EP1335937A2 - Fibrilles melangees - Google Patents

Fibrilles melangees

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Publication number
EP1335937A2
EP1335937A2 EP01997501A EP01997501A EP1335937A2 EP 1335937 A2 EP1335937 A2 EP 1335937A2 EP 01997501 A EP01997501 A EP 01997501A EP 01997501 A EP01997501 A EP 01997501A EP 1335937 A2 EP1335937 A2 EP 1335937A2
Authority
EP
European Patent Office
Prior art keywords
fibril
fibrils
peptides
peptide
ttr
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.)
Withdrawn
Application number
EP01997501A
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German (de)
English (en)
Inventor
Christopher Martin Dobson
Catherine Macphee
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.)
Oxford University Innovation Ltd
Original Assignee
Oxford University Innovation Ltd
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Filing date
Publication date
Priority claimed from GBGB0028710.2A external-priority patent/GB0028710D0/en
Application filed by Oxford University Innovation Ltd filed Critical Oxford University Innovation Ltd
Publication of EP1335937A2 publication Critical patent/EP1335937A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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

Definitions

  • This invention relates to mixed fibrils.
  • the aggregation of proteins and peptides in the form of stable and highly ordered amyloid fibrils is most commonly associated with pathological conditions such as Alzheimer's disease and the transmissible spongiform encephalopathies.
  • amyloid fibrils can be formed consisting of two or more distinct molecular species that co-assemble within the ⁇ -sheet airay that makes up the core of these structures.
  • amyloid fibrils can be "doped” by the incorporation of peptide like molecules or ones with different side chains.
  • the present invention provides an amyloid fibril comprising two or more different peptides. Typically the fibril is free of other protein.
  • the present invention further provides: a plastic or gel comprising mixed fibrils of the invention; a support for the growth of cells, said support comprising mixed fibrils, a plastic or a gel of the invention;
  • an amyloid fibril of the invention which comprises preparing a solution of the peptides, said solution being in a state so that nucleation and fibril growth will occur over an acceptable time and allowing nucleation and fibril growth to take place; - an amyloid fibril when made by a process of the invention;
  • a fibril of the invention as a plastic or in electronics or catalysis; and a fibril, plastic, gel or support of the invention for use in a method of treatment of the human or animal body by therapy.
  • Figure 1 shows the fluorescence emission spectrum for F5M-TTR 10 . 19 incorporated into TTR 10 . 19 fibrils (dashed line) compared with the spectrum for free F5M- TTR 10 . 19 (solid line).
  • Figure 1(b) shows the spectrum for dansyl-TTR 105 . 115 incorporated into TTRio 5 -ii 5 fibrils (dashed line) compared with that for free dansyl-TTR 105 . 115 (solid line).
  • Figure 2 shows the circular dichroism spectra for TTR 10S . 115 fibrils (dashed line) and TTRio M is fibrils containing 1% (w/w) F5M-TTR 10 . 19 (solid line).
  • Figure 3 shows that the binding of anti-fluorescein IgG to insulin-TTR 10 . 19 mixed fibrils is periodic.
  • Figure 3(a) shows a histogram of the distance in nm between colloidal gold particles bound to TTR 105 . 115 fibrils contai-ning F5M-TTR ⁇ 0 . 19 .
  • Figure 3 (b) shows the a histogram of the distance between colloidal gold particles bound to insulin fibrils containing F5M-TTR 10 - ⁇ 9 .
  • Figure 4(a) shows the conductivity of unmodified fibrils .
  • Figure 4(b) shows the conductivity of fibrils contai-tiing 2% w/w of fluorescently labeled peptide.
  • SEQ ID No. 1 shows the amino acid sequence of the peptide TTR 10 - ⁇ 9 .
  • SEQ ID No. 2 is the amino acid sequence of the peptide TTR 105 . 115 .
  • protein as used herein, it is meant one or more proteins, protein fragments, polypeptides or peptides.
  • the protein is any protein capable of forming fibrils and may be a pharmaceutically active protein.
  • amyloid fibrils consisting of two or more unrelated species can be formed.
  • none of the peptides or proteins examined is directly implicated in any naturally-occurring disease (although such peptides and proteins are not excluded and may in particular be used in embodiments where the fibrils formed are not to be used in vivo) yet the resulting structures resemble closely those formed in vivo in pathological states.
  • the mixed fibrils may comprise two, three, four or more different types of peptide species.
  • amyloid fibrils of the invention can provide a rigid scaffold that can be covalently modified by the conjugation of fully-folded and active proteinaceous enzymes or cofactors, or non-protein materials such as metals, chelating agents or optically active compounds.
  • fibril assembly is a fundamental property of the polypeptide chain, this raises the possibility of employing these fibrillar arrays as structural building blocks in the development of novel biomaterials and nanostructures.
  • artificial proteins can be engineered with virtually absolute control of composition, chain length, sequence and stereochemical purity.
  • the proteins forming the fibril are unrelated, typically they do not have a high degree of sequence homology.
  • the proteins possess the same amino acid sequence, or ones which are very similar such as homologous sequences e.g. insulin from a pig and from a human, (and are therefore related) and differ in that one possesses a pendant functional group, for example, one peptide can be labeled and the other is unlabelled. More generally, one of the proteins possess a pendant functional group and is referred to as having been functionalised.
  • Functional groups which can be incorporated in this way include metals, such as gold, silver, copper or heavy metals, radioisotopes, chelating groups and optically active groups including fluorescent groups such as dansyl chloride and fluorescein-5-maleimide, other proteins, drugs and biotin or magnetic particles.
