WO1997007402A1 - Methode de controle de l'assemblage des fibrilles de proteine - Google Patents

Methode de controle de l'assemblage des fibrilles de proteine Download PDF

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WO1997007402A1
WO1997007402A1 PCT/CA1996/000555 CA9600555W WO9707402A1 WO 1997007402 A1 WO1997007402 A1 WO 1997007402A1 CA 9600555 W CA9600555 W CA 9600555W WO 9707402 A1 WO9707402 A1 WO 9707402A1
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peptide
fluorophore
donor
alzheimer
denaturant
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PCT/CA1996/000555
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Avi Chakrabartty
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The Ontario Cancer Institute
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the invention is a method for in vitro monitoring of protein fibril assembly at physiologically relevant concentrations.
  • the invention is readily adaptable for in vi tro monitoring of fibril assembly processes associated with various amyloidosis disorders, such as Alzheimer's disease, multiple myeloma, rheumatoid arthritis, diabetes, and prion disorders.
  • the invention has particular utility as a screening assay for potential inhibitors of fibril formation, which in turn may be candidates for treatment of Alzheimer's disease or other amyloidosis disorders.
  • Alzheimer's disease is a neurodegenerative disorder of the elderly. While neurodegeneration in Alzheimer's disease begins slowly, its progression follows an exponential course, and it finally culminates in dementia and death (Muller-Hill and Beyreuther, 1989) .
  • Key pathological features of Alzheimer's disease found in the brains of afflicted patients include the following: a) senile plaques, which are extracellular amyloid deposits in close contact with neurons, b) neurofibrillary tangles, which are intraneuronal deposits of paired helical filaments, and c) cerebrovascular amyloid, which are amyloid deposits in the walls of cortical and meningeal vessels (Selkoe, 1991; Katzman and Saitoh, 1991) .
  • the link between the symptoms of Alzheimer's disease and its pathology arises from the plaques and neurofibrillary tangles being concentrated around the hippocampus, a region of the brain responsible for memory and learning.
  • a major component of senile plaques and cerebrovascular amyloid is Alzheimer's /S-amyloid peptide (A / S) , a 39-43 residue peptide that folds into a / 3-sheet structure and assembles into fibrils 60-90A in diameter (Fraser et al., 1993) .
  • A3 is a fragment of a much larger integral membrane protein called amyloid precursor protein (APP) .
  • APP amyloid precursor protein .
  • APP amyloid precursor protein
  • A/3 is derived from residues 672 to 715 of APP, and it encompasses the C-terminal region of the extracellular domain of APP and the N-terminal half of the transmembrane domain of APP.
  • Other proteins found in senile plaques include: serum complement proteins (Clq, C3d, and C4d) , serum amyloid P, ⁇ -antichymotrypsin, Apo-E, and heparan sulfate proteoglycans. The relative amounts of these proteins in senile plaques are, however, much lower than that of A3.
  • Alzheimer's disease The other characteristic feature of Alzheimer's disease are neurofibrillary tangles, which are intraneuronal deposits of paired helical filaments. Paired helical filaments are not as well characterized as senile plaques (Crowther, 1991) .
  • the protein components of paired helical filaments discovered so far include: neurofilament protein, a microtubule associated protein (MAP) called tau, MAP2, and MAP5.
  • MAP microtubule associated protein
  • Alzheimer's disease The molecular pathogenesis of Alzheimer's disease is not well established. However, there is accumulating evidence that deposition of A/3-fibrils in senile plaques may account for much of the neurotoxicity of Alzheimer's disease (Selkoe, 1993) . Neurons which contact A/3-fibrils are dystrophic. Patients that have three copies of the APP gene (i.e. Down's syndrome patients) produce a greater number of senile plaques and develop Alzheimer's disease when they reach their late thirties. Mutations in the APP gene are often seen in cases of familial Alzheimer's disease, and related genetic diseases (Goate et al. , 1991) .
  • Aj ⁇ -fibrils are toxic to neurons in culture (Yankner et al., 1989; Pike et al., 1993).
  • Alzheimer-type neuropathology is induced in transgenic mice overexpressing a mutant form of APP (Games et al. , 1995) .
  • These findings all point to A/3 as the primary neurotoxic agent of Alzheimer's disease.
  • the mechanism of A/3 neurotoxicity is also not well understood, but it appears to be related to calcium ion homeostasis in neurons (Mattson et al., 1993).
