U.S. patent application number 11/656542 was filed with the patent office on 2007-06-14 for peptides and methods using same for diagnosing and treating amyloid-associated diseases.
This patent application is currently assigned to Tel Aviv University Future Technology Development L.P.. Invention is credited to Ehud Gazit.
Application Number | 20070135334 11/656542 |
Document ID | / |
Family ID | 31990574 |
Filed Date | 2007-06-14 |
United States Patent
Application |
20070135334 |
Kind Code |
A1 |
Gazit; Ehud |
June 14, 2007 |
Peptides and methods using same for diagnosing and treating
amyloid-associated diseases
Abstract
A peptide comprising at least 5 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 as well as pharmaceutical
compositions, kits and methods for diagnosing and treating amyloid
associated diseases.
Inventors: |
Gazit; Ehud;
(Ramat-HaSharon, IL) |
Correspondence
Address: |
Martin D. Moynihan;PRTSI, Inc.
P.O. Box 16446
Arlington
VA
22215
US
|
Assignee: |
Tel Aviv University Future
Technology Development L.P.
Tel-Aviv
IL
|
Family ID: |
31990574 |
Appl. No.: |
11/656542 |
Filed: |
January 23, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10235852 |
Sep 6, 2002 |
|
|
|
11656542 |
Jan 23, 2007 |
|
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Current U.S.
Class: |
514/21.1 ;
514/17.8; 514/6.9 |
Current CPC
Class: |
C07K 5/0819 20130101;
C07K 14/4711 20130101; A61K 38/00 20130101; C07K 5/1016 20130101;
C07K 5/1021 20130101; C07K 5/1008 20130101 |
Class at
Publication: |
514/009 ;
514/015; 514/016; 514/017; 514/014 |
International
Class: |
A61K 38/12 20060101
A61K038/12; A61K 38/10 20060101 A61K038/10; A61K 38/08 20060101
A61K038/08 |
Claims
1. A method of inhibiting amyloid formation, the method comprising
contacting a biological sample with a peptide having at least 5
amino acid residues and less than 15 amino acid residues, said
peptide comprising an amino acid sequence as set forth in SEQ ID
NO:. 7, thereby inhibiting amyloid formation and/or occurrence of
said amyloid in the biological sample.
2. The method of claim 1, wherein said amino acid sequence is
selected from the group consisting of SEQ ID NO: 4, 12 and 13.
3. The method of claim 1, wherein said peptide further comprises at
least two serine residues attached to a C-terminus thereof.
4. The method of claim 1, wherein said peptide is a linear or
cyclic peptide.
5. A method of disaggregating amyloid, the method comprising
contacting a biological sample which comprises the amyloid with a
peptide having at least 5 amino acid residues and less than 15
amino acid residues, said peptide comprising an amino acid sequence
as set forth in SEQ ID NO: 7, thereby disaggregating the amyloid in
the biological sample.
6. The method of claim 5, wherein said amino acid sequence is
selected from the group consisting of SEQ ID NO: 4, 12 and 13.
7. The method of claim 5, wherein said peptide further comprises at
least two serine residues attached to a C-terminus thereof.
8. The method of claim 5, wherein said peptide is a linear or
cyclic peptide.
9. A method of screening test compounds useful in prevention or
disaggregation of amyloid deposits, the method comprising: (a)
contacting the test compounds with amyloid aggregates which
comprise a labeled peptide having at least 5 amino acid residues
and less than 15 amino acid residues, said peptide comprising an
amino acid sequence as set forth in SEQ ID NO: 7; and (b)
monitoring displacement of said labeled peptide by a test compound
of the test compounds, said test compound being useful in
prevention or disaggregation of amyloid deposits.
10. The method of claim 9, wherein said amino acid sequence is
selected from the group consisting of SEQ ID NO: 4, 12 and 13.
11. The method of claim 9, wherein said peptide further comprises
at least two serine residues attached to a C-terminus thereof.
12. The method of claim 9, wherein said peptide is a linear or
cyclic peptide.
Description
RELATED APPLICATION
[0001] This Application is a continuation of pending U.S. patent
application Ser. No. 10/235,852 filed Sep. 6, 2002, the contents of
which are hereby incorporated by reference.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to peptides, which can be used
to diagnose, prevent, and treat amyloid-associated diseases, such
as Type II diabetes mellitus.