  • TTR 10 - 19 and TTR 105 . ⁇ i 5 both derived from the sequence of the human plasma protein transthyretin can be used for co-fibril formation. Each corresponds to a ⁇ -strand in the native structure of this extensively ⁇ -sheet protein. Both peptides readily assemble into fibrils with all of the structural characteristics that are diagnostic of amyloid.
  • TTR ⁇ legal- 19 has a single cysteine residue at position 1 and this can be chemically modified with the fluorophore fluorescein-5-maleimide (F5M-TTR 10 . 19 ) or other suitable groups.
  • TTR 105 - 115 does not contain any amino groups other than the terminal ⁇ -amino group; this group can be coupled to the fluorescent group dansyl chloride (dansyl- TTR 105 - 115 ) or other suitable groups.
  • the functional group can be attached by reaction of the peptide, if desirable after modification of a function of the peptide, with a molecule comprising the desired functional group.
  • a cysteine residue e.g. the N-terminal cysteine group
  • a phosphine such as tri-(2- carboxyethyl) phosphine
  • This thiol group can be reacted with, say, fluorescein-5-maleimide.
  • Other groups which can be functionalised include amino, carboxyl, hydroxyl and amino groups. The functionalisation can be carried out using standard reactions. Thus dansyl chloride can be reacted with an amino group.
  • the peptides may also be functionalised using bifunctional or multifunctional linkers.
  • the bifunctional linkers comprise two active groups which can be used to conjugate the linker to other molecules. In the case of a hetero-bifunctional linker these groups are different and preferably such linkers are used. Typically, one of the groups of the linker is reacted with the peptide, whilst the other group is reacted with a second molecule.
  • the linker may be conjugated to the peptide before the peptide is used to form fibrils and then the second group conjugated to the other molecule after, or during, fibril formation.
  • the second molecule may be a peptide in another fibril or a separate polypeptide, nucleic acid or therapeutic molecule.
  • the second group may be conjugated to a virus, cell or synthetic surface.
  • the linker may be cleavable. This may be used to release the conjugated substances from the fibril or for networks or meshes of cross-linked fibrils to be broken up or dissembled, when exposed to a suitable enzyme or chemical capable of cleaving the linker.
  • suitable bifunctional linkers which may be used include:
  • APDP - N(4-Azidosalicylamido)Butyl-3'(2 , Pyridyldithio)Propionamide
  • cleavable, second site is non-specifically photoreactive
  • ASIB (l-(P-Azidosalicylamido)-4-(Iodoacetamido)Butane)
  • ASIB (l-(P-Azidosalicylamido)-4-(Iodoacetamido)Butane)
  • MPBH (4-(4-N-Maleimidophenyl) Butyric Acid Hydrazide Hydrochloride) non-cleavable, second site is reactive towards carbohydrates;
  • SIAB N-Succinimidyl(4-Iodoacetyl) Aminobenzoate
  • non-cleavable, second site is reactive towards primary amines, 10.6 A spacer arm
  • SMCC Succinimidyl-4-(N-Maleimidomethyl)
  • non-cleavable, second site is reactive towards primary amines, 11.6 A spacer arm
  • SMPT (4-Succinimidyloxycarbonyl-Methyl-(2-Pyridyldithio)-Toluene
  • non-cleavable, second site is reactive towards primary amines, 11.2 A spacer arm
  • SPDP ⁇ -Succin--midyl-3-(2-Pyridyldithio) Propionate
  • cleavable, second site is reactive towards primary amines, 6.8 A spacer arm.
  • the hetero-bifunctional linker may be used to cross link fibrils and in a preferred embodiment a linker comprising an amine reactive NHS ester and a photoreactive arylazide is used.
  • the linker is first joined to one of the peptide species and a suitable amount of the linked peptide introduced into fibrils. The fibrils are then subsequently exposed to UN light to cross link them and hence may be used to generate a plastic.
  • the peptide species comprising the fibrils may be chosen for their ability to form fibrils and/or their functional properties. In one embodiment, one of the polypeptides is chosen for its ability to form fibrils and the other for its functional properties such as its therapeutic properties or for ease of functionalisation.
  • the structural polypeptide may be chosen so that the fibril disassociates at a particular temperature, ionic strength, or pH or when it comes into contact with a specific substance or cell type. This may mean that the rate at which the fibril disassociates may be specifically designed by varying the amount and/or type of each subunit present. This may be used to allow the controlled release of a drug at a set rate.
  • the individual peptides themselves may have activities.
  • the peptides may be an agonist, an antagonist, an inhibitor or modulator such as of a receptor.
  • the peptide may be a toxin, an enzyme, a fluorescent protein (such as green fluorescent protein), a hormone, a drug, a single chain antibody, a receptor, an epitope, or an inflammatory modulator such as an cytosine or interleukin, a cofactor, or clotting factor.
  • the peptide may typically adopt its active conformation once released from the fibril, but typically not before.
  • the fibrils are comprised of two or more types of peptides may allow pairs of drugs or therapeutic molecules to be delivered to the same location at the same time. In one embodiment this may allow both an enzyme and its substrate to be delivered to a location and the enzyme to only begin to act on a substrate once the two are released. Prior to, and during fibril formation the enzyme may be kept in an inactive state due to, for example, the conditions employed to make the fibrils.
  • the relative amounts of the two peptides in the fibril may also be chosen to optimize the structural and functional properties of the fibrils.
  • the ratio of the two subunits may be altered to define the amount of therapeutic peptide released or the intensity of the fluorescence. This may allow low dose or high dose fibrils to be produced.
