WO2003063760A2 - Peptides antibodies directed thereagainst and methods using same for diagnosing and treating amyloid-associated diseases - Google Patents
Peptides antibodies directed thereagainst and methods using same for diagnosing and treating amyloid-associated diseases Download PDFInfo
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- WO2003063760A2 WO2003063760A2 PCT/IL2003/000079 IL0300079W WO03063760A2 WO 2003063760 A2 WO2003063760 A2 WO 2003063760A2 IL 0300079 W IL0300079 W IL 0300079W WO 03063760 A2 WO03063760 A2 WO 03063760A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- 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
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- 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
- C07K14/575—Hormones
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- 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
- C07K14/575—Hormones
- C07K14/585—Calcitonins
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0819—Tripeptides with the first amino acid being acidic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1021—Tetrapeptides with the first amino acid being acidic
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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
Definitions
- the present invention relates to peptides and antibodies directed thereagainst which can be used to diagnose, prevent, and treat amyloid-associated diseases, such as Type II diabetes mellitus.
- Amyloid material deposition (also referred to as amyloid plaque formation) is a central feature of a variety of unrelated pathological conditions including Alzheimer's disease, prion-related encephalopathies, type II diabetes mellitus, familial amyloidosis and light-chain amyloidosis.
- Amyloid material is composed of a dense network of rigid, nonbranching proteinaceous fibrils of indefinite length that are about 80 to 100 A in diameter. Amyloid fibrils contain a core structure of polypeptide chains arranged in antiparallel ⁇ -pleated sheets lying with their long axes perpendicular to the long axis of the fibril [Both et al. (1997) Nature 385:787-93; Glenner (1980) N. Eng. J. Med. 302:1283- 92].
- amyloid fibril proteins Approximately twenty amyloid fibril proteins have been identified in-vivo and correlated with specific diseases. These proteins share little or no amino acid sequence homology, however the core structure of the amyloid fibrils is essentially the same. This common core structure of amyloid fibrils and the presence of common substances in amyloid deposits suggest that data characterizing a particular form of amyloid material may also be relevant to other forms of amyloid material and thus can be implemented in template design for the development of drugs against amyloid-associated diseases such as type II diabetes mellitus, Alzheimer's dementia or diseases and prion-related encephalopathies.
- amyloid deposits do not appear to be inert in vivo, but rather are in a dynamic state of turnover and can even regress if the formation of fibrils is halted [Gillmore et al. (1997) Br. J. Haematol. 99:245-56].
- therapies designed to inhibiting the production of amyloid polypeptides or inhibiting amyloidosis may be useful for treating amyloid associated diseases.
- Inhibition of the production of amyloid polypeptides Direct inhibition of the production of amyloid polypeptides may be accomplished, for example, through the use of antisense oligonucleotides such as against human islet amyloid polypeptide messenger RNA (mRNA).
- mRNA messenger RNA
- the addition of antisense oligonucleotides or the expression of antisense complementary DNA against islet amyloid polypeptide mRNA increased the insulin mRNA and protein content of cells, demonstrating the potential effectiveness of this approach [Kulkarni et al. (1996) J. Endocrinol. 151:341-8;
- Amyloid fibrils - Amyloid contains potential stabilizing or protective substances, such as serum amyloid P component, apolipoprotein E, and perlecan. Blocking their binding to developing amyloid fibrils could inhibit amyloidogenesis [Kahn et al. (1999) Diabetes 48:241-53], as could treatment with antibodies specific for certain parts of an amyloidogenic protein [Solomon et al. (1997) Proc. Natl. Acad. Sci. USA 94:4109-12].
- Destabilizing compounds - Heparin sulfate has been identified as a component of all amyloids and has also been implicated in the earliest stages of inflammation-associated amyloid induction.
- Kisilevsky and co-workers described the use of low molecular weight anionic sulfonate or sulfate compounds that interfere with the interaction of heparin sulfate with the inflammation-associated amyloid precursor and the ⁇ peptide of Alzheimer's disease (AD).
- Heparin sulfate specifically influences the soluble amyloid precursor (SAA2) to adopt an increased ⁇ -sheet structure characteristic of the protein-folding pattern of amyloids.
- SAA2 soluble amyloid precursor
- These anionic sulfonate or sulfate compounds were shown to inhibit heparin accelerated A ⁇ fibril formation and were able to disassemble preformed fibrils in vitro, as monitored by electron micrography.
- these compounds substantially arrested murine splenic inflammation-associated amyloid progression in vivo in acute and chronic models.
- the most potent compound i.e., poly- (vinylsulfonate)] showed acute toxicity.
- Antioxidants Another proposed therapy has been the intake of antioxidants in order to avoid oxidative stress and maintain amyloid proteins in their reduced state (i.e., monomers and dimers).
- the use of sulfite was shown to lead to more stable monomers ofthe TTR both in vitro and in vivo [Altland (1999) Neurogenetics 2:183- 188].
- a complete characterization of the antioxidant effect is still not available and the interpretation of results concerning possible therapeutic strategies remains difficult.
- a peptide comprising at least 3 amino acid residues and less than 15 amino acid residues, the peptide including an amino acid sequence as set forth in SEQ ID NO: 7.
- amino acid sequence further includes a polar uncharged amino acid selected from the group consisting of serine, threonine, asparagine, glutamine and natural derivatives thereof.
- the amino acid sequence further includes at least one positively charged amino acid and at least one negatively charged amino acid.
- the at least one positively charged amino acid is selected from the group consisting of lysine, arginine and natural and synthetic derivatives thereof.
- the at least one negatively charged amino acid is selected from the group consisting of aspartic acid, glutamic acid and natural and synthetic derivatives thereof.
- amino acid sequence is selected from the group consisting of SEQ ID NO: 4, 12-19 and 27-45.
- the peptide is selected from the group consisting of SEQ ID NOs. 4, 12-19 and 27-45.
- a peptide comprising at least 3 amino acid residues and less than 15 amino acid residues, the peptide including an amino acid sequence selected from the group consisting of SEQ
- a peptide selected from the group consisting of SEQ ID NOs: 8, 10-11, 21 -22 and 25.
- a method of treating or preventing an amyloid-associated disease in an individual comprising providing to the individual a therapeutically effective amount of a peptide having at least 3 amino acid residues and less than 15 amino acid residues, the peptide including an amino acid sequence as set forth in SEQ ID NO: 7.
- a method of treating or preventing an amyloid-associated disease in an individual comprising providing to the individual therapeutically effective amount of a peptide having at least 3 amino acid residues and less than 15 amino acid residues, the peptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12-19 and 27-45.
- the peptide is an active ingredient of a pharmaceutical composition which also includes a physiologically acceptable carrier.
- a method of treating or preventing an amyloid-associated disease in an individual comprising providing to the individual a therapeutically effective amount of a peptide selected from the group consisting of SEQ ID NOs: 8, 10-11, 21-22 and 25, wherein the peptide is an active ingredient of a pharmaceutical compositions which also includes a physiologically acceptable carrier.
- a method of treating or preventing an amyloid-associated disease in an individual comprising providing to the individual a therapeutically effective amount of a peptide selected from the group consisting of SEQ ID NOs: 4, 12-19 and 27-45.
- a method of treating or preventing an amyloid-associated disease in an individual comprising providing to the individual therapeutically effective amount of a peptide having at least 3 amino acid residues and less than 15 amino acid residues, the peptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 10-11, 21-22 and 25, wherein the peptide is an active ingredient of a pharmaceutical composition which also includes a physiologically acceptable carrier.
- the peptide is expressed from a nucleic acid construct.
- a pharmaceutical composition for treating or preventing an amyloid-associated disease comprising as an active ingredient a peptide having at least 3 amino acid residues and less than 15 amino acid residues, the peptide including an amino acid sequence as set forth in SEQ ID NO: 7 and a pharmaceutically acceptable carrier or diluent.
- a pharmaceutical composition for treating or preventing an amyloid-associated disease comprising as an active ingredient a peptide selected from the group consisting of SEQ ID NOs: 8, 10-11, 21-22 and 25 and a pharmaceutically acceptable carrier or diluent.
- a pharmaceutical composition for treating or preventing an amyloid-associated disease comprising as an active ingredient a peptide having at least 3 amino acid residues and less than 15 amino acid residues, the peptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 10-11, 21-22 and 25 and a pharmaceutically acceptable carrier or diluent.