[0003] 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.
[0004] Amyloid material is composed of a dense network of rigid,
nonbranching proteinaceous fibrils of indefinite length that are
about 80 to 100 .ANG. in diameter. Amyloid fibrils contain a core
structure of polypeptide chains arranged in antiparallel
.beta.-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].
[0005] Approximately twenty amyloid fibril proteins have been
identified in-vivo and correlated with specific diseases. Amyloid
proteins share little or no amino acid sequence homology, however
the core structure of the amyloid fibrils is essentially the
same.
[0006] The 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.
[0007] Type II diabetes mellitus is a heterogeneous and
multifactorial disease characterized by abnormalities in the action
of insulin (i.e., insulin resistance) and the secretion of insulin
(i.e., beta-cell failure). The relative contribution of each
abnormality varies among patients as well as during the course of
the disease [Ferrannini (1998) Endocr. Rev. 19:477-90].
[0008] Apparently, deposition of islet amyloid is involved in the
pathogenesis of type II diabetes. Islet amyloidosis in patients
with type II diabetes is associated with a reduced mass of
insulin-producing beta cells and is most likely an important factor
in the development of beta cell failure. Patients who require
insulin treatment have the greatest reduction in islet mass and the
most prominent amyloid deposits, indicating that the degree of
islet amyloidosis may be related to the severity of the disease
[Westermark (1994) Amyloid 1:47-60]. A link between islet
amyloidosis and type II diabetes is further supported by the
finding of islet amyloid in other animal species in which type II
diabetes occurs, notably monkeys and cats [Westermark et al. (1990)
Proc. Natl. Acad. Sci. USA 87:5036-40].
[0009] The building block of islet amyloid fibrils is a 37 amino
acid residue peptide known as islet amyloid polypeptide [IAPP,
Johnson et al. (1989) N. Eng. J. Med. 321:513-8]. The nucleotide
sequence of the gene indicates that the islet amyloid polypeptide
in normal subjects is identical to the islet amyloid polypeptide in
amyloid deposits in diabetic patients. This finding suggests that a
change in the amino acid sequence of islet amyloid polypeptide is
not the pathogenic mechanism that leads to the formation of islet
amyloid fibrils [Mosselman et al. (1988) FEBS Lett.
239:227-32].
[0010] However, comparison of the amino acid sequences of islet
amyloid polypeptide among various animal species, in some of which
islet amyloid does not develop, in combination with experiments
involving in vitro formation of fibrils from synthetic islet
amyloid polypeptide molecules has led to the identification of an
"amyloidogenic" region within the human islet amyloid polypeptide
molecule that is essential for the formation of fibrils [Johnson et
al. (1989) N. Eng. J. Med. 321:513-8; Moriarty et al. (1999)
Biochemistry 38:1811-8].
[0011] As suggested supra, islet amyloidosis is involved in the
loss of up to 50% of beta cell mass in the pancreatic tissue of
patients with type II diabetes as well as in diabetic cats and
transgenic mice that produce human islet amyloid polypeptide
[Hoppener et al. (2000) N. Eng. J. Med. 343:411-19].
[0012] Preventing or arresting the process of amyloid-related
beta-cell failure at an early stage of type II diabetes might
preserve endogenous insulin production and prevent or at least
delay hyperglycemia.
[0013] Furthermore, it was found that mutations in the IAPP gene
are correlated with predisposition and early onset of type II
diabetes [Seino (2001) Diabetologia 44:906-9]. While Type II
diabetes usually occurs at the age of 50 and higher, individuals
with the genetic predisposition may be diagnosed with diabetes at
their 30s. Therefore, prevention of amyloid formation may serve as
a prophylactic treatment for such individuals (e.g., about 1% of
the Far Asian population (China, Korea, Japan, and Taiwan).
[0014] 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].
[0015] Thus, therapies designed to inhibiting the production of
human islet amyloid polypeptide or inhibiting amyloidosis may be
useful for treating type II diabetes mellitus.