  • the ratio of fluorochrome labeled, therapeutic, or functionalised peptide to the other peptide may be from 100:1 to 10:1, preferably from 50:1 to 5:1 and more preferably from 10: 1 to 2: 1, alternatively the amount of the other peptide may be equal or greater than the functionalised, therapeutic or labeled peptide and hence the ratio may be from 1 : 1 to 1 : 100,000, typically from 1 :2 to 1 : 10,000, preferably from 1 :5 to 1 : 100, more preferably from 1 : 10 to 1: 100, but will depend on the particular use the fibril is being put to. By altering the ratio of the constituent peptides this may allow the temperature at which the fibril breaks down to be modified or to alter the rate of disassembly.
  • the peptide species used to form the fibrils may have different binding sites so that the fibril has several specificities.
  • the peptides may have binding or recognition sites for two different cell types and facilitate the bringing together of the two types of cells.
  • the fibrils may also comprise binding sites to allow them to localise specifically to a particular cell type, organ or tissue. Fibrils containing a binding site for a particular substance may be used to purify or extract that substance, for example, fibrils with comprising a particular epitope may be used to affinity purify antibodies specific for the epitope.
  • the peptides employed in the invention are preferably synthetic. Alternatively, they may be, or be derived from, recombinant proteins. In some embodiments, they may be purified naturally occurring proteins. The fibrils may be formed in vivo such as in a non-human transgenic animal and subsequently harvested. In addition to the 20 naturally occurring amino acids, manipulation of bacterial expression systems has allowed the incorporation of amino acids with unnatural side- chains into polypeptide chains, thereby expanding the repertoire of chemical functionalities available for exploitation. Any of these unnatural amino acids may be incorporated in the polypeptides of the invention.
  • the fibrils of the present invention can be obtained in a similar manner to that described in WO 00/17328 but using a solution of the different proteins, the solution being in a state so that nucleation of the protein and fibril growth will occur over an acceptable time, and allowing nucleation and fibril growth to take place.
  • nucleation is meant the initiation of processes that lead to fibril formation. Fibril formation from a solution involves, successively, protein self- association, formation of aggregates and fibril growth. Thus, desirably, the initiation solution is on the verge of instability. Nucleation and growth are slow processes and conditions are normally chosen so that fibril formation occurs over a period of hours or days. It will be appreciated that if nucleation occurs too rapidly then this will often have an adverse affect on fibril formation. Fibrils for use in vivo are typically larger than aggregates.
  • Nucleation can be caused by a variety of means including variations in solvents, concentration, salt, ligand, temperature and pH.
  • fibrils from TTR 1(H9 and TTR 105 . 115 can be obtained from an aqueous solution at pH about 2.
  • Fibrils from bovine insulin can be obtained from an aqueous solution at pH about 2.5. Shaking, agitation and exposure to certain surfaces, for example the surface of a glass or plastic vessel, may cause local denaturation and thereby initiate fibril formation.
  • the solution comprising a protein may comprise any solvent or mixture of solvents in which nucleation can occur.
  • the solution may comprise DMSO, dioxin and/or water.
  • the solution is an aqueous solution.
  • organic solvents which can promote nucleation and fibril growth may be incorporated into the solution.
  • the organic solvent is generally water-miscible and is preferably an alcohol or an aliphatic nitrile such as acetonitrile.
  • the alcohol is typically a C x . 6 alkanol which may be substituted or unsubstituted for example by one or more halogen atoms, especially fluorine atoms. Examples include methanol, ethanol, propenyl or butanol, or fluorinated alcohols such as trifluoroethanol or hexafluoroisopropanol.
  • the alcohol is trifluoroethanol.
  • the concentration of alcohol is typically from 5 to 40% v/v and preferably about 25%v/v.
  • the concentration of aliphatic nitrile can vary between wide limits and is typically from 5 to 95% v/v.
  • the concentration of protein in the solution is not limited in any way but it must be such that nucleation can occur. Generally the concentration is from 0. ImM to lOmM, preferably from 0.5mM to 5mM and more preferably the concentration of protein is about ImM.
  • the temperature of the solution is generally from 0°C to 100°C.
  • the temperature is from 0 ° C to 70 ° C, more preferably from 0 ° C to 40 ° C and most preferably from 5°C to 30°C.
  • the pH of the solution is any pH suitable for nucleation, typically from pH 1 to pH 10.
  • the solution is acidic and more preferably the pH of the solution is from 1 to 6.5, for example 1.5 to 3.5.
  • the solution may be seeded with, for example, previously formed particles of aggregated protein; this can greatly speed up the process.
  • the fibrils of the present invention are suitably isolated by centrifugation, filtration or evaporation of solvent.
  • the fibrils thus obtained may then be washed and dried and, if necessary, resuspended.
  • the physical properties of the fibrils, such as solubility, can be altered in this way.
  • the polypeptides can be tagged so that they can be removed. If a tagged mixed fibril can be formed and readily removed problems such as beta-2M aggregation can be minimised.
  • the functional group present in the fibrils may be used to facilitate their purification or recovery.
  • TTR 10 . 1 and TTR 105 . 115 can be incorporated into fibrils assembled primarily from an unrelated protein sequence, bovine insulin, which consists of two disulfide-linked chains in its largely helical native fold.
  • bovine insulin which consists of two disulfide-linked chains in its largely helical native fold.
  • the formation of fibrils from bovine insulin is well established and can readily be achieved by heating the protein to high temperatures (60°C) at low pH e.g. 2.5.