  • Aj ⁇ is a product of normal cells (Haass et al., 1992) , and that it is present in cerebrospinal fluid and blood plasma of healthy individuals (Seubert et al. , 1992) .
  • Experimental evidence suggests that there may be multiple primary causes that ultimately lead to A/3 deposition in senile plaques and Alzheimer's disease (Selkoe, 1993) . While certain individuals may develop Alzheimer's disease by producing greater amounts of A/3, others might develop the disease by producing a form of A ⁇ that is especially prone to fibril formation.
  • the concentration of A ⁇ in the brain as well as its intrinsic tendency to aggregate are both integral to the process of A/3-fibril deposition in Alzheimer's disease.
  • a ⁇ fibril formation by A ⁇ may account for the neurotoxicity in Alzheimer's disease
  • molecules that inhibit fibril formation should be good candidates for therapeutic agents for the treatment of Alzheimer's disease.
  • One critical research tool for evaluating therapeutic inhibitors is an assay for A ⁇ -fibril formation.
  • Current methods that monitor A/3 fibril formation employ various biophysical techniques, such as electron microscopy, X-ray diffraction, fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. Studies utilizing these biophysical techniques have shown that A ⁇ fibrils possess remarkable intrinsic stability. Even after altering much of the sequence of A/3, fibril formation still occurs (Fraser et al.
  • Asp23 of A ⁇ is a unique residue; since when it is replaced with Lys a significant reduction in the stability of the fibril is observed (Fraser et al., 1994) .
  • Two regions of the A ⁇ sequence show a high propensity to form /3-structure: residues 9 to 28 (Fraser et al., 1991) and residues 34 to 42 (Halverson et al., 1990) .
  • fibrillogenesis is pH; at low ( ⁇ 3) and high (>10) pH, fibrillogenesis by A ⁇ analogues is both slower and less stable (Fraser et al., 1991; 1992). It appears that hydrogen bond, hydrophobic, and electrostatic interactions all act in concert to stabilize the A ⁇ fibril; however, a high resolution structural model of the A/3 fibril has yet to be developed.
  • Fibril assembly is a bimolecular reaction: A ⁇ + (A/3) n « ⁇ (A/3) n+1 .
  • a ⁇ + (A/3) n « ⁇ (A/3) n+1 One distinguishing feature of bimolecular reactions is that both their kinetics and thermodynamics are concentration-dependent. It follows, therefore, that the concentration of A/3 in the brain and cerebrovasculature controls the rate of amyloid fibril formation as well as the intrinsic stability of amyloid plaques. Consequently, the mechanism of fibril assembly at low physiological concentrations may differ from the mechanism that operates at the high concentrations required by the above mentioned biophysical techniques. Any attempt to model Alzheimer's disease in vitro should take into consideration the relatively low concentrations of A ⁇ in the brain and cerebrovasculature.
  • the invention provides a very sensitive fluorescence technique for monitoring fibrillogenesis by A/3 at concentrations close to the physiological concentration of A/3 in the brain.
  • This fluorescence technique can be adapted into a rapid, technically simple, standardized assay to simultaneously screen many compounds that could potentially inhibit fibril formation.
  • the invention provides a method for in vitro monitoring of peptide or protein fibril assembly, comprising the steps of:
  • Figure 1 shows fluorescence emission spectra of fibrils composed of Trp-A/3(9-25) and AEDANS -A ⁇ (9-25) .
  • Figure 2 is a graph showing pH dependence of fibril formation monitored by fluorescent fibrillogenesis assay, and attenuation of the titration curve in the presence of 0.5 M NaCl.
  • Figure 3 is a graph of the kinetics of monomer fibril exchange.
  • Figure 4 is a graph showing the promotion of fibril formation by heparin as monitored by the fluorescence assay of the invention.
  • the invention will be described in relation to a specific assay for A/3 fibril assembly.
  • the following description while specific for A ⁇ fibrillogenesis illustrates the general principles of the invention which may be used to assay fibril assembly processes in other amyloidosis disorders.
  • Fluorescence spectroscopy is a very sensitive technique, and depending on the quantum yield of the fluorophore, the technique may be sensitive down to the nanomolar concentration range.