- a pharmaceutical composition for treating or preventing an amyloid-associated disease comprising as an active ingredient a peptide having at least 3 amino acid residues and less than 15 amino acid residues, the peptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12-19 and 27-45 and a pharmaceutically acceptable carrier or diluent.
- compositions for treating or preventing an amyloid-associated disease comprising as an active ingredient a peptide selected from the group consisting of SEQ
- nucleic acid construct comprising a polynucleotide segment encoding a peptide having at least 3 amino acid residues and less than 15 amino acid residues, the peptide including an amino acid sequence as set forth in SEQ ID NO: 7.
- a nucleic acid construct comprising a polynucleotide segment encoding a peptide selected from the group consisting of SEQ ID NOs: 8, 10-11, 21-22 and 25. According to still a further aspect of the present invention there is provided a nucleic acid construct comprising a polynucleotide segment encoding a peptide selected from the group consisting of SEQ ID NOs: 4, 12-19 and 27-45.
- an antibody or an antibody fragment comprising an antigen recognition region capable of binding a peptide including at least 3 amino acid residues and less than 15 amino acid residues, the peptide including an amino acid sequence as set forth in SEQ ID NO: 7.
- a pharmaceutical composition for treating or preventing an amyloid-associated disease comprising as an active ingredient an antibody or an antibody fragment having an antigen recognition region capable of binding a peptide including at least 3 amino acid residues and less than 15 amino acid residues, the peptide including an amino acid sequence as set forth in SEQ ID NO: 7.
- a method of treating or preventing an amyloid-associated disease in an individual comprising providing to the individual therapeutically effective amount of an antibody or an antibody fragment having an antigen recognition region capable of binding a peptide including at least 3 amino acid residues and less than 15 amino acid residues, the peptide including an amino acid sequence as set forth in SEQ ID NO: 7.
- the peptide further comprising at least two serine residues at a C-terminus thereof.
- the peptide is a linear or cyclic peptide.
- the peptide further includes at least one beta-breaker amino acid.
- beta-beaker amino acid is proline.
- the present invention successfully addresses the shortcomings ofthe presently known configurations by providing novel peptides, compositions and methods, which can be used to diagnose and treat amyloid associated diseases such as type II Diabetes mellitus.
- FIG. 1 is a schematic illustration depicting the self-assembly ability and hydrophobicity of a group of peptides from a number of amyloid proteins as deduced using Kyte and Dolittle scale. Note, that no correlation is observed between hydrophobicity and the amyloidogenic potential of the analyzed peptides. The only apparent indication for potential amyloid fibril formation in this group of peptide is a combination of aromatic nature and minimal length.
- FIGs. 2a-c are schematic illustrations of amyloid binding with the inhibitory aromatic reagents: Ro 47-1816/001 (Figure 2a), Thioflavin T ( Figure 2b) and CR dye ( Figure 2c).
- FIGs. 3a-c are schematic illustrations of a primary sequence comparison between human and rodent LAPP and the synthetic peptides of the present invention.
- Figure 3a is a sequence alignment of human and rodent LAPP.
- a block indicates a seven amino acid sub-sequence illustrating the major inconsistencies between the sequences.
- the "basic amyloidogenic unit" is presented by bold letters and underlined.
- Figure 3b illustrates the chemical structure ofthe wild type LAPP peptide (SEQ ID NO: 1).
- Figure lc illustrates the primary sequences and SEQ ID NOs. of the peptides derived from the basic amyloidogenic unit.
- FIGs. 4a-b are graphs illustrating light absorbance at 405 nm as a function of time during fibril formation thus reflecting the aggregation kinetics of IAPP-derived peptides. The following symbols are used: closed squares - N1A, opened circles -
- G3A closed circles - wild type, opened triangles - L6A, opened squares - I5A and closed triangles - F2A.
- FIG. 5 is a histogram depicting mean particle size of assembled LAPP peptide and derivatives as measured by light scattering. Each column represents the results of 3-5 independent measurements.
- FIGs. 6a-n are photomicrographs illustrating Congo Red binding to pre- assembled LAPP peptides. Normal field and polarized field micrographs are shown respectively for each of the following aged peptide suspensions: N1A peptide ( Figures 6a-b), F2A peptide ( Figures 6c-d), G3A peptide ( Figures 6e-f), wild type peptide ( Figures 6g-h), 15 A peptide ( Figures 6i-j) and L6A ( Figures 6k-l).
- FIGs. 7a-f are electron micrographs of "aged" LAPP peptide and derivatives. N1A peptide (Figure 7a), F2A peptide (Figure 7b), G3A peptide ( Figure 7c), wild type peptide (Figure 7d), I5A peptide ( Figure 7e) and L6A ( Figure If).
- the indicated scale bar represents 100 nm.
- FIG. 8a is a nucleic acid sequence alignment of wild type hlAPP and a corresponding sequence modified according to a bacterial codon usage. Modified bases are underlined.
- FIG. 8b is a schematic illustration of the pMALc2x-NN vector which is used for cytoplasmic expression of the 48 kDa MBP-IAPP protein.
- the V8 Ek cleavage site and the (His)6 tag are fused C-terminally to the malE tag vector sequence.
- a factor Xa cleavage site for removal ofthe MBP tag is indicated.
- FIG. 9 is a protein gel GelCode Blue staining depicting bacterial expression and purification of MBP and MBP-IAPP fusion protein.
- Bacterial cell extracts were generated and proteins were purified on an amylose resin column. Samples including 25 ⁇ g protein were loaded in each of Lanes 1-3 whereas 5 ⁇ g protein were loaded on each of lanes 4-5. Proteins were resolved on a 12 % SDS-PAGE and visualized with GelCode Blue staining. A molecular weight marker is indicated on the left.
- Lane 1 - 0.5 mM IPTG-induced soluble extract of MBP.
- Lane 2 - 0.1 mM IPTG-induced soluble extract of MBP-IAPP.
- Lane 3 - 0.5 mM IPTG-induced soluble extract of MBP-IAPP.
- An arrow marks the MBP-IAPP.
- FIGs. lOa-b are a dot-blot image ( Figure 10a) and densitometric quantitation thereof (Figure 10b) depicting putative amyloidogenic sequences in hlAPP.
- FIG. 11 is a graphic illustration depicting light absorbance at 405 nm as a function of time during fibril formation thus reflecting the aggregation kinetics of LAPP-derived peptides (SEQ ID NOs. 14-19). The following symbols are used: closed squares - FLVHSS, opened circles - FLVHS, closed diamonds - NFLVHSS, opened triangles - NFLVHSSNN, opened squares - FLVH and closed triangles - NFLVH.
- FIGs. 12a-f are photomicrographs illustrating Congo Red binding to pre- assembled IAPP peptides. Polarized field micrographs are shown for each of the following one day aged peptide suspensions: NFLVHSSNN peptide ( Figures 12a),
- FIGs. 13a-f are electron micrographs of "aged” LAPP peptides.
- NFLVHSSNN peptide Figures 13a
- NFLVHSS Figure 13b
- FLVHSS Figure 13c
- NFLVH Figure 13d
- FLVHS Figure 13e
- FLVH Figure 13f
- the indicated scale bar represents 100 nm.
- FIGs. 14a-f are graphs showing secondary structures in the insoluble LAPP aggregates as determined by Fourier transformed infrared spectroscopy.
- NFLVHSSNN peptide Figures 14a
- NFLVHSS Figure 14b
- FLVHSS Figure 14a
- FIG. 15 is a chemical structure of a previously reported amyloidogenic peptide fragment of Medin [Haggqvist (1999) Proc. Natl. Acad. Sci. U S A 96:8669-
- FIGs. 16a-b are graphs illustrating light absorbance at 405 nm as a function of time during fibril formation thus reflecting the aggregation kinetics of Medin-derived peptides.
- Figure 16a illustrates a short-term kinetic assay.
- Figure 16b illustrates a long-term kinetic assay .
- FIGs. 17a-f are electron micrographs of "aged" Medin-derived peptides. NFGSVQFA - Figures 17a, NFGSVQ - Figure 17b, NFGSV - Figure 17c, FGSVQ -
- FIGs. 18a-f are photomicrographs illustrating Congo Red binding to pre- assembled Medin-derived peptides. Polarized field micrographs are shown for each of the following aged peptide suspensions: NFGSVQFA - Figures 18a, NFGSVQ -
- FIGs. 19a-c depict the effect of an alanine mutation on the amyloidogenic features of the hexapeptide amyloidogenic fragment of Medin.