  • the rate of fibril formation by the insulin molecules is significantly faster than that observed for TTR 10 . 19 and TTR 105 - 115 : 7 hours for completion of the process, whereas both peptides remain soluble for 48 hours under similar conditions.
  • the peptides alone at the concentration used for co-fibril formation remained fully soluble over a period of 3 weeks under identical conditions.
  • the fibrils of the present invention may also be formed in vivo.
  • nucleic acid sequences encoding one, several or all of the polypeptides may be introduced into cells, such as eukaryotic and in particular mammalian cells, and expressed.
  • the sequences are introduced into the cells of an organism suffering from, or a model for, a fibril associated disease. Fibrils will then be formed comprising both the endogenous polypeptide associated with the disease and the heterologous polypeptide. This may allow the fibrils to be labelled or the targeting of cells or therapeutic substances to the fibrils. This may be useful in studying the disease state or in therapy and/or diagnosis.
  • the organism may be a non-human transgenic animal into which the heterologous sequences have been introduced.
  • nucleic acid sequences are targeted to, or comprise sequences which ensure the expression in, the same cell type which is synthesizing the endogenous peptide.
  • their expression may be inducible rather than constitutive.
  • incorporation into a fibril may result in a change in the properties of the label.
  • the label may be a fluorochrome and incorporation into the fibril of the labeled peptide may result in a change its emission spectra. Such changes may be used to monitor fibril formation' and breakdown both in vitro and in vivo.
  • the shape of the fibril and the periodicity of the doped peptide may be altered. This may allow peptides with a particular shape, such as a helical twist or a kink, which are hollow or ribbon shaped to be produced or to set the distance between functional groups.
  • Non-native self-assembling peptide polymers have previously been synthesized, following careful design principles based on side-chain interactions, to form reversible and pH-dependent hydrogels, ⁇ -sheet peptide nanotubes that can act as non-specific pores within membranes, ⁇ -sheet "nanotapes" and lamellar crystals.
  • the formation of amyloid fibrils of the present invention typically requires no explicit design process since it is a fundamental property of the polypeptide backbone.
  • the solubility, stability and conformational properties of monomeric precursors will, however, vary with the specific sequence allowing exploitation of a wide range of environments for fibril assembly.
  • amyloid fibrils as tubular structures with a hollow core, flat ribbon-like morphologies, twisted fibrils where the periodicity of the twist is dictated by the identity of the protein or peptide precursor and these can be chosen to tailor the fibril to its specific use.
  • the identity of the protein precursor influences the average diameter and homogeneity of the resulting fibril population. Fibril assembly can be reversed on modulation of the solvent environment the fibrils of this invention can find utility as chemically reversible and biodegradable gels. Indeed, it seems likely that the amyloid fibrils from native proteins and peptides are closely related to some of these artificially designed systems. This is, therefore, a valuable indication that nanostructures and devices could be made if desired.
  • the fibrils of the inventions may be cross-linked, typically by using bifunctional linkers and preferably a hetero-bifunctional linker.
  • one of the peptides used to form the fibrils may be reacted with a linker, then incorporated into a fibril, and the fibrils subsequently cross-linked using the second of the linker's functional groups.
  • a bifunctional linker comprising an a ine reactive NHS ester and a photoreactive arylazide may be used with the peptide of choice being conjugated to the linker via the free amino group and kept in the dark.
  • the modified peptide can then be incorporated into fibrils and afterwards the fibrils cross-linked by exposure to near UN light to activate the photoreactive arylaride.
  • Fibril cross-linking can be used to produce a stronger, more robust network or mesh of fibrils.
  • the cross-lmking can, in effect, produce a plastic.
  • the degree of doping can be adjusted to alter the amount of cross-linking and hence the strength of the plastic produced.
  • the plastic may be formed in a particular shape, such as a tubular structure.
  • the plastics can be used in a number of devices or structures and may also be used to coat or cover devices.
  • the plastics may be used to form or cover a stent, valve, patch or support. They may be used to cover wounds or in bandages or plasters.
  • the plastics may be used as a support on which cells can be grown on and the whole structure or the cells can then be used as a graft or transplant.
  • the plastic may be used to grow skin cells on, or islets cells.
  • the linker may also include a cleavage site to allow the cross-linked mesh to be broken down. This may be used, to remove the plastic whilst leaving the cells intact. This may be done in vitro or in vivo and in particular in vivo.
  • the cleavage may be carried out chemically or enzymatically and is typically done in such a way as to cause mini al damage to the surrounding tissues. This may allow shaped arrangements of cells to be produced such as in a pouch, bladder or vessel.
  • the peptides of the present invention may be conjugated to nucleic acids or nucleic acid analogues such as DNA, RNA or PNA.
  • a bifunctional linker is used to link the nucleic acid to the fibrils.
  • the nucleic acid molecule may be modified to include a group through which it can be conjugated or bound to a peptide either directly or through a linker.
  • the nucleic acid may posses a free sulfhydryl group and be conjugated to the peptide through that.
  • the peptide may be conjugated with the nucleic acid before, after or during fibril formation, but preferably before.
  • the nucleic acid may be joined to the fibril through a cleavable linker so that it can be subsequently released.
  • the nucleic acid molecules may be either double or single stranded and may range in size from a short oligonucleotide to considerably longer, such as from 10 to 5,000 bases, preferably from 20 to 1,000 bases, more preferably from 25 to 50 bases in length.
  • the peptide-nucleic acid may be immobilized on a surface such as a membrane or in silico and may be in the form of a micro-array or chip. These may be used to detect the presence and quantity of a specific target sequence in a test solution. This may be employed in diagnosis or genotyping.