  • the physiological concentration of A/3 in the brain is estimated to be in the nanomolar range; therefore, the sensitivity of fluorescence spectroscopy is in principle sufficient to detect physiological concentrations of A/3.
  • fluorescence cannot be used to examine fibril formation by native A ⁇ because the only intrinsic fluorophore present in A/3 is the phenolic side chain of TyrlO, and its very low quantum yield is inadequate for sensitive measurements.
  • Extrinsic fluorescent groups can be chemically attached to A/3, and the technique of nonradiative fluoresence energy transfer can be used to measure fibril assembly by A ⁇ in the nanomolar concentration range.
  • the principle of fluorescence energy transfer is that when appropriate donor and acceptor fluorophores are close in space, light energy absorbed by the donor can be transferred to the acceptor, and the efficiency of energy transfer depends on the distance separating the donor and acceptor (see Fairclough and Cantor, 1978) .
  • the following example outlines the general method of energy transfer measurements.
  • the fluorescent amino acid tryptophan absorbs light of 281 nm, and after absorption it emits light of 337 nm.
  • the fluorescent compound 5-acetylethyl- diaminonaphthalene-1-sulfonic acid (AEDANS) absorbs light of 281 nm very poorly but it absorbs light of 337 nm very strongly; after absorption AEDANS emits light of 490 nm. Since the absorption band of AEDANS coincides with the emission band of Trp, Trp and AEDANS can be used as donor and acceptor, respectively, in a fluorescence energy transfer experiment. To illustrate the method consider a solution containing a mixture of Trp and AEDANS, if the mixture is irradiated with light of 281 nm then the wavelength of the emitted light depends on the distance separating the Trp and AEDANS fluorophores.
  • Trp and AEDANS as the donor-acceptor pair, a method for monitoring fibril formation by A/3 using nonradiative fluorescence energy transfer was devised.
  • the fluorescent fibrillogenesis assay of the invention uses a fragment of A/3 corresponding to residues 9 to 25.
  • This fragment of A/3 forms fibrils that display similar morphology, pH-dependence, X-ray diffraction pattern, and secondary structure as the full length A/3 molecule (Fraser et al., 1994).
  • Adapting the assay procedure to the full length A ⁇ molecule is very straightforward and simple.
  • Trp-A/3(9-25) when Trp-A/3(9-25) is mixed with AEDANS-A/3(9-25) , fibrils composed of both forms of A ⁇ will assemble, and in the fibril state the Trp and AEDANS groups will be closer in space than in the nonfibril state. Since fluorescence energy transfer between Trp and AEDANS increases when the two fluorophores are close in space, the efficiency of energy transfer between Trp and AEDANS will increase as Trp-A/3 (9-25) and AEDANS-A3 (9-25) assemble into fibrils. At a separation distance of 22 A energy transfer between Trp and AEDANS is easily measurable (Fairclough and Cantor, 1978; the upper limit of energy transfer is ⁇ 40A) .
  • Trp-A/3(9-25) Ac- GGYEVHHQKLVFFAEDVG-CONH 2 (SEQ ID NO:2)
  • Trp and AEDANS are relatively large chemical structures. There is a possibility, therefore, that introduction of these large groups into A ⁇ may disrupt or alter fibril formation through steric mechanisms. For this reason, it was decided to place the fluorescent groups at the N-terminus of the A ⁇ (9-25) sequence rather than at an internal position, because placement in the interior could potentially disrupt certain interactions which are known to stabilize fibril formation (Halverson et al., 1990; Hilbich et al. , 1992; Fraser et al. , 1994). In addition, a glycine residue was inserted between the fluorescent group and the rest of the A/3 (9-25) sequence.
  • Glycine is the smallest amino acid, it lacks a side chain, and it should act like a flexible tether for the fluorescent group; therefore, as the fluorescent-labeled A ⁇ (9-25) molecule assembles into fibrils the flexible glycine tether will act like a hinge and allow the fluorescent group to be pushed out of the way if steric clashes occur with other parts of the fibril.
  • the fluorescent-A/3 molecules must be prevented from forming fibrils before they are mixed together.
  • One approach to achieving this objective is to mix the peptides in solid lyophilized form before dissolving them in buffer. The shortcoming of this method, however, is that the relative amounts of each peptide will have to be determined using dry weights, which is a very inaccurate method.
  • An alternative approach is to first dissolve the two peptides in a solution containing a denaturant that prevents fibril formation, and then to initiate the fibril assembly process by diluting out the denaturant.
