bonds.
  • the peptide molecule has the formula: X1 -Arg-Tyr-Tyr-Glu-X6-X7-X8-X9-X10-X11-Tyr-Pro-Asp wherein X6 and X7, which may be the same or different from each other, represent aminoacid residues selected from the group consisting of: Ser, Cys.
  • X8 is Leu
  • X1 represents an aminoacid residue selected from the group consisting of: His, Arg, Lys, Phe, Tyr, Trp
  • X9 represents an aminoacid residue selected from the group consisting of Lys, Thr, Met
  • Glu represents an aminoacid residue selected from the group consisting of Pro
  • Ala represents an aminoacid residue selected from the group consisting of Ala, Val, Thr, Gin, Tyr, Trp.
  • X8 is Val
  • X1 represents an aminoacid residue selected from the group consisting of: His, Arg, Gly
  • X9 represents an aminoacid residue selected from the group consisting of Glu
  • Met represents an aminoacid residue selected from the group consisting of Pro
  • Ala represents an aminoacid residue selected from the group consisting of Ala and Gin.
  • X8 is Thr
  • X1 represents an aminoacid residue selected from the group consisting of: His, Phe, Pro,
  • Tyr represents an aminoacid residue selected from the group consisting of Glu, Gin, Lys and Met
  • X10 represents an aminoacid residue selected from the group consisting of Pro
  • Ala Ser
  • X11 represents an aminoacid residue selected from the group consisting of Trp, Ala, Val, Tyr.
  • X8 is Pro
  • X1 represents an aminoacid residue selected from the group consisting of: Phe
  • His represents an aminoacid residue selected from the group consisting of
  • Asp, Glu, Thr, X10 represents an aminoacid residue selected from the group consisting of Pro, Ala and X11 represents an aminoacid residue selected from the group consisting of Ala, Trp.
  • X8 is Glu
  • X1 represents an aminoacid residue selected from the group consisting of: His, Phe, Tyr
  • X9 represents an aminoacid residue selected from the group consisting of Asn, Gin, Asp and Met
  • X10 represents an aminoacid residue selected from the group consisting of Pro, Ala, lie
  • X11 represents an aminoacid residue selected from the group consisting of Trp, Ala, Thr.
  • the peptide molecule comprises the sequence: His-Arg-Tyr-Tyr-Glu-X6-X7-Leu-Glu-Pro-Trp-Tyr- Pro-Asp wherein X6 and X7, which may be the same or different from each other, represent aminoacid residues selected from the group consisting of: Ser, Cys.
  • the peptide molecule has the formula: X1 -Arg-Tyr-Tyr-Glu-X6-X7-Leu-Glu-Pro-X11 -X12-X13 wherein X6 and X7, which may be the same or different from each other, represent aminoacid residues selected from the group consisting of: Ser, Cys..
  • X1 represents an aminoacid residue selected from the group consisting of Phe, Tyr, Trp
  • X11 represents an aminoacid residue selected from the group consisting of Phe, Trp, Tyr
  • X12 represents an aminoacid residue selected from the group consisting of Trp, Pro
  • Asp represents an aminoacid residue selected from the group consisting of Asp, Glu, lie, Trp.
  • the peptide molecule includes one of the following sequences:
  • X6 and X7 which may be the same or different from each other, represent aminoacid residues selected from the group consisting of: Ser, Cys.
  • R is a peptide molecule as in any of the previously described formula or forms;
  • X can be an amino acid with at least two aminic functional groups, more preferably X can be Lysine, ornitine, nor-lysine or amino alanine. Alternatively, X can be an aspartic acid or glutamic acid. Alternatively X can be propylene glycol, succinic acid, diisocyanates or diamines.
  • the present invention is based on a number of observations, both in-vitro and in-vivo, indicating that the use of MAP can increase the efficiency of peptide mimotopes of the nicotinic receptor binding site, when used as receptor competitors for snake venom ⁇ -neurotoxin binding or for the detection of snake ⁇ -neurotoxins.
  • a synthetic peptide mimicking the neurotoxin binding site of nicotinic receptor which we had selected from a peptide combinatorial library, when synthesized in the MAP form retains a similar affinity, in terms of affinity of each peptide measured by the BIACORE (Fig.1), than the monomeric peptide.
  • the MAP peptide retains the ability of the monomeric peptide to compete with the receptor for ⁇ -bgt binding (Fig. 3).
  • MAP4p6.7 some minutes after the injection of a 100% lethal dose of ⁇ -bgt, completely neutralizes ⁇ - bgt lethality and protects animals from any visible symptoms.
  • the correspondent monomeric peptide does not neutralize toxin lethality, even at a dose of 5mg/animal.
  • the NMR structure of the complex between the peptide p6.7 and ⁇ -bgt was compared to that formed by the same neurotoxin with available structural models for the nicotinic receptor neurotoxin-binding site, like the Acetylcholine-Binding Protein and a peptide reproducing the ligand site of neuronal ⁇ 7 nicotinic receptor subunit.
  • the central loop of the peptide p6.7 fits with the structure of the Acetylcholine-Binding Protein and ⁇ 7 peptide ligand sites, while peptide terminal residues seems to be less involved in toxin binding