  • the mixed fibrils conjugated with DNA or other nucleic acids may also be used as a delivery system for the nucleic acid into an organism for gene therapy purposes.
  • the nucleic acid may drive the expression of a protein or alternatively may be an antisense molecule employed to block the expression of a target gene.
  • the nucleic acid may be a catalytic nucleic acid.
  • the fibrils of the invention may display conductivity and in particular those comprising fluorescent functional groups may do so, and display particularly good conductivity.
  • the ability to carry current may be important in the use of the fibrils in nanotechnology and biocomputers.
  • hollow fibrils may be used.
  • the fibrils conductance may change according to the surrounding conditions and/or the binding of target molecules. This will also allow the use of the fibrils in sensors or determine the presence or amount of a particular analyte.
  • the fibrils are typically linked to a means for detecting the change in conductivity on binding.
  • the fibrils may be used to treat diseases associated with amyloid deposition.
  • diseases which may be treated, prevented or ameliorated include diabetes, senility and dementia and in particular Alzheimer's and Parkinson's.
  • Prion disorders such as Creutzfield- Jakob disease (CJD), BSE, scrapie, fatal familial insomnia (FFI), and kuru may be also treated and in particular new variant CJD.
  • Diabetes may also be treated and in particular late onset diabetes.
  • amyloid protein A amyloidosis in mice can be accelerated by the injection of preformed amyloid fibrils assembled from a number of peptide sources, including residues 20-29 of islet amyloid polypeptide (IAPP) and the sequences TTR ⁇ 0 . 19 and TTR 105 . 115
  • injection of fibrillar material can provide a seeding mechanism for the deposition of amyloid protein A.
  • the ability to incorporate a different polypeptide and hence to form a mixed fibril in vivo could be used to block fibril formation, to incorporate a drag or introduce a drag binding site. It could also be used as a diagnostic or therapy for the fibril associated condition.
  • the mixed fibrils may be used to deliver drugs to individuals suffering from non- fibril associated diseases such as cardiovascular disorder or a cancer such as a soft tissue cancer, sarcoma, leukemia or lymphoma.
  • non- fibril associated diseases such as cardiovascular disorder or a cancer such as a soft tissue cancer, sarcoma, leukemia or lymphoma.
  • the particular fibril employed may be chosen so that its characteristics such as the rate it disassembles, the temperature, pH or location it disassembles at, or the dosage and combination of drags it releases are tailored to the needs of the patient.
  • the fibrils may also be used to facilitate the bringing together of two entities such as different types of cells or a tissue and a cell due to the presence of bringing sites for each cell. Labeled fibrils may also be used in imaging.
  • the fibrils may be formulated with standard pharmaceutically acceptable carriers and/or excipients as is routine in the pharmaceutical art.
  • a suitable substance may be dissolved in physiological saline or water for injections.
  • the exact nature of a formulation ill depend upon several factors including the particular fibrils to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Eastern Pennsylvania, 17 th Ed. 1985, the disclosure of which is included herein of its entirety by way of reference.
  • the fibrils may be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, topical or other appropriate administration routes.
  • a therapeutically effective amount of the fibrils is administered to a patient.
  • the dose of the fibril may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen.
  • a physician will be able to determine the required route of administration and dosage for any particular patient.
  • a typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific modulator, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration.
  • daily dosage levels are from 5 mg to 2 g.
  • the fibrils of the invention may also be used to form gels to encapsulate drags or to make gels impregnated or carrying a drag.
  • the gel typically breaks down to release the drug either because the capsule is broken or the impregnated drag released.
  • the breakdown of the gel is typically the result of the disassembly of the fibrils. The breakdown may be made temperature or pH dependent and may occur slowly or rapidly to ensure the desired release rate of the drag.
  • Such gels may be used to produce pills or capsules or alternatively implants.
  • the gels may also be used to coat devices introduced into the body such as stems or patches to slowly release the drug at that site.
  • TTR 1(H9 SEQ ID No: 1 - CPLMVKVLDA
  • TTR 105 . 115 SEQ ID No:2 - YTIAALLSPYS
  • Applied Biosystems 430A automated peptide synthesizer using standard Fmoc chemistry, within the Oxford Centre for
  • the peptides were purified by RP- HPLC on a Beckman System Gold HPLC using either an analytical Brownlee 4.6x30 mm C8 reverse-phase column or a preparative Nydac 10x250 mm C-18 reverse phase column. Fractions eluting from the column were analysed by MALDI-TOF mass spectrometry, and fractions of the desired molecular weight pooled and lyophilised.
  • TTR 10 _ 19 labeling with fluorescein.
  • the ⁇ -terminal cysteine residue of TTR 10 . 19 was the target for labeling by the thiol-reactive fluorophore fluorescein-5-maleimide (F5M; Molecular Probes, Eugene, OR).
  • F5M thiol-reactive fluorophore fluorescein-5-maleimide
  • TTR 10 . 19 was resuspended at 1 mg/ml in H 2 O, and the cysteine residue reduced by the addition of a 10-fold molar excess of tris-(2-carboxyethyl) phosphine (TCEP; Molecular Probes, Eugene, OR) and incubation for 1 h at room temperature.