  • This method will involve: a) preparing concentrated stock solutions of Trp-A/3 and AEDANS-A3 in denaturant and accurately determining the peptide concentrations by UV absorbance using the absorption coefficients of Trp and AEDANS, b) mixing appropriate amounts of each peptide solution to produce equimolar mixtures of both peptides in denaturant solution, c) initiate fibril assembly by diluting out the denaturant, and d) simultaneously starting to monitor the evolution of fluorescence energy transfer in the fluorimeter.
  • This method should be very rapid and it should allow evaluation of the kinetics and thermo ⁇ dynamics of fibril assembly. The kinetic measurement will be limited only by the dead-time required for diluting out the denaturant> which for manual mixing is around 10 seconds. If the kinetics of fibril assembly occur too rapidly using the manual mixing method, then this method can easily be adapted into a stopped-flow fluorimetric method which has a dead-time of 30 milliseconds.
  • formic acid which is known to prevent fibril assembly (Halverson et al., 1990). Initiating fibril assembly, however, would then require neutralization of the acid, which in turn would cause an increase in ionic strength of the solution. Thus fibril assembly at low ionic strengths cannot be explored using an acid as the denaturant.
  • formic acid is not an ideal denaturant for the purposes of the invention.
  • the organic solvent 40% trifluoroethanol has been shown to maintain A/3 in a soluble monomeric helical state at very high peptide concentrations and at pH values between 1.3 and 10 (Barrow and Zagorski, 1991) . Accordingly, this solvent was chosen as the preferred denaturant because it does not cause any harmful side reactions, it is nonviscous, so mixing is efficient; and it is volatile allowing for easy removal.
  • Trp-A/3(9-25) and AEDANS-A/3(9-25) are dissolved separately in 40% trifluoroethanol at millimolar concentrations. Under these conditions the A/3 analogues will adopt monomeric helical conformations. 2) After determining the concentration of the stock peptide solutions by UV absorbance, an equimolar mixture of the peptides at micromolar concentration is prepared.
  • Fibrils composed of Trp-A/3(9-25) alone, AEDANS-A/3(9-25) alone, and equimolar mixtures of the two peptides were formed by diluting concentrated solutions of the peptides dissolved in 40% TFE into aqueous buffer.
  • the fluorescence emission spectrum of these peptide preparations are shown in Figure 1.
  • the emission of the Trp fluorophore is significantly quenched in the mixed fibril preparation compared to the Trp-emission of fibrils composed of Trp-A / ⁇ (9-25) alone.
  • the AEDANS-emission is significantly enhanced in the mixed fibril preparation, relative to the emission of fibrils composed of AEDANS-A/3(9-25) alone.
  • Donor quenching of Trp and the sensitized emission of AEDANS are typical features of energy transfer.
  • Fibril formation reached a maximum at pH 5 and decreased both towards more acidic and more basic pH (Figure 2) .
  • Basal acceptor/donor ratios occurred below pH 2 in the acidic limb and above pH 8 in the basic limb of the titrations suggesting very few fibrils were present beyond these values.
  • the pH dependence of fibril formation was modelled as a sequential equilibrium between 4 ionic species, and the data were fit to the following equation (I) which relates the acceptor/donor ratio to the concentrations of each ionic species and 3 pKa constants:
  • Fibrils composed of Trp-A / ⁇ (9-25) alone and AEDANS-A/3(9-25) alone were formed separately, aged for 24 hrs, mixed, and then fluorescence emission spectra of the mixed fibril preparation were acquired at timed intervals for the next 8 hrs.
  • the acceptor/donor ratio as a function of time after mixing is shown in Figure 3.
  • the successful development of a fluorescent fibrillogenesis assay depends on two prerequisites: a) the extrinsic fluorescent groups must not interfere with the process of fibril formation, and b) significant energy transfer must occur between donor and acceptor fluorophores in the fibril state.
  • the data presented show that both prerequisites have been met.
  • the fluorescent-labelled A/3(9-25) peptides formed fibrils, as observed by electron microscopy, that had very similar morphology to fibrils formed by native A/3 peptides and A ⁇ fragments (Fraser et al., 1991; Fraser et al., 1994).
  • the apparent pKa of 3.98 is suggestive of carboxyl groups on Asp and Glu.