- Figure 19a - is a graph illustrating light absorbance at 405 nm as a function of time during fibril formation thus reflecting the aggregation kinetics of Medin-derived alanine mutant;
- Figure 19b is an electron micrograph of "aged" Medin- derived alanine mutant, The scale bar represents 100 nm;
- Figure 19c - is a photomicrograph illustrating Congo
- FIGs. 20a-b are the amino acid sequence of human Calcitonin ( Figure 20a) and chemical structure of an amyloidogenic peptide fragment of human Calcitonin
- FIGs. 21a-d are electron micrographs of "aged" Calcitonin-derived peptides. DFNKF - Figure 21a, DFNK - Figure 21b, FNKF - Figure 21c and DFN - Figure 21d. The indicated scale bar represents 100 nm.
- FIGs. 22a-d are photomicrographs illustrating Congo Red binding to pre- assembled Calcitonin-derived peptides. Polarized field micrographs are shown for each of the following aged peptide suspensions: DFNKF - Figure 22a, DFNK - Figure 22b, FNKF - Figure 22c and DFN - Figure 22d.
- FIG. 23 is a graphic illustration showing secondary structures in the insoluble
- Calcitonin aggregates as determined by Fourier transformed infrared spectroscopy.
- FIGs. 24a-c depict the effect an alanine mutation on the amyloidogenic features ofthe pentapeptide amyloidogenic fragment of Calcitonin.
- Figure 24a is an electron micrograph of "aged" Calcitonin-derived alanine mutant. The scale bar represents 100 nm;
- Figure 24b - is a photomicrograph illustrating Congo Red binding to pre-assembled Calcitonin-derived peptide mutant;
- Figure 24c is a graph showing secondary structures in the mutant peptide as determined by Fourier transformed infrared spectroscopy.
- FIG. 25 is an electron micrograph depicting self-assembly of "aged" Lactotransferrin-derived peptide.
- the scale bar represents 100 nm.
- FIG. 26 is an electron micrograph depicting self-assembly of "aged" Serum amyloid A protein-derived peptide.
- the scale bar represents 100 nm.
- FIG. 27 is an electron micrograph depicting self-assembly of "aged” BriL- derived peptide.
- the scale bar represents 100 nm.
- FIG. 28 is an electron micrograph depicting self-assembly of "aged” Gelsolin- derived peptide.
- the scale bar represents 100 nm.
- FIG. 29 is an electron micrograph depicting self-assembly of "aged" Serum amyloid P-derived peptide.
- the scale bar represents 100 nm.
- FIG. 30 is an electron micrograph depicting self-assembly of "aged" Immunoglobulin light chain-derived peptide.
- the scale bar represents 100 nm.
- FIG. 31 is an electron micrograph depicting self-assembly of "aged” Cystatin C-derived peptide. The scale bar represents 100 nm.
- FIG. 32 is an electron micrograph depicting self-assembly of "aged” Transthyretin-derived peptide. The scale bar represents 100 nm.
- FIG. 33 is an electron micrograph depicting self-assembly of "aged” Lysozyme-derived peptide.
- the scale bar represents 100 nm.
- FIG. 34 is an electron micrograph depicting self-assembly of "aged"
- Fibrinogen-derived peptide The scale bar represents 100 nm.
- FIG. 35 is an electron micrograph depicting self-assembly of "aged" Insulin- derived peptide.
- the scale bar represents 100 nm.
- FIG. 36 is an electron micrograph depicting self-assembly of "aged" Prolactin-derived peptide.
- the scale bar represents 100 nm.
- FIG. 37 is an electron micrograph depicting self-assembly of "aged" Beta 2 microglobulin-derived peptide.
- the scale bar represents 100 nm.
- FIG. 38 is a graphic representation of the effect of an inhibitory peptide on LAPP self-assembly. Squares - wild type (wt) LAPP peptide; triangles - wt-IAPP + inhibitory peptide; circles - no peptides.
- FIG. 39 is a graphic illustration depicting light absorbance at 405 nm as a function of time during fibril formation thus reflecting the aggregation kinetics of IAPP-derived peptides (SEQ ID NOs. 46-49).
- FIG. 40 is a histogram representation illustrating turbidity of LAPP analogues following seven day aging.
- FIG. 41a-f are electron micrographs of "aged" LAPP analogues.
- the indicated scale bar represents 100 nm.
- Figure 42a shows short exposure of the bound peptide-a ⁇ ay.
- Figure 42b shows long exposure of the bound peptide-array.
- Figure 42c shows quantitation of the short exposure ( Figure 42a) using densitometry and arbitrary units. DESCRIPTION OF THE PREFERRED EMBODIMENTS
- the present invention is of novel peptides antibodies directed thereagainst, compositions including same and methods of utilizing each for diagnosing or treating amyloid associated diseases such as type II Diabetes mellitus.
- amyloid associated diseases such as type II Diabetes mellitus.
- amyloid associated diseases such as II diabetes mellitus
- the present inventor has identified a sequence characteristic of amyloid forming peptides which directs fibril formation. This finding suggests that ordered amyloidogenesis involves a specific pattern of molecular interactions rather than the previously described mechanism involving non-specific hydrophobic interactions [Petkova (2002) Proc. Natl. Acad. Sci. U S A 99:16742-16747]. As is further illustrated hereinbelow and in the Examples section which follows, the present inventor attributed a pivotal role for aromatic residues in amyloid formation.
- ⁇ -stacking interactions are non-bonded interactions which are formed between planar aromatic rings.
- the steric constrains associated with the formation of those ordered stacking structures have a fundamental role in self-assembly processes that lead to the formation of supramolecular structures.
- Such ⁇ -stacking interactions which are probably entropy driven, play a central role in many biological processes such as stabilization of the double-helix structure of DNA, core-packing and stabilization ofthe tertiary structure of proteins, host-guest interactions, and porphyrin aggregation in solution [for further review on the possible role of ⁇ -stacking interaction in the self-assembly of amyloid fibrils see Gazit (2002) FASEB J. 16:77-83].
- SEQ ID NO: 7 includes at least one aromatic amino acid residue which, as is shown by the results presented in the Examples section, is pivotal to the formation of amyloid fibrils. It will be appreciated that aromaticity rather than hydrophobicity of the aromatic amino acid is the prevailing chemical feature in amyloid self-assembly as illustrated in Examples 36-39 of the Examples section.
- the aromatic amino acid can be any naturally occurring or synthetic aromatic residue including, but not limited to, phenylalanine, tyrosine, tryptophan, phenylglycine, or modificants, precursors or functional aromatic portions thereof.
- aromatic residues which can be used by the present invention are provided in Table 2 below. As is demonstrated by the results provided in the Examples section which follows, the present invention facilitates the design of peptides exhibiting varying degrees of self-aggregation kinetics and aggregate structure.
- self-aggregation refers to the capability of a peptide to form aggregates (e.g. fibrils) in an aqueous solution.
- aggregates e.g. fibrils
- the ability of a peptide to self-aggregate and the kinetics and type of such self-aggregation determines a use for the peptide in treating or diagnosing amyloid diseases.
- the present invention encompasses both longer peptides (e.g., 10-50 amino acids) which include the sequences set forth in SEQ ID NOs: 4, 8, 10-19, 21-22, 25 or 27-45, or shorter peptides (2-10 amino acid residues) including any of these sequences. Due to their self-aggregating nature these peptides can be used as potent diagnostic reagents.
- the peptides of the present invention preferably further include at least one polar and uncharged amino acid including but not limited to serine, threonine, asparagine, glutamine or natural or synthetic derivatives thereof (see Table 2).
- the peptides of the present invention may further include at least one pair of positively charged (e.g., lysine and arginine) and negatively charged (e.g., aspartic acid and glutamic acid) amino acids (e.g., SEQ ID NOs. 27-29).
- Such amino acid composition may be preferable, since as shown in Examples 21 ofthe Examples section, it is likely that electrostatic interactions between opposing charges may direct the formation of ordered antiparallel structure. Since the present inventors have identified the sequence characteristics governing fibril formation, the teachings ofthe present invention also enable design of peptides which would not aggregate into fibrils and be capable of either preventing or reducing fibril formation or disrupting preformed fibrils and thus can be used as a therapeutic agents. For example, a peptide encompassed by SEQ ID NO: 9, 10, 11, 17, 19, 25 or
- peptide encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.
- Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is inco ⁇ orated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.
- Natural aromatic amino acids, Trp, Tyr and Phe may be substituted for synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (Nol), ring- methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
- the peptides ofthe present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
- amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
- amino acid includes both D- and L-amino acids.