  • TCEP tris-(2-carboxyethyl) phosphine
  • F5M in DMSO was added to the peptide solution to give a 10-fold molar excess of fluorophore over peptide in a final concentration of 10% DMSO. Under these conditions TTR 10 . 19 was fully soluble. The mixture was incubated overnight at 4°C to ensure maximum labeling. The labeling mixture was then applied to a disposable SepPak C-18 reverse-phase chromatography column (Waters, Milford, MA) and washed with H 2 O/ CH 3 C ⁇ (90: 10 v/v). TCEP and unreacted label eluted in the wash.
  • the peptide was then eluted from the column with H 2 O/ CH 3 CN (40:60 v/v), and a mixture of labeled and unlabelled peptide was detected by MALDI-TOF mass spectrometry.
  • the labeling efficiency was estimated at ⁇ 70% by mass spectrometry, and also by absorption measurements assuming an extinction coefficient of 83,000 at 492 nm.
  • TTR 105 . 115 labeling with dansyl chloride.
  • the N-terminal amino group of TTR 105 . 115 was the target for labeling with dansyl chloride (Molecular Probes, Eugene, OR).
  • TTR 105 - 115 was resuspended at 1 mg/ml in H 2 O/ CH 3 CN (80:20 v/v).
  • Dansyl chloride in DMSO was added to the peptide solution to give a 10-fold molar excess of fluorophore over peptide in a final concentration of 10% DMSO.
  • the pH was adjusted manually to 9.0 with the addition of ammonia. Under these conditions TTR 105 -. 115 was fully soluble.
  • the mixture was incubated for 2 h at room temperature to ensure maximum labeling.
  • the labeling mixture was then applied to a disposable SepPak C-18 reversed phase chromatography column (Waters, Milford, MA) and washed with H 2 O. There was no detectable elution of material during the wash step.
  • the peptide was then eluted from the column with H 2 O/ CH 3 CN (50:50 v/v), and a mixture of labeled and unlabelled peptide was detected by MALDI-TOF mass spectrometry. Unreacted dansyl chloride did not elute from the column under these conditions.
  • the labeling efficiency was estimated at ⁇ 80% by mass spectrometry, and also by absorption measurements assuming an extinction coefficient of -4000 at 370 nm.
  • amyloid fibrils containing a mixture of labeled and unlabelled peptides of the same amino acid sequence. After incubation for 7 days the resulting fibrils were examined by ultracentrifugation, electron microscopy (EM), circular dichroism (CD) spectroscopy and fluorescence methods (see below).
  • EM electron microscopy
  • CD circular dichroism
  • TTR ⁇ 0 - ⁇ 9 fibrils in the presence and absence of F5M-TTR 10 . 19 , were generated by resuspending the peptide at 10 mg/ml in H 2 O at pH 2.0 and incubating at room temperature for 5 days. In fibrils containing the labeled species, the molar ratio of unlabelled to labeled peptide was 100:1.
  • TTR 105 -. ⁇ i 5 fibrils in the presence and absence of dansyl-TTR 105 . ⁇ 5 , were generated by resuspending the peptide at 10 mg/ml in CH 3 CN/ H 2 O (20:80 v/v) at pH 2 and incubating at 37°C for 7 days. In fibrils containing the labeled species, the molar ratio of unlabelled to labeled peptide was 100:1.
  • Bovine insulin Sigma Chemical Co., MO
  • fibrils were generated by resuspending the protein at 11.5 mg/ ml (2 mM) in H 2 O at pH 2.5 and incubating the suspension at 70°C for 1 h.
  • the protein solution was plunged into liquid N 2 , and then returned to 70°C for a further 4 h. Mixed fibril formation.
  • Both TTR ⁇ 0 . 19 fibrils containing dansyl-TTR ⁇ 05 . 115 and TTR ⁇ s-ns fibrils containing F5M-TTR 1( 9 were prepared at a molar ratio of unlabelled to labeled peptide of 100:1.
  • the stock solution containing a total of 10 mg/ml peptide was prepared in CH 3 CN/ H 2 O (10: 1 v/v) adjusted to pH 2 with H 2 O and incubated at 37°C for 10 days.
  • Bovine insulin was prepared as a stock solution of 9.7 mg/ml in H 2 O at pH 2.7, containing a molar ratio of insulin to labeled peptide species (dansyl-TTR 105 . n5 or F5M-TTR 10 . 19 ) of 40: 1. The solution was incubated at 55°C for 10 days. Fibril formation was assessed by CD spectroscopy and by EM.
  • Comparison of CD spectra acquired over the 10-day incubation period indicates the transition from the predominantly ⁇ -helical structure of bovine insulin to the ⁇ -sheet structure typical of amyloid fibril formation.
  • the resulting fibrils display a characteristic helical twist along the long axis of the fibril, repeating every ⁇ 60 nm as determined by electron microscopy.
  • the fluorescence properties of purified cofibrils containing dansyl-TTR 105 . 115 were also consistent with insertion of the fluorophore into the insulin fibrils, with a 32 nm blue shift in the wavelength of maximum emission to 470 nm, and an increase in anisotropy to 0.19 (Table 1). More than 70% of the peptide was found in the pellet on centrifugation of the fibrils .
  • Circular dichroism spectroscopy CD spectra were acquired on a Jasco model J-720 spectropolarimeter using a 0.1 cm pathlength quartz cuvette. The temperature was kept constant at 25°C by use of a circulating water bath. Each spectrum represents the average of 4 scans acquired at 0.5 nm intervals between 200-250 nm, with a response time of 4 s. The data are expressed in millidegrees rather than mean residue ellipticity due to the difficulty associated with accurately determining the absolute concentration of an aggregated peptide. For this reason, it was not possible to accurately determine the amount of ⁇ -sheet structure within the complexes. Furthermore, in all cases the absorption of the sample was monitored concurrently to the circular dichroism signal, to avoid possible distortion of the spectrum due to light scattering or absorption flattening phenomena.