  • the apparent pKa's of 5.86 and 7.62 is suggestive of imidazole groups on His.
  • fibrillogenesis in A ⁇ (9-25) is maximal when Hisl3, His 14, Glull, Glu22 and Asp23 are in their ionized states.
  • the involvement of Lysl6 cannot be assessed because fibril formation does not occur around pH 10 where titration of Lys sidechain occurs.
  • the pH dependence could be caused by either isoelectric precipitation or formation of salt bridges.
  • the maximal fibrillogenesis at pH 5.00 ( Figure 2) is a result of the peptide showing a greater tendency to associate at its isoelectric point.
  • the pH dependence could be caused by formation of imidazole-carboxylate salt bridges between His sidechains and sidechains of Asp and/or Glu. To differentiate between these two possibilities the effect of counter ion screening by NaCl was examined.
  • amyloid- enhancing factors to promote fibril formation under physiological conditions.
  • a ⁇ fibrillogenesis can be investigated at the physiological pH of 7.00.
  • increasing the amount of heparin present results in an increased level of fluorescence energy transfer as evidenced by a decrease in donor fluorescence.
  • the data shown in Figure 4 were obtained using a concentration of Trp/AEDANS-A/3(9-25) of 100 nM, and the buffer was phosphate-buffered saline, pH 7.00.
  • the invention is a new technique for monitoring fibrillogenesis by A/3. Using this technique, it has been demonstrated that fibril formation by Trp-A/3(9-25) and AEDANS-Aj ⁇ (9-25) is stabilized by 2 imidazole-carboxylate salt bridges. It has also been demonstrated that monomers that compose the fibril are not kinetically trapped in the fibril state, and that they exchange with the soluble fraction. The exchange kinetics are multiphasic. This information will be extremely valuable when designing pharmacological agents that inhibit fibril formation. By obtaining a greater understanding of the fibril assembly process under physiological conditions, a better idea of which features of the process are the most susceptible to attack can be obtained and exploited.
  • the standardized fluorescence assay for fibrillogenesis of the invention should also be of immediate value for screening compounds that have potential for being inhibitors of fibril formation.
  • Peptides were synthesized, by the solid phase method on the Milligen 9050TM peptide synthesizer, as peptide- amides using Rink-resin (Advanced Chemtech) .
  • An active ester coupling procedure employing pentafluorophenyl esters of 9-fluorenylmethoxycarbonyl amino acids, was used.
  • the N-termini was acetylated with acetic anhydride.
  • the peptides were cleaved from the resin with 95:2.5:2.5 trifluoroacetic acid: thioanisole: ethanedithiol mixture.
  • the peptides were purified by C 18 reverse phase chromatography, and peptide identity was confirmed by FAB mass spectrometry and amino acid analysis. Peptide purity was assessed by analytical C 18 reverse phase chromatography using the Pharmacia FPLC system.
  • AEDANS 5-Acetylethyldiaminonaphthalene-1-sulfonic acid labelling was performed by incubation of 1 mM Cys-containing peptide with 5 mM (1,5) -IAEDANS (Molecular Probes) in 50 mM Tris-HCl, 6 M guanidine hydrochloride, pH 8.0 for 18 hrs. AEDANS-labelled peptide was separated from unreacted peptide and 1,5-IAEDANS by C 18 reverse phase chromatography.
  • Trp-A/3(9-25) was dissolved in a 2 ml solution of 40% (v/v) trifluoroethanol (TFE) containing 10 mM acetic acid.
  • TFE trifluoroethanol
  • This stock solution was diluted 1:10 with 7.5 M guanidine hydrochloride (GdnHCl) and the peptide concentration was determined via tryptophan absorbance at 280 nm with an absorbance coefficient (e) of 5690 M ⁇ cm '1 (Edelhoch, 1967) .
  • Lyophilised AEDANS -A ⁇ (9-25) was dissolved in a 2ml solution of 40% (v/v) TFE adjusted to pH 9 with ammonium hydroxide.
  • e for AEDANS in 40% ethanol is 6500 M ⁇ cm "1 and the absorbance ⁇ max is 338.0 nm. It was found that the difference in absorbance between AEDANS in ethanol and TFE is less than 1%, well within experimental error; therefore, an e of 6500 M ⁇ c ⁇ r 1 were used in all concentration determinations of AEDANS-A/3(9-25) in 40% TFE.