- Tables 1 and 2 below list naturally occurring amino acids (Table 1) and non- conventional or modified amino acids (Table 2) which can be used with the present invention.
- the present peptides are preferably utilized in therapeutics or diagnostics which require the peptides to be in soluble form
- the peptides ofthe present invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.
- the peptides of the present invention preferably further include at least one beta-sheet breaker amino acid residue such as proline (e.g., proline),
- SEQ ID NO. 45 see background section
- SEQ ID NO. 45 which is characterized by a limited phi angle of about -60 to +25 rather than the typical beta sheet phi angle of about -120 to -140 degrees, thereby disrupting the beta sheet structure ofthe amyloid fibril.
- the peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclization does not severely interfere with peptide characteristics, cyclic forms ofthe peptide can also be utilized. Cyclic peptides can either be synthesized in a cyclic form or configured so as to assume a cyclic form under desired conditions (e.g., physiological conditions).
- the present invention provides conclusive data as to the identity of the structural determinant of amyloid peptides, which directs fibril assembly.
- the present invention enables design of a range of peptide sequences, which can be utilized for prevention/treatment or diagnosis of amyloidosis. It will be appreciated that the present inventor could identify the consensus aromatic sequence of the present invention (SEQ ID NO: 7) in numerous amyloid related proteins (see Examples 6-35 of the Examples section). Thus, the present invention enables accurate identification of amyloidogenic fragments in essentially all amyloidogenic proteins. Furthermore, the fact that small aromatic molecules, such as Ro 47-1816/001
- peptides of the present invention allows for the generation of antibodies directed thereagainst, which may be used to dissociate or prevent the formation of amyloid plaques (U.S. Pat. No. 5,688,561).
- antibody refers to intact antibody molecules as well as functional fragments thereof, such as Fab, F(ab') 2 , and Fv that are capable of binding to macrophages.
- functional antibody fragments are defined as follows: (i) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (ii) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (iii) (Fab') 2 , the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds; (iv) Fv, defined as a genetically engineered fragment
- Antibodies may be generated via any one of several methods known in the art, which methods can employ induction of in vivo production of antibody molecules, screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed [Oriandi D.R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837, Winter G. et al. (1991) Nature 349:293-299] or generation of monoclonal antibody molecules by continuous cell lines in culture.
- Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
- Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
- antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
- This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
- an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
- Fv fragments comprise an association of V H and V L chains. This association may be noncovalent, as described in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659- 62, 1972. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker.
- sFv single- chain antigen binding proteins
- the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
- the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow and
- CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry, Methods, 2: 106-10, 1991.
- the antibodies ofthe present invention are preferably humanized.
- Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
- Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) ofthe recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
- CDR complementary determining region
- Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
- Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
- the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
- the humanized antibody optimally also will include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
- Fc immunoglobulin constant region
- a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature
- humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
- humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
- Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol.
- Preferred individual subjects according to the present invention are mammals such as canines, felines, ovines, porcines, equines, bovines, humans and the like.
- treating refers to reducing or preventing amyloid plaque formation, or substantially decreasing plaque occurrence in the affected tissue.
- amyloid plaque refers to fibrillar amyloid as well as aggregated but not fibrillar amyloid, hereinafter “protofibrillar amyloid", which may be pathogenic as well.
- protofibrillar amyloid an aggregated but not necessarily fibrillar form of LAPP was found to be toxic in culture.
- protofibrillar LAPP like protofibrillar ⁇ -synucelin, which is implicated in Parkinson's disease pathogenesis, permeabilized synthetic vesicles by a pore-like mechanism.
- Amyloid-associated diseases treated according to the present invention include, but are not limited to, type TJ diabetes mellitus, Alzheimer's disease (AD), early onset Alzheimer's disease, late onset Alzheimer's disease, presymptomatic Alzheimer's disease, Perkinson's disease, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, medullary carcinoma, aortic medical carcinoma, Insulin injection amyloidosis, prion-systematic amyloidosis, choronic inflammation amyloidosis, Huntington's disease, senile systemic amyloidosis, pituitary gland amyloidosis, Hereditary renal amyloidosis, familial British dementia, Finnish hereditary amyloidosis, familial non-neuropathic amyloidosis [Gazit (2002) Curr.
- prion diseases including scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith and Wells (1991) Cu ⁇ Top Microbiol Immunol 172: 21-38] and human prion diseases including (i) kuru, (ii) Creutzfeldt-Jakob Disease (CJD), (iii) Gerstmann-Streussler-Sheinker Disease (GSS), and (iv) fatal familial insomnia (FF1) [Gajdusek (1977) Science 197: 943-960; Medori, Tritschler et al. (1992) N Engl J Med 326: 444-449].
- CJD Creutzfeldt-Jakob Disease
- GSS Gerstmann-Streussler-Sheinker Disease
- FF1 fatal familial insomnia
- the method includes providing to the individual a therapeutically effective amount of the peptide of the present invention.
- the peptide can be provided using any one of a variety of delivery methods. Delivery methods and suitable formulations are described hereinbelow with respect to pharmaceutical compositions.
- the peptide of the present invention when utilized for treatment of amyloid diseases, includes an amino acid sequence suitable for preventing fibril formation, reducing fibril formation, or disaggregating formed aggregates by competitive destabilization of the preformed aggregate.
- SEQ ID NO: 45 can be utilized for treatment of amyloid diseases, particularly type II diabetes mellitus since as shown in Example 35 of the Examples section which follows, such a sequence interferes with LAPP self-assembly as demonstrated by the decreased ability of the amyloidogenic peptide to bind thioflavin T in the presence of an inhibitory peptide.
- the peptides set forth in SEQ ID NOs: 10 or 11 can be used as potent inhibitors of type II diabetes since as shown in the Examples section which follows, substitution of either leucine or isoleucine in the peptide elicits very slow kinetics of aggregation. Since amyloid formation in vivo is a very slow process, it is conceivable that under physiological conditions no fibrilization will occur upon the substitution of isoleucine or leucine to alanine in the context ofthe full length LAPP. Alternatively, self-aggregating peptides such as those set forth in SEQ ID NOs.
- the peptides of the present invention are preferably synthesized from D- isomers of natural amino acids [i.e., inverso peptide analogues, Tjernberg (1997) J.
- the peptides of the present invention include retro, inverso and retro-inverso analogues thereof. It will be appreciated that complete or extended partial retro-inverso analogues of hormones have generally been found to retain or enhance biological activity. Retro-inversion has also found application in the area of rational design of enzyme inhibitors (see U.S. Pat. No. 6,261,569).
- retro peptide refers to peptides which are made up of L- amino acid residues which are assembled in opposite direction to the native peptide sequence.
- Retro-inverso modification of naturally occurring polypeptides involves the synthetic assembly of amino acids with ⁇ -carbon stereochemistry opposite to that of the corresponding L-amino acids, i.e., D- or D-allo-amino acids in inverse order to the native peptide sequence.
- a rerto inverso analogue thus, has reversed termini and reversed direction of peptide bonds, while essentially maintaining the topology of the side chains as in the native peptide sequence.
- inhibitory peptides preferably include N-methylated amino acids which constrain peptide-backbone due to steric effects [Kapurniotu (2002) 315:339-350].
- aminoisobutyric acid (Aib or methyl alanine) is known to stabilize an ⁇ - helical structure in short natural peptides.
- the N-methylation also affects the intermolecular NH to CO H-bonding ability, thus suppressing the formation of multiplayer ⁇ -strands, which are stabilized by H-bonding interactions.
- the antibodies ofthe present invention may also be used to treat amyloid-associated diseases.
- the peptides and/or antibodies of the present invention can be provided to an individual per se, or as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier.
- a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
- the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
- active ingredient refers to the peptide or antibody preparation, which is accountable for the biological effect.
- pharmaceutically acceptable carrier refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties ofthe administered compound.
- An adjuvant is included under these phrases.
- One of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979).
- excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
- excipients examples include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
- Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
- oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
- transmucosal especially transnasal, intestinal or parenteral delivery
- intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
- one may administer a preparation in a local rather than system
- compositions ofthe present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
- compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically.
- the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
- physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
- penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
- the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
- Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
- Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
- Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpy ⁇ olidone (PVP).
- disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
- Dragee cores are provided with suitable coatings.
- concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl py ⁇ olidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
- Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
- compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
- the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
- the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
- stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
- compositions may take the form of tablets or lozenges formulated in conventional manner.