  • FIG. 2 shows the circular dichroism spectroscopy of TTR 105 . ⁇ 5 fibrils in the presence and absence of 1%> (w/w) F5M-TTR 10 . 19 .
  • TTRi 05 . n5 fibrils (dashed line) were generated by resuspending the peptide at 10 mg/ml in 20% CH 3 CN at pH 2 and incubating for 24 hours at 37°C, followed by 7 days incubation at room temperature.
  • Fibrils containing 1% (w/w) F5M-TTR 10 . ⁇ 9 solid line
  • the 10 mg/ml peptide stock solution was incubated for 10 days at 37°C in solution containing 10% CH 3 CN. This shows the formation of extensive ⁇ -sheet structure.
  • Electron microscopy Electron microscopy. Electron micrographs of amyloid fibrils were acquired on a Jeol JEM-1010 transmission electron microscope at 80 kN excitation voltage. 2 ⁇ l of peptide at a concentration of 10-60 ⁇ g/ml was applied to formvar- and carbon-coated copper grids. The fibrils were washed by the successive addition of three 10 ⁇ l aliquots of water, each wash step followed by drying with filter paper. The fibrils were stained with by the addition of 10 ⁇ l uranyl acetate (1% w/v), and immediately dried with filter paper. Electron micrographs of :
  • Fluorescence Spectroscopy Fluorescence Spectroscopy. Fluorescence spectroscopic measurements were performed on a Perkin-Elmer LS50B luminescence spectrometer equipped with a fast filter accessory to enable anisotropy measurements. Quartz cuvettes with a 1 cm pathlength were employed throughout. The temperature was kept constant at 25°C by use of a circulating water bath. Slit widths were 2.5 nm and 10 nm unless otherwise stated. Samples containing fluorescein were excited at 490 nm, with emission detected between 500-600 nm. Samples containing dansyl chloride were excited at 370 nm, and the emission measured between 400-550 nm.
  • Panel a F5M-TTR ⁇ 0 - ⁇ 9 incorporated into TTR I0 - ⁇ 9 fibrils (dashed line) is compared with F5M-TTR 10 - 19 (solid line) in 20 mM sodium phosphate buffer, pH 7.0 at a final labeled peptide concentration of 0.3 ⁇ M.
  • Panel b dansyl-TTR 105 - ⁇ 5 incorporated into TTR 105 .
  • 115 fibrils (dashed line) is compared with dansyl-TTR 105 . 115 (solid line) in water at a final labeled peptide concentration of 0.9 ⁇ M.
  • the fluorescence spectra have been normalised.
  • the fluorescence emission of the fluorescein moiety of F5M-TTR ⁇ 0 . 19 undergoes a red-shift of 9 nm when incorporated into TTR 10 . 19 fibrils, and the anisotropy of the fluorescein increases from 0.03 for the monomeric peptide in aqueous solution to 0.17 on formation of the fibrils ( see Table 1).
  • This increase in the anisotropy of the fluorescent moiety indicates incorporation of the fluorophore into large complexes that exhibit slow rotational motion.
  • the dansyl moiety of dansyl-TTR 105 The dansyl moiety of dansyl-TTR 105 .
  • the fibrils were pelleted by centrifugation at 9500 g for 15 min, the pellet resuspended in H 2 O pH 2 and deposited on a glass slide and imaged using fluorescence light microscopy. Fluorescence light microscopy of this pelleted material demonstrated a green fluorescence, consistent with insertion of the probe into high molecular weight structures. Similarly, 10 mg/ml TTR ⁇ 05 - ⁇ s containing only 1% by weight of F5M-TTR 10 . 19 incubated for 10 days at 37°C in 10% ⁇ CH 3 CN formed extensive ⁇ -sheet structure as indicated by CD.
  • the shape of the CD spectrum is, however, substantially different to that of TTR 105 _ 115 alone: a prominent negative band previously observed at ⁇ 235 nm, which we ascribe to the formation of a turn structure in the assembled fibrils, decreases in magnitude ( Figure 2). This suggests a substantial change in the secondary structure of the peptides in the fibrillar array.
  • the fibrils observed by EM are different in appearance from the almost crystalline structures formed by TTR ⁇ o 5 - 115 alone, and we observe cofibrils with a regular periodic twist in the electron micrographs.
  • Fluorescence spectroscopy indicates little change in the spectral properties of the fluorescein moiety, which exhibits a red shift of only ⁇ 2 nm, but the anisotropy increases substantially to 0.22.
  • the 5 nm gold particles were observed to be associated with the various fibrils, whereas TTR ⁇ 0 . l9 fibrils alone did not elicit a reaction to the antibody.
  • Measurement of the distances between successive gold clusters bound to the fibril indicates that the gold particles are distributed on average every 40-50 nm, but at irregular intervals along the fibril length.
  • the hydrogen-bonding distance between peptides along the long axis of the fibril is 4.7 A, so the average distance between clusters suggests that the antigenic label is incorporated every 80-100 peptides.
  • the fluorescently labeled peptide which makes up approximately one in every hundred of the peptide precursors, appears to be incorporated randomly into the growing fibril.
  • the secondary antibody, colloidal gold-labeled goat anti-rabbit IgG (Sigma Chemical Co) was diluted 1/10 in HBSS, and applied to the grids for 30 min.