  • a series of peptides were synthesized where terminal residues of p6.7 were progressively deleted and peptide binding to ⁇ -bgt was analysed by Surface Plasmon Resonance (Fig 5a and 5b). Once determined the minimal binding sequence, 10-fold affinity maturation of the lead peptide was achieved by the systematic addition of all the L- aminoacids in each position flanking the minimal binding sequence, through an iterative process followed by selection of binding sequences on the basis of their off (Fig 6).
  • Figure 1 shows kinetic constants and KA measurement of ⁇ -bgt binding to p6.7 and MAP4p6.7 by SPR in BIACORE.
  • the affinity of each toxin-binding site of the MAP4p6.7 is practically identical to that of p6.7.
  • Figure 2 A and B show 125" l ⁇ -bgt binding to monomeric and MAP peptides by a solid phase Radio Immuno Assay. MAP peptides binding efficacy is higher than that of correspondent monomeric peptides.
  • Figure 3 shows inhibition of 125' l ⁇ -bgt binding to nAchR by Map4p6.7 and p6.7. The half-maximal inhibition constant IC 5 0 of MAP4p6.7, expressed as molar concentration, is about 4 times lower than IC50 of the monomeric p6.7.
  • Figure 4 shows the neutralization of toxin lethality in-vivo by
  • Figure 5 Definition of p6.7 minimal ⁇ -bgt-binding sequence, ⁇ - bgt binding of peptide from p6.7 sequence, progressively shortened at N- (Fig.5A) and C-terminus (Fig.5B), was analysed in BIACORE on a ⁇ -bgt- biotin SA sensor chip.
  • Solid-phase synthesis was carried out by a Syro MultiSynTech peptide synthesizer (Witten, D), employing a 4-(2',4'-Dimethoxyphenyl- Fmoc-aminomethyl)-phenoxyacetamido-norleucyl-(4- methylbenzhydrylamine) (Rink-MBHA) resin and fluorenylmethoxycarbonyl (Fmoc) chemistry.
  • the deprotection reaction was carried out by adding 40% piperidine in N-methylpyrrolidone and in situ prepared N- hydroxybenzotriazole esters of Fmoc-amino acids were used for the coupling reaction.
  • Peptides were cleaved from the resin and simultaneously deprotected using a trifluoroacetic acid/thioanisole/ethanedithiol/water (93/2/3/2) mixture for 3 hours at room temperature. Peptides were purified by reverse-phase HPLC on a semipreparative Vydac C18 column (10 ⁇ m, 100 x 250 mm) using a 30-min gradient of 0% to 100% buffer B (Buffer A: 0.1 % trifluoroacetic acid/water; Buffer B: 0.1 % trifluoroacetic acid/methanol).
  • MAP4 4-branch Multiple Antigen Peptide
  • Fmoc 4 -K 2 -K- ⁇ A Wang resin employing Fmoc chemistry.
  • MAP peptides were cleaved from the support using standard techniques and purified by reverse-phase HPLC (20-21). Peptides were controlled by Mass Spectrometry.
  • EXAMPLE 2 Receptor purification Muscle nAchR was extracted from electric organs of Torpedo marmorata by Triton X-100 and affinity-purified through a sepharose- ⁇ - cobratoxin column as described by Lindstrom et al. (22, 19).
  • EXAMPLE 3 Receptor purification Muscle nAchR was extracted from electric organs of Torpedo marmorata by Triton X-100 and affinity-purified through a sepharose- ⁇ - cobratoxin column as described by Lindstrom et al. (22, 19).
  • MAP4p6.7 peptide was immobilized over the carboxymethylated dextran matrix of a CM5 sensor chip (Biacore, AB) using standard amine coupling chemistry, ⁇ -bgt (SIGMA) diluted in HBS, was injected over the MAP4- dextran matrix at concentration ranging from 10 to 0.1 ⁇ g/mL with a flow rate of 10 ⁇ Umin.
  • Association and dissociation kinetic rate constants (kon and k otf ) and the equilibrium association constant KA were calculated using the BIAevaluation 3.0 software.
  • EXAMPLE 4 Binding of 125 l-a-bgt to monomeric and MAP peptides (Fig.
  • Microtiter plates (Falcon 3912, Becton Dickinson, Oxnan, CA, USA) were coated over night at 4°C with monomeric and MAP peptides (1 ⁇ g/mL in 0.05M carbonate buffer pH9.6), blocked with 3% BSA in phosphate buffer saline (PBS) pH7.4 for 2 hrs at room temperature and then incubated for 1 hour with 125" l ⁇ -bungarotoxin ranging from 2 x 10 5 to 2.5 x 10 4 cpm. ⁇ -bgt binding to peptides was revealed measuring radioactivity with a ⁇ -counter.
  • PBS phosphate buffer saline
  • Microtiter plates (Falcon 3912, Becton Dickinson, Oxnan, CA, USA) were coated over night at 4°C with affinity purified muscle nAchR (5 ⁇ g/mL in 0.05M carbonate buffer ⁇ H9.6) and then blocked with 3% BSA in phosphate buffer saline (PBS) pH7.4 for 1 h at room temperature.
  • affinity purified muscle nAchR 5 ⁇ g/mL in 0.05M carbonate buffer ⁇ H9.6
  • PBS phosphate buffer saline
  • Peptides at concentrations ranging from 54 ⁇ M to 5.4pM were incubated together with 10 5 cpm of 125 ⁇ - ⁇ -bungarotoxin (Amersham Italia s.r.l.) for 1 hour at room temperature.
  • MAP4p6.7 was tested for a-bgt lethality neutralization in-vivo. Experiments were performed on 15g Swiss mice with subcutaneous injections. The MAP is able to neutralize the toxicity of a ⁇ -bgt dose twice the IC 50 (10 ⁇ g) in mice. Monomeric peptide was injected at a dose of 0.5mg and 5mg of peptide/mouse. At 0.5 mg monomeric peptide resulted completely ineffective in toxin lethality neutralization. When mice were injected with 5mg of monomeric peptide/mouse, the animals survived for 4-6 hrs accordingly to different monomeric peptides previously described (15-16).
  • MAP4p6.7 peptide injected at the dose of 0.1 mg/mouse resulted to completely protect mice from toxin lethality when administrated 5 minutes after the toxin.
  • MAP4p6.7 peptide was injected 5 minutes after the toxin at the dose of 0.05mg mouse, 13 mice over 30 survived showing some symptoms, while among the remaining 17, 16 died in 4-8 hrs and 1 died in 20 hrs.