- the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
- the dosage unit may be determined by providing a valve to deliver a metered amount.
- Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix ofthe compound and a suitable powder base such as lactose or starch.
- compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
- Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
- the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions.
- Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
- Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
- the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
- the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
- a suitable vehicle e.g., sterile, pyrogen-free water based solution
- the preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
- compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
- the therapeutically effective amount or dose can be estimated initially from in vitro assays.
- a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
- Toxicity and therapeutic efficacy ofthe active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
- the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
- the dosage may vary depending upon the dosage form employed and the route of administration utilized.
- the exact formulation, route of administration and dosage can be chosen by the individual physician in view ofthe patient's condition. [See e.g., Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p.l].
- dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution ofthe disease state is achieved.
- compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment ofthe prescribing physician, etc.
- compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
- compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
- the pack may, for example, comprise metal or plastic foil, such as a blister pack.
- the pack or dispenser device may be accompanied by instructions for administration.
- the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency ofthe form ofthe compositions or human or veterinary administration.
- Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
- the peptides or antibodies of the present invention can also be expressed from a nucleic acid construct administered to the individual employing any suitable mode of administration, described hereinabove (i.e., in-vivo gene therapy).
- the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e., ex-vivo gene therapy).
- the nucleic acid construct of the present invention further includes at least one cis acting regulatory element.
- cis acting regulatory element refers to a polynucleotide sequence, preferably a promoter, which binds a trans acting regulator and regulates the transcription of a coding sequence located downstream thereto.
- the promoter utilized by the nucleic acid construct of the present invention is active in the specific cell population transformed.
- cell type-specific and/or tissue-specific promoters include promoters such as albumin that is liver specific [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al.
- the nucleic acid construct of the present invention can further include an enhancer, which can be adjacent or distant to the promoter sequence and can function in up regulating the transcription therefrom.
- the constructs of the present methodology preferably further include an appropriate selectable marker and/or an origin of replication.
- the construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in cells, or integration in a gene and a tissue of choice.
- the construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
- prefe ⁇ ed in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
- viral or non-viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
- Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].
- the most prefe ⁇ ed constructs for use in gene therapy are viruses, most preferably adenoviruses,
- a viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
- Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct.
- LTRs long terminal repeats
- such a construct typically includes a signal sequence for secretion of the peptide or antibody from a host cell in which it is placed.
- the signal sequence for this purpose is a mammalian signal sequence.
- the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
- a signal that directs polyadenylation will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
- Other vectors can be used that are non- viral, such as cationic lipids, polylysine, and dendrimers.
- peptides ofthe present invention can also be used as potent detectors of amyloid fibrils/plaques in biological samples. This is of a special significance to amyloid- associated diseases such as Alzheimer's disease wherein unequivocal diagnosis can only be made after postmortem examination of brain tissues for the hallmark neurofibrillary tangles (NFT) and neuritic plaques.
- NFT neurofibrillary tangles
- the method is effected by incubating the biological sample with a peptide of the present invention capable of co-aggregating with the amyloid fibril and detecting the peptide, to thereby detect the presence or the absence of amyloid fibril in the biological sample.
- a peptide of the present invention capable of co-aggregating with the amyloid fibril and detecting the peptide, to thereby detect the presence or the absence of amyloid fibril in the biological sample.
- a variety of peptide reagents which are capable of recognizing conformational ensembles are known in the art some of which are reviewed in Bursavich (2002) J. Med. Chem. 45(3): 541-58 and in Baltzer Chem Rev. 101(10):3153-63.
- the biological sample utilized for detection can be any body sample such as blood (serum or plasma), sputum, ascites fluids, pleural effusions, urine, biopsy specimens, isolated cells and/or cell membrane preparation.
- tissue biopsies and body fluids from mammals are well known in the art.
- the peptide of the present invention is contacted with the biological sample under conditions suitable for aggregate formation (i.e., buffer, temperature, incubation time etc.); suitable conditions are described in Example 2 of the Examples section.
- Measures are taken not to allow pre-aggregation of peptides prior to incubation with the biological sample. To this end freshly prepared peptide stocks are preferably used.
- Protein complexes within a biological sample can be detected via any one of several methods known in the art, which methods can employ biochemical and/or optical detection schemes.
- the peptides of the present invention are highlighted preferably by a tag or an antibody. It will be appreciated that highlighting can be effected prior to, concomitant with or following aggregate formation, depending on the highlighting method.
- tag refers to a molecule, which exhibits a quantifiable activity or characteristic.
- a tag can be a fluorescent molecule including chemical fluorescers such as fluorescein or polypeptide fluorescers such as the green fluorescent protein (GFP) or related proteins (www.clontech.com). In such case, the tag can be quantified via its fluorescence, which is generated upon the application of a suitable excitatory light.
- a tag can be an epitope tag, a fairly unique polypeptide sequence to which a specific antibody can bind without substantially cross reacting with other cellular epitopes.
- epitope tags include a Myc tag, a Flag tag, a His tag, a leucine tag, an IgG tag, a streptavidin tag and the like.
- aggregate detection can be effected by the antibodies of the present invention.
- this aspect of the present invention provides a method of assaying or screening biological samples, such as body tissue or fluid suspected of including an amyloid fibril. It will be appreciated that such a detection method can also be utilized in an assay for uncovering potential drugs useful in prevention or disaggregation of amyloid deposits.
- the present invention may be used for high throughput screening of test compounds.
- the co-aggregating peptides of the present invention are radiolabeled, to reduce assay volume.
- a competition assay is then effected by monitoring displacement of the label by a test compound [Han (1996) J. Am. Chem. Soc. 118:4506-7 and Esler (1996) Chem. 271:8545-8].
- the peptides of the present invention may also be used as potent detectors of amyloid deposits in-vivo.
- a designed peptide capable of binding amyloid deposits, labeled non-radioactively or with a radio-isotope, as is well known in the art can be administered to an individual to diagnose the onset or presence of amyloid-related disease, discussed hereinabove.
- the binding of such a labeled peptide after administration to amyloid or amyloid-like deposits can be detected by in vivo imaging techniques known in the art.
- the peptides of the present invention can be included in a diagnostic or therapeutic kit.
- peptide sets of specific disease related proteins or antibodies directed thereagainst can be packaged in a one or more containers with appropriate buffers and preservatives and used for diagnosis or for directing therapeutic treatment.
- the peptides can be each mixed in a single container or placed in individual containers.
- the containers include a label.
- Suitable containers include, for example, bottles, vials, syringes, and test tubes.
- the containers may be formed from a variety of materials such as glass or plastic.
- additives such as stabilizers, buffers, blockers and the like may also be added.
- kits can also be attached to a solid support, such as beads, array substrate (e.g., chips) and the like and used for diagnostic purposes.
- a solid support such as beads, array substrate (e.g., chips) and the like and used for diagnostic purposes.
- kits or immobilized to substrates may be conjugated to a detectable label such as described hereinabove.
- the kit can also include instructions for determining if the tested subject is suffering from, or is at risk of developing, a condition, disorder, or disease associated with amyloid polypeptide of interest. Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
- Pancreatic amyloid is found in more than 95 % of type II diabetes patients.
- Pancreatic amyloid is formed by the aggregation of a 37 amino acid long islet amyloid polypeptide (LAPP, GenBank Accession No. gi:4557655), the cytotoxicity thereof being directly associated with the development ofthe disease.
- LAPP amyloid formation follows a nucleation-dependent polymerization process, which proceeds through conformational transition of soluble LAPP into aggregated ⁇ -sheets.
- NFGATL hexapeptide (22-27) (NFGATL, SEQ ID NO: 111) of IAPP, also termed as the "basic amyloidogenic unit" is sufficient for the formation of ⁇ -sheet-containing amyloid fibrils [Konstantinos et al. (2000) J. Mol. Biol. 295:1055-1071].
- alanine scan was performed. Amino-acids were replaced with alanine in order to specifically change the molecular interface of the peptides, without significantly changing their hydrophobicity or tendency to form ⁇ -sheet structures, alanine-scan was preformed in the context of the block that is unique to human LAPP (Figure 3a). This block includes two serine residues that follow the NFGATL motif in the full-length polypeptide. These eight amino-acid peptide sequences were used since the shorter peptides are hydrophobic and as s such less soluble.
- Figure 3b shows a schematic representation ofthe chemical structure of the wild-type peptide while Figure 3c indicates the amino-acid substitutions in the different mutant peptides that were generated.