  • the grids were washed three times for 10 min in blocking solution, fixed in 0.5 % (v/v) glutaraldehyde for 10 min, washed with H 2 O for 1 min and stained with 0.5 % (w/v) uranyl acetate.
  • Insulin fibrils containing F5M-TTR 10 - ⁇ 9 were negatively stained with uranyl acetate and electron micrographs taken, these showed the regular nature of the helical twist along the length of the fibril.
  • Immunogold labelling of the bovine insulin fibrils containing 1% (w/w) F5M-TTR 10 - 19 showed gold clusters associated with the fibrils, indicating the incorporation of the labeled peptide, and there was no non-specific binding of F5M-TTR 10 . 19 on incubation of soluble peptide with preformed insulin fibrils.
  • Figure 3 shows that the binding of anti-fluorescein IgG to insulin-TTR 10 - 19 cofibrils is periodic.
  • Panel a Histogram showing the distance in nm between colloidal gold particles bound to TTR 105 . ⁇ i 5 fibrils containing F5M-TTR 10 - ⁇ 9 from the immunogold labeling images obtained above.
  • Panel b Histogram showing the distance in nm between colloidal gold particles bound to insulin fibrils containing F5M-TTR 10 - ⁇ 9 from the immunogold labeling images obtained above.
  • the binding of gold-labeled antibody to insulin-TTR cofibrils appears to exhibit a degree of regularity.
  • the periodicity is presented as a histogram in Figure 3b, which indicates that the labeling occurs at -60 and -125 nm intervals.
  • a possible interpretation of this result is the regular insertion of the peptide into the growing insulin fibrils: the helical twisting of insulin fibrils every 60 nm along the fibre axis suggests at least one structural phenomenon that repeats at regular intervals.
  • the periodic binding of gold particles may represent insertion of the peptide into the growing insulin fibril only on fibril twisting.
  • amyloid fibrils provide compelling support for a generic structure of these fibrils that is dictated not by sidechain-specific interactions but by the formation of a network of hydrogen bonds between the invariant groups that make up the polypeptide backbone.
  • the backbone is involved in a series of stabilising interactions and thus amyloid fibril formation is observed only following partial denaturation or destabilisation of a stable tertiary structure or the physiologically inappropriate generation of unstructured peptide fragments in vivo.
  • the identity of the amino acid sidechains will affect the structure and stability of the protein precursor, but will not dictate the fundamental ⁇ -sheet hydrogen-bonding network of the assembled fibril. Different sidechains will pack together in different ways within the assembled sheet structure, as indeed is observed in globular structures. It is likely that for some sequences particularly favorable contacts will be made between different sidechains and result in more favorable packing interactions. Such sequences may therefore show high propensities to form fibrillar structures. But these propensities, and the stability of the fibrils under specific solution conditions, will also depend on the intrinsic properties of the soluble form of the polypeptide chain, such as its interaction with solvent and the stability of the globular structure.
  • the identity of the amino acid sidechains will also influence the interactions between the unstructured regions that are thought to have an effect on the self-assembly of protofilaments into amyloid fibrils.
  • different protein species may give rise to amyloid fibrils with differences in the detailed morphology, but with a common underlying hydrogen-bonding ⁇ -sheet network.
  • the present invention has wide applicability.
  • Fibril cross-linking Peptide was modified by the addition of a hetero-bifunctional cross- linker consisting of an amine reactive NHS ester and a photoreactive arylazide in the dark.
  • the cross-linker conjugates to the peptide via free amino groups, leaving the reactive arylazide, leaving the reactive arylazide.
  • About 10% w/w of the peptide was incorporated into amyloid fibrils as described above and then kept in the dark. The mature fibrils were then exposed to near UN light which resulted in aligned and cross-linked sheets of fibrils which chemically resembled naturally occurring plastics. Electron micrographs of the fibrils before UN cross -linking, stored in the dark and after UN cross-linking confirmed that a dense cross-linked network resulted from the exposure to UN light.
  • Peptide-D ⁇ A mixed fibrils Single stranded D ⁇ A was chemically conjugated to a peptide by exploiting thiol chemistry. 1% of the peptide was subsequently introduced into amyloid fibrils comprising predominately the same peptide but without the conjugated D ⁇ A. The successful introduction of the D ⁇ A conjugated peptides into the fibrils was demonstrated by immunogold labeling. Transmission electron micrographs for amyloid fibrils including the D ⁇ A conjugated peptide, negatively stained with uranyl acetate and immunogold labeling demonstrated the successful incorporation of the D ⁇ A-peptide subunits into the fibrils.

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Abstract

L'invention concerne des fibrilles comprenant au moins deux peptides différents, lesdits peptides pouvant être apparentés ou non. Lesdites fibrilles peuvent être utilisées comme biomatériaux et nanomatériaux.
EP01997501A 2000-11-24 2001-11-23 Fibrilles melangees Withdrawn EP1335937A2 (fr)

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US7851434B2 (en) 2006-03-15 2010-12-14 The Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth Near Dublin Amyloid and amyloid-like structures
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JANCIAUSKIENE S. ET AL: "Alzheimer's peptide Abeta1-42 binds to two beta sheets of alpha1-antichemotrypsin and transforms it from inhibitor to substrate", J. BIOL. CHEM., vol. 273, no. 43, 23 October 1998 (1998-10-23), pages 28360 - 28364 *
MA J. ET AL: "Alzheimer Abeta neurotoxicity: promotion by antichymotrypsin, Apo4; inhibition by Abeta-related peptides", NEUROBIOLOGY OF AGING, vol. 17, no. 5, 1996, pages 773 - 780 *
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