  • Negatively stained fibrils were prepared by floating charged pioloform, carbon-coated grids on peptide solutions (0.025 mg/mL Trp :A ⁇ (9-25) and 0.025 mg/mL AEDANS:A/3(9-25) , pH2-pH8) . These solutions were pre-aged 24 h and 1 week. To control pH, the peptide solutions were made using a buffer of 1 mM borate, 1 mM citrate and 1 mM phosphate. After the grids were blotted and air-dried, the samples were stained with 1% (w/v) phosphotungstic acid which was prepared using the same borate, citrate, phosphate buffer and pH adjusted to correspond to the respective samples. The Hitachi H-7000TM instrument used for visualization was operated at 75 kV. The samples were inspected and images of representative fibrils were recorded.
  • Amyloid /3-protein is produced by cultured cells during normal metabolism. Nature 359, 322-325.

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Abstract

Méthode permettant de contrôler in vitro l'assemblage des fibrilles de peptide ou de protéine. Pour obtenir une sensibilité à des concentrations nanomolaires de l'ordre de celles observées in vivo, on recourt dans cette méthode à un transfert d'énergie de fluorescence entre des fluorophores donneur et receveur étroitement juxtaposés. Le procédé consiste à fixer un fluorophore donneur à un peptide ou une protéine fibrillogène, et fixer un fluorophore receveur à un deuxième peptide ou une deuxième protéine fibrillogène. Les fluorophores donneur et receveur sont situés sur les premier et second peptides ou protéines, et sont ainsi juxtaposés de façon à permettre le transfert d'énergie entre eux lors de la formation de fibrilles. Les peptides ou protéines contenant le fluorophore sont mélangés en solution à une concentration physiologique normale, et on suit la formation de fibrilles en observant le transfert d'énergie de fluorescence entre les fluorophores donneur et receveur. Le mélange est réalisé de préférence par l'apport en quantités équimolaires de peptides ou de protéines dans une solution dénaturante; on commence le contrôle de la formation de fibrilles en diluant le dénaturant.
PCT/CA1996/000555 1995-08-17 1996-08-16 Methode de controle de l'assemblage des fibrilles de proteine WO1997007402A1 (fr)

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Cited By (12)

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FR2766927A1 (fr) * 1997-07-31 1999-02-05 Centre Nat Rech Scient Methodes et kits pour le diagnostic de la maladie d'alzheimer
EP1003539A1 (fr) * 1997-08-14 2000-05-31 The Regents Of The University Of California PEPTIDES AMYLOIDES A$g(b) FLUORESCENTS ET UTILISATION DE CES DERNIERS
US6329356B1 (en) 1998-04-10 2001-12-11 Neurochem, Inc. Phosphono-carboxylate compounds for treating amyloidosis
WO2002042321A2 (fr) * 2000-11-24 2002-05-30 Isis Innovation Limited Fibrilles melangees
US6399314B1 (en) 1999-12-29 2002-06-04 American Cyanamid Company Methods of detection of amyloidogenic proteins
US6562836B1 (en) 1999-05-24 2003-05-13 Queen's University Of Kingston Methods and compounds for inhibiting amyloid deposits
US6703212B1 (en) 1997-07-25 2004-03-09 Centre National De La Recherche Scientifique Methods and kits for diagnosing Alzheimer's disease from a blood sample
FR2871464A1 (fr) * 2004-06-15 2005-12-16 Centre Nat Rech Scient Collections de composes tracables et leurs utilisations
US7105307B2 (en) 1997-08-30 2006-09-12 Cyclacel, Ltd. Compositions and methods for screening for modulators of enzymatic activity
EP2330113A1 (fr) 2002-04-19 2011-06-08 The Governing Council Of The University Of Toronto Méthodes immunologiques et compositions pour le traitement de la maladie d'Alzheimer
US8835654B2 (en) 2004-12-22 2014-09-16 Bhi Limited Partnership Method and compositions for treating amyloid-related diseases
US9499480B2 (en) 2006-10-12 2016-11-22 Bhi Limited Partnership Methods, compounds, compositions and vehicles for delivering 3-amino-1-propanesulfonic acid

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EP0552108A2 (fr) * 1992-01-17 1993-07-21 The University Of Maryland At Baltimore Essai immunologique utilisant transfert d'énergie fluorescent

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