- Methods and Reagents - Peptide synthesis was performed by PeptidoGenic
- EXAMPLE 2 Kinetics of aggregation of IAPP peptide fragment and mutant derivatives as monitored by turbidity measurements To study self-assembly of the LAPP peptide derived fragments, aggregation and insolubilization kinetics were monitored using turbidity measurements at 405 nm.
- peptide stock solutions were prepared by dissolving lyophilized form of the peptides in DMSO, a disaggregating solvent, at a concentration of 100 mM. To avoid any pre-aggregation, fresh stock solutions were prepared prior to each and every experiment. Peptide stock solutions were diluted into assay buffer and plated in 96-well plates as follows: 2 ⁇ l of peptides stock solutions were added to 98 ⁇ l of 10 mM Tris pH 7.2, resulting in a 2 mM final concentration of the peptide in the presence of 2% DMSO. Turbidity data was measured at 405 nm. A buffer solution including 2 % DMSO was used as a blank. Turbidity was measured at room temperature over several time points.
- wild-type peptide fragment (SEQ LD NO: 1) showed an aggregation kinetic profile that was very similar to those previously reported for non-seeded hlAPP hexapeptide [Tenidis et al. (2000) J. Mol. Biol 295:1055-71]. Such a profile is strongly indicative of a nucleation-dependent polymerization mechanism [Ja ⁇ ett and Lansbury (1992) Biochemistry 31:6865-70]. Following a lag-time of 20 minutes, wild type peptide self-assembled into insoluble fibrils. Peptide G3A (SEQ LD NO: 4) showed essentially the same profile as that of wild type peptide.
- the NIA peptide (SEQ LD NO: 2) mediated higher kinetics of aggregation, albeit with different kinetic profile as compared to that of wild-type peptide. Interestingly, the aggregation of NIA seemed to be less nucleation- dependent. Substitution of the isoleucine or leucine to alanine (peptides I5A, SEQ LD NO: 5 and L6A, SEQ LD NO: 6 respectively) reduced the kinetics of aggregation but did not abolish it completely. Substitution ofthe phenylalanine residue to alanine (peptide F2A, SEQ LD NO:3) led to a total loss of peptide ability to aggregate.
- the F2A peptide was completely soluble in the aqueous assay buffer. Altogether, kinetic aggregation studies of the amyloidogenic fragments suggested a major role to the phenylalanine residue in the process of amyloid formation by the LAPP active fragment.
- the average size ofthe aggregates, formed by the various peptides was determined using dynamic light scattering (DLS) experiments.
- Method - Freshly prepared peptide stock solutions at a concentration of 10 mM were diluted in 10 mM Tris buffer pH 7.2 and further filtrated through a 0.2 ⁇ m filter to a final concentration of 100 ⁇ M peptide and 1% DMSO.
- Particle size measurement was conducted with a laser-powered ALV-NLBS/HPPS non-invasive backscattering instrument. Autoco ⁇ elation data was fitted using the ALV- NLBS/HPPS software to derive average apparent hydrodynamic diameters.
- Congo red (CR) staining combined with polarization microscopy was utilized to test amyloidogenicity of the peptides of the present invention.
- the fibrillogenic potential of the various peptides was assessed by electron microscopy analysis.
- G3A peptides could be explained by the fast kinetics of formation of these fibrils (see Example 2). More distinct structures and longer fibrils, albeit less frequent, were observed with peptides 15 A and L6A. These longer fibrils may be a result of a slower kinetics, which allow for a more ordered fibril organization.
- Bacterial strains - E. coli strain TG-1 (Amersham Pharmacia, Sweden) was used for molecular cloning and plasmid propagation.
- the bacterial strain BL21(DE3) (Novagen, USA) was used for protein overexpression.
- fusion sequence was then constructed using the LAPP synthetic template, which was amplified using primer YAR2 (SEQ LD NO: 60) and primer YAR1 (SEQ LD NO. 59), thereby introducing a V8 Ek cleavage site and a (His) 6 tag at the N-terminus of LAPP.
- the two primers included a Not I and an Nco I cloning sites, respectively.
- the resultant PCR product was digested with Nco I and Not I and ligated into the pMALc2x-NN expression vector.
- the pMALc2x-NN expression vector was constructed by cloning the polylinker site of pMALc-NN 19 into pMALc2x (New England Biolabs, USA) [BACH (2001) J. Mol. Biol. 312:79-93].
- Cell extracts were prepared in 20 mM Tric-HCl (pH 7.4), 1 mM EDTA, 200 mM NaCl and a protease inhibitors cocktail (Sigma) using a freeze-thaw followed by a brief sonication as previously described [Gazit (1999) J. Biol. Chem. 274:2652- 2657]. Protein extracts were clarified by centrifugation at 20,000 g and stored at 4° C MBP-IAPP fusion protein was purified by passing the extract over an amylose resin column (New England Biolabs, USA) and recovered by elution with 20 mM maltose in the same buffer.
- MBP-IAPP Purified MBP-IAPP was stored at 4 °C Protein concentration was determined using the Pierce Coomassie plus reagent (Pierce, USA) with BSA as a standard. MBP and MBP-IAPP protein fractions were analyzed on SDS/12 % polyacrylamide gels, which were stained with GelCode Blue (Pierce, USA).
- MBP and MBP-IAPP proteins were reacted with 5 equivalents of N-iodoacetyl-N'-(8- sulfo-1 -naphthyl) ethylenediamine (IAEDANS) (Sigma, Rehovot, Israel) for overnight at room temperature in the dark. Free dye was separated from labeled protein by gel filtration chromatography on a QuickSpin G-25 Sephadex column. MBP and MBP-IAPP fluorescence was then determined.
- IAEDANS N-iodoacetyl-N'-(8- sulfo-1 -naphthyl) ethylenediamine
- MBP-IAPP was purified to near-homogeneity at a yield of 80 mg/1 of cells.
- an additional His-Tag was also included ( Figure 8b). The His-Tag could be removed by Ek V8 cleavage at the N-terminal Lys residue of the LAPP sequence, resulting in the release of wild type LAPP.
- IAPP peptide array construction - Decamers corresponding to consecutive overlapping sequences of hIAPP 1-37 SEQ LD NOs. 61-88) were synthesizes on a cellulose membrane matrix using the SPOT technique (Jerini AG, Berlin, Germany).
- the peptides were covalently bound to a Whatman 50 cellulose support (Whatman,
- N-terminal acetylation was used for peptide scanning because of higher stability to peptide degradation, and better representation of the native recognition motif.
- Peptides Synthesis - Peptide synthesis was effected using solid-phase synthesis methods performed by Peptron, Inc. (Taejeon, Korea). Co ⁇ ect identity ofthe peptides was confirmed by ion spray mass-spectrometry using a HP 1100 series LC/MSD [Hewlett-Packard Company, Palo Alto, CA]. The purity of the peptides was confirmed by reverse phase high-pressure liquid chromatography (RP-HPLC) on a C 18 column, using a 30 minute linear gradient of 0 to 100% acetonitrile in water and 0.1% trifluoroacetic acid (TFA) at flow rate of 1 ml/min.
- RP-HPLC reverse phase high-pressure liquid chromatography
- Binding studies The cellulose peptide array was initially blocked with 5 % (V/N) non fat milk in Tris buffered saline (TBS, 20 mM Tris pH 7.5 , 150 mM ⁇ aCl). Thereafter, cellulose membrane was incubated in the presence of 10 ⁇ g/ml MBP- IAPP ⁇ -3 at 4°C for 12 h in the same blocking buffer. The cellulose membrane was then washed repeatedly with 0.05 % Tween 20 in TBS. MBP-LAPP 1-37 bound to the cellulose membrane was detected with an anti MBP monoclonal antibody (Sigma, Israel). HRP-conjugated goat anti mouse antibodies (Jackson Laboratories, USA) were used as a secondary antibody.
- Binding analysis ofthe recombinant MBP-bJAPP fusion protein to the hlAPP peptide array identified a putative self-assembly domain within the central part ofthe hlAPP protein.
- Table 3 illustrates the examined peptides.