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Abstract

L'invention concerne un système MAP qui comprend: a) un noyau dendritique constitué essentiellement d'au moins une molécule bifonctionnelle; b) au moins deux molécules peptidiques capables de se lier avec des alpha-neurotoxines de serpent, en cas d'injection à un mammifère, et de neutraliser la toxicité des neurotoxines de serpent. Le noyau et les molécules en question sont liés par des liaisons covalentes.
PCT/IT2002/000580 2001-09-14 2002-09-12 Systeme de peptides antigeniques multiples (map) servant d'antidote contre une intoxication par des neurotoxines de serpent WO2003025001A1 (fr)

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IT2001RM000563A ITRM20010563A1 (it) 2001-09-14 2001-09-14 Peptidi antigenici multipli come antidoti nell'intossicazione da veleno di serpente.
ITRM2001A000563 2001-09-14

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005095444A3 (fr) * 2004-03-30 2006-02-09 Uni Degli Studi De Siena Peptides de synthese utilises en tant qu'antidotes contre la toxine de l'anthrax

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WO1990011778A1 (fr) * 1989-04-12 1990-10-18 The Rockefeller University Polymere dendritique d'un systeme de peptides d'antigenes multiples utile en tant que vaccin anti-paludeen

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005095444A3 (fr) * 2004-03-30 2006-02-09 Uni Degli Studi De Siena Peptides de synthese utilises en tant qu'antidotes contre la toxine de l'anthrax

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