- Kinetic Aggregation Assay freshly prepared peptide stock solutions were generated by dissolving the lyophilized form of the peptides in dimethyl sulfoxide (DMSO) at a concentration of 100 mg/ml. To avoid any pre-aggregation, fresh stock solutions were prepared for each experiment. Peptide stock solutions were diluted into the assay buffer in enzyme-linked immunosorbent assay (ELISA) plate wells as follows: 8 ⁇ L of peptide stock solutions were added to 92 ⁇ L of 10 mM Tris, pH 7.2 (hence the final concentration of the peptide was 8 mg/ml in the presence of 8% DMSO). Turbidity data were collected at 405 nm. Buffer solution containing the same amount of DMSO as the tested samples was used as blank, which was subtracted from the results. Turbidity was measured continuously at room temperature using THERMOmax ELISA plate reader (Molecular Devices, Sunnyvale CA). Results
- Turbidity assay was performed in-order to determine the ability of the various peptides (Table 3) to aggregate in an aqueous medium. Fresh stock solutions of the different peptide fragments were made in DMSO, and then diluted into a Tris buffer solution and turbidity, as a hallmark of protein aggregation, was monitored for two hours. As shown in Figure 11, the peptides NFLVHSS, FLVHSS and FLVHS exhibited high turbidity.
- Congo red (CR) staining combined with polarization microscopy was utilized to test amyloidogenicity of the peptides of the present invention.
- Amyloid fibrils bind CR and exhibit gold/green birefringence under polarized light [Puchtler (1966)
- Congo Red Staining and Birefringence A 10 ⁇ L suspension of 8 mg/ml peptide solution in 10 mM Tris buffer, pH 7.2 aged for at least one day was allowed to dry overnight on a glass microscope slide. Staining was performed by the addition of a 10 ⁇ L suspension of saturated Congo Red (CR) and NaCl in 80% ethanol (v/v) solution as previously described [Puchtler (1966) Supra]. The solution was filtered via 0.45 ⁇ m filter. The slide was then dried for few hours. Birefringence was determined with a SZX-12 Stereoscope (Olympus, Hamburg, Germany) equipped with cross polarizers.
- EXAMPLE 10 Ultrastructural analysis of the fibrillogenic hlAPP peptides The fibrillogenic potential of the various peptides was assessed by electron microscopy analysis.
- the NFLVHSS peptide spectrum exhibited major minimum band at 1929 cm “1 1675 cm '1 , this spectrum is classical for an anti-parallel ⁇ -sheet structure ( Figure 14b).
- a similar spectrum was observed for the peptide FLVHS with a major minimum at 1625 cm “1 and a minor minimum at 1676 cm “1 ( Figure 14e).
- the spectrum of FLVHSS peptide showed also a major minimum at 1626 cm “1 .
- the spectrum had also some minor minima around 1637-1676 cm “1 but those were shaped more like noise than signal (Figure 14c).
- EXAMPLE 12 Identification ofthe minimal amyloidogenic peptide fragment of Medin Background Medin (GenBank Accession No. gi:5174557) is the main constitute of aortic medial amyloid deposits [Haggqvist (1999) Proc. Natl. Acad. Sci. USA. 96:8674- 8669]. Previous studies found aortic medial amyloid in 97% of the subjects above the age of 50 [Mucchiano (1992) Am. J. Pathol. 140:811-877]. However, the pathological role of those amyloid deposits is still unknown.
- CR staining was effected as described in Example 9. A CR staining was effected to determine whether the structures formed by the various Medin-derived peptides show a typical birefringence. As shown in Figure 18b, the NFGSVQ hexapeptide bound CR and exhibited a characteristic bright and strong green-gold birefringence. The NFGSVQFV octapeptide also exhibited significant birefringence ( Figure 18a), although less typical than that observed with the hexapeptide. The gel-forming NFGSV peptide deposits exhibited very low degree of birefringence (Figure 18c).
- EXAMPLE 16 The effect of phenylalanine substitution on the self-assembly of Medin T elucidate a possible role for the phenylalanine residue in the process of amyloid fibrils formation by the minimal amyloid-forming hexapeptide, the phenylalanine amino acids was replaced with an alanine.
- the alanine-substituted peptide was prepared and examined in the same way as described for the various fragments of Medin. As shown in Figure 19a, a significantly lower turbidity was observed with the alanine-substituted peptide as compared to the wild-type hexapeptide.
- Human Calcitonin (hCT, GenBank Accession No. gi: 179880) is a 32 amino acid long polypeptide hormone that is being produced by the C-cells of the thyroid and is involve in calcium homeostasis [Austin and Health (1981) N. Engl. J. Med. 304:269-278; Copp (1970) Annu. Rev. Physiol. 32:61-86; Zaidi (2002) Bone 30:655- 663].
- Amyloid fibrils composed of hCT were found to be associated with medullary carcinoma of the thyroid [Kedar (1976) Isr. J. Sci. 12:1137; Berger (1988) Arch. A. Pathol. Anat. Histopathol.
- Circular dichroism (CD) studies have shown that in water monomeric hCT has little ordered secondary structure at room temperature [Arvinte (1993) Supra]. However, studies of hCT fibrils using circular dichroism, fluorescence, and infrared spectroscopy revealed that fibrillated hCT molecules have both ⁇ -helical and ⁇ -sheet secondary structure components [Bauer (1994) Supra]. NMR spectroscopy studies have shown that in various structure promoting solvents like TFE/H 2 O, hCT adopts an amphiphilic ⁇ -helical conformation, predominantly in the residue range 8-22 [Meadows (1991) Biochemistry 30:1247-1254; Motta (1991) Biochemistry 30:10444- 10450]. In DMSO/H 2 O, a short double-stranded antiparallel ⁇ -sheet form in the central region made by residues 16-21 [Motta (1991) Biochemistry 30:2364-71].
- the shorter DFNK tetrapeptide also formed fibrillar structures ( Figure 21b). However, the structures formed were less ordered as compared to those formed by the DFNKF pentapeptide. The amount of fibrillar structures formed by DFNK was also lower as compared to the DFNKF peptapeptide. No clear fibrils could be detected using solutions that contained the FNKF tetrapeptide and the DFN tripeptide, in spite of extensive search. In the case of the FNKF tetrapeptide only amo ⁇ hous aggregates could be found (Figure 21c). The DFN tripeptide formed more ordered structures ( Figure 2 Id) that resembled the structure formed by gel-forming tripeptide [Lyon (2001) Supra].
- CR staining was effected as described in Example 9. A CR staining was effected to determine whether the structures formed by the various hCT-derived peptides show a typical birefringence. As shown in Figures 22a-d, all the studies peptides showed some degree of birefringence. However, the green birefringence, which was observed with the DFNKF-pentapeptide was clear and strong ( Figure 22a). The level of birefringence that was observed with the other peptides was lower but significant since no birefringence could be detected using control solutions which did not contain the peptides.
- EXAMPLE 20 Secondary structure ofthe aggregated hCT-derived peptides FT-LR spectroscopy was effected as described in Example 11. Amyloid deposits are characteristic of fibrils rich with ⁇ -pleated sheet structures. To get a quantitative information regarding the secondary structures that were formed by the various peptide fragments FT-LR spectroscopy was used. Aged peptide solutions were dried on CaF 2 plates forming thin films as described in Example 11.
- the DFNKF pentapeptide exhibited a double minima (at 1639 cm “1 and 1669 cm “1 ) an amide I FT-LR spectrum that is consistent with anti-parallel ⁇ -sheet structure and is remarkably similar to the spectrum of the amyloid-forming hexapeptide fragment of the islet amyloid polypeptide [Tenidis (2000) Supra].
- the amide I spectrum observed with the DFNK tetrapeptide ( Figure 23) was less typical of a ⁇ -sheet structure. While it exhibited a minimum at 1666 cm-1 that may reflect an anti-parallel ⁇ -sheet it lacked the typical minimum around 1620-1640 cm '1 that is typically observed with ⁇ -sheet structures.
- the FNKF tetrapeptide exhibited a FT-LR spectrum that is typical of a non-ordered structure ( Figure 23) and is similar to spectra of the short non-amyloidogenic fragments of the islet amyloid polypeptide [Tenidis (2000) Supra].
- the DFN tripeptide exhibited a double minima (at 1642 cm “1 and 1673 cm “1 , Figure 23) amide I FT-LR spectrum that is consistent with a mixture of ⁇ -sheet and random structures. This may further indicate that the structures observed by EM visualization may represent some degree of ordered structure composed of predominantly ⁇ -sheet structural elements.
- the FT-LR spectrum of the DANKA pentapeptide was similar to that of the FNKF tetrapeptide and other short non-amyloidogenic peptide, typical of non-ordered structures [Tenidis (2000) Supra].
- the effect ofthe phenylalanine to alanine substitution is very similar to the effect of such a change in the context of a short amyloid-forming fragment ofthe islet amyloid polypeptide [Azriel (2001) Supra].
- the hCT fragment seems to be the pentapeptide with the highest amyloidogenic potential similar to the potent amyloidogenic fragment of the ⁇ - amyloid (A ⁇ ) polypeptide, KLVFFAE [Balbach (2000) Biochemistry 39:13748-59]. It is possible that electrostatic interactions between the opposing charges on the lysine and aspartic acids direct the formation of ordered antiparallel structure. Interestingly, the DFNK polypeptide exhibited a significantly lower amyloidogenic potential as compared to the DFNKF peptide. It is possible that a pentapeptide is a lower limit for potent amyloid former. This is consistent with recent results that demonstrate that two pentapeptides of LAPP, NFLVH and FLVHS, can form amyloid fibrils, but their common denominator, the tetrapeptide FLVH, could not form such fibrils (see
- Amyloid fibril formation by lactotransferrin (GenBank Accession No. gi:24895280) is associated familial subepithelial corneal amyloid formation [Sacchettini and Kelly (2002) Nat Rev Drug Discov 1:267-75]. Based on the proposed role of aromatic residues in amyloid self-assembly, the amyloidogenic features of a Lactotransferrin-derived peptide, LFNQTG (SEQ LD NO: 32) were studied.
- Lactotransferrin is important for the polypeptide self-assembly. These results further substantiate the ability of the present invention to predict amyloidogenic peptide sequences.
- the human BRI gene is located on chromosome 13.
- the amyloid fibrils ofthe BriL gene product (GenBank Accession No. gi: 12643343) are associated with neuronal dysfunction and dementia (Vidal et al (1999) Nature 399, 776-781).
- the amyloidogenic features of a BriL-derived peptide, FENKF (SEQ LD NO: 34) were studied.
- Amyloid fibril formation by beta-amyloid is promoted by interaction with serum amyloid-P (GenBank Accession No. gi:2144884). Based on the proposed role of aromatic residues in amyloid self-assembly, the amyloidogenic features of a Serum amyloid P-derived peptide, LQNFTL (SEQ LD NO: 36) were studied.
- EXAMPLE 27 Identification of an amyloidogenic peptide from Immunoglobulin light chain Amyloid fibrils formation by Immunoglobulin light chain (GenBank Accession No. gi:625508) is associated with primary systemic amyloidosis [Sacchettini and Kelly (2002) Nat Rev Drug Discov 1:267-75]. Based on the proposed role of aromatic residues in amyloid self-assembly, the amyloidogenic features of an Immunoglobulin light chain-derived peptide, TLLFGG (SEQ LD NO: 37) were studied.
- Amyloid fibril formation by Cystatin C (GenBank Accession No. gi:4490944) is associated with hereditary cerebral amyloid angiopathy [Sacchettini and Kelly
- Amyloid fibril formation by Transthyretin (GenBank Accession No. gi:72095) is associated with familial amyloid polyneuropathy (Sacchettini and Kelly (2002) Nat
- Transthyretin is important for the polypeptide self-assembly. These results further substantiate the ability of the present invention to predict amyloidogenic peptide sequences.
- Amyloid fibril formation by Lysozyme (GenBank Accession No. gi:299033) is associated with familial non-neuropathic amyloidosis [Sacchettini and Kelly (2002)
- GTFQLN (SEQ LD NO: 40) were studied.
- Amyloid fibril formation by Fibrinogen (GenBank Accession No. gi:l 1761629) is associated with hereditary renal amyloidosis (Sacchettini and Kelly (2002) Nat Rev
- Fibrinogen is important for the polypeptide self-assembly. These results further substantiate the ability of the present invention to predict amyloidogenic peptide sequences.
- Amyloid fibril formation by Insulin (GenBank Accession No. gi:229122) is associated with injection-localized amyloidosis [Sacchettini and Kelly (2002) Nat Rev
- Amyloid fibrils formation by prolactin (GenBank Accession No. gi:4506105) is associated with pituitary-gland amyloidosis (Sacchettini and Kelly (2002) Nat Rev
- Amyloid fibrils formation by beta-2-microtublin (GenBank Accession No. gi:70065) is associated haemodialysis-related amyloidosis (Sacchettini and Kelly (2002) Nat Rev Drug Discov 1:267-75). Based on the proposed role of aromatic residues in amyloid self-assembly, the amyloidogenic features of a beta-2-microtublin
- amyloidogenic peptide identified according to the teachings of the present invention The ability of amyloidogenic peptides of LAPP, identified according to the teachings of the present invention to inhibit amyloid formation by the full-length polypeptide was tested by the addition of beta-breaker proline residues to the recognition sequence as set forth in the peptide sequence NFLVHPP (SEQ ID NO: 45).
- the degree of amyloid fibrils formation with and without the inhibitor was assessed using thioflavin T (ThT) as molecular indicator.
- ThT thioflavin T
- the degree of fluorescence of the ThT dye is directly co ⁇ elated with the amount of amyloid fibrils in the solution [LeVine H 3rd. (1993) Protein Sci. 2:404-410.
- LAPP solutions (4 ⁇ M hIAPPin 10 mM Tris buffer pH 7.2), were incubated in the presence or absence of 40 ⁇ M of the modified peptide (i.e., NFLVHPP) at room temperature.
- Fibril formation was determined by a ten fold dilution of the solutions into a solution that contained 3 ⁇ M thioflavin T (ThT) in 50 mM sodium phosphate pH 6.0 and determination of fluorescence at 480 nm with excitation at 450 nm using a LS50B spectroflurimeter (Perkin Elmar, Wellesley, MA).
- ThT thioflavin T
- As a control 10 mM Tris buffer pH 7.2 were diluted into the ThT solution and fluorescence was determined as described.
- hydrophobic amino acids are similar or even slightly more hydrophobic than phenylalanine [Wolfenden (1981) Biochemistry 20:849-855; Kyte (1982) J. Mol. Biol. 157:105-132; Radzicka (1988) ], they are not aromatic. Furthermore, valine and isoleucine, are considered to be very strong ⁇ -sheet formers [Chou (1974) Biochemistry 13:211-222; Chou (1978) Annu. Rev. Biochem. 47:251-276], which is assumed to be important to the formation of ⁇ -sheet rich amyloid fibrils.
- EXAMPLE 38 Ultrastructural analysis of hydrophobically modified hlAPP peptide fragments Electron microscopy analysis was effected as described in Example 10. An ultrastructural visualization of any possible structures formed by the various analogous peptides was effected following five days of incubation. This structural analysis represents the most sensitive method since various aggregates were visualized individually. For that aim, the occurrence and characteristics of the formed structures were studied by electron microscopy using negative staining, with the same of peptide solution which were incubated in the aggregation assay (Example 32). As expected, well-ordered fibrils were observed with the wild-type peptide NFGAILSS peptide fragment ( Figures 41a-b).
- NFGAILSS peptide as mentioned above, these aggregates were quite rare and did not have ordered structures ( Figures 41c-f). Those irregular and sporadic structures are consistent with some degree of non-specific aggregation as expected after long incubation of rather hydrophobic molecules.
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EP1638517A2 (en) * | 2003-06-30 | 2006-03-29 | Tel Aviv University Future Technology Development L.P. | Peptides antibodies directed thereagainst and methods using same for diagnosing and treating amyloid-associated diseases |
WO2007126111A1 (en) * | 2006-04-28 | 2007-11-08 | Kagoshima University | PEPTIDE CAPABLE OF INHIBITING AMYLOID-β FIBROSIS |
WO2008104580A1 (en) | 2007-03-01 | 2008-09-04 | Probiodrug Ag | New use of glutaminyl cyclase inhibitors |
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US7781396B2 (en) | 2002-01-31 | 2010-08-24 | Tel Aviv University Future Technology Development L.P. | Peptides directed for diagnosis and treatment of amyloid-associated disease |
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- 2003-01-30 KR KR10-2004-7011868A patent/KR20040081165A/en not_active Application Discontinuation
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Also Published As
Publication number | Publication date |
---|---|
EP1534310A4 (en) | 2006-05-31 |
CA2473987C (en) | 2013-11-19 |
CA2473987A1 (en) | 2003-08-07 |
KR20040081165A (en) | 2004-09-20 |
JP2006506942A (en) | 2006-03-02 |
EP1534310A2 (en) | 2005-06-01 |
AU2003207973A1 (en) | 2003-09-02 |
WO2003063760A3 (en) | 2005-02-10 |
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