[0016] Inhibition of the production of islet
amyloidpolypeptide--Both human islet amyloid gene and insulin share
common promoter elements [Mosselman et al. (1988) FEBS Lett.
239:227-32]. Thus, the design of drugs, which inhibit the
expression of the islet amyloid polypeptide gene without
simultaneously inhibiting the expression of the insulin gene has
not been attempted. Nevertheless, direct inhibition of the
production of islet amyloid polypeptide may be accomplished through
the use of antisense oligonucleotides against human islet amyloid
polypeptide messenger RNA (mRNA). In vitro, 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; Novials et al. (1998) Pancreas 17:182-6].
However, no experimental results demonstrating the in vivo
effectiveness of such antisense molecules have been
demonstrated.
[0017] Inhibition of the formation of amyloid fibrils--Amyloid,
including islet 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].
[0018] The following summarizes current attempts to engineer drugs
having the capability of destabilizing amyloid structures.
[0019] 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 (Mature Med. 1:143-148, 1995) 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 .beta.
peptide of Alzheimer's disease (AD). Heparin sulfate specifically
influences the soluble amyloid precursor (SAA2) to adopt an
increased .beta.-sheet structure characteristic of the
protein-folding pattern of amyloids. These anionic sulfonate or
sulfate compounds were shown to inhibit heparin accelerated AP
fibril formation and were able to disassemble preformed fibrils in
vitro, as monitored by electron micrography. Moreover, these
compounds substantially arrested murine splenic
inflammation-associated amyloid progression in vivo in acute and
chronic models. However, the most potent compound [i.e.,
poly-(vinylsulfonate)] showed acute toxicity. Similar toxicity has
been observed with another compound, IDOX (Anthracycline
4'-iodo-4'-deoxy-doxorubicin), which has been observed to induce
amyloid resorption in patients with immunoglobin light chain
amyloidosis (AL) [Merlini et al. (1995) Proc. Natl. Acad. Sci.
USA].
[0020] Destabilizing antibodies--Anti-.beta.-amyloid monoclonal
antibodies have been shown to be effective in disaggregating
.beta.-amyloid plaques and preventing .beta.-amyloid plaque
formation in vitro (U.S. Pat. No. 5,688,561). However, no
experimental results demonstrating the in vivo effectiveness of
such antibodies have been demonstrated.
[0021] Destabilizing peptides--The finding that the addition of
synthetic peptides that disrupt the .beta.-pleated sheets
(".beta.-sheet breakers") dissociated fibrils and prevented
amyloidosis [Soto et al. (1998) Nat. Med. 4:822-6] is particularly
promising from a clinical point of view. In brief, a penta-residue
peptide inhibited amyloid beta-protein fibrillogenesis,
disassembled preformed fibrils in vitro and prevents neuronal death
induced by fibrils in cell culture. In addition, the beta-sheet
breaker peptide significantly reduced amyloid beta-protein
deposition in vivo and completely blocked the formation of amyloid
fibrils in a rat brain model of amyloidosis.
[0022] While reducing the present invention to practice, the
present inventors have demonstrated that contrary to the teachings
of U.S. Pat. No. 6,359,112 to Kapurniotu, peptide aggregation into
amyloid fibrils is governed by aromatic interactions. Such findings
enable to efficiently and accurately design peptides which can be
used to diagnose and treat amyloid-associated diseases.
SUMMARY OF THE INVENTION
[0023] According to one aspect of the present invention there is
provided a peptide comprising at least 5 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.
[0024] According to another aspect of the present invention there
is provided a peptide comprising at least 5 amino acid residues and
less than 15 amino acid residues, the peptide including an amino
acid sequence as set forth SEQ ID NO: 13, wherein the peptide is
capable of forming self-aggregates under physiological
conditions.
[0025] According to yet another aspect of the present invention
there is provided a peptide selected from the group consisting of
SEQ ID NOs: 8, 9, 10 and 11.
[0026] According to still another aspect of the present invention
there is provided a peptide having an amino acid sequence set forth
in SEQ ID NO: 13.
[0027] According to an additional aspect of the present invention
there is provided a method of treating or preventing an
amyloid-associated disease in an individual, the method comprising
providing to the individual a therapeutically effective amount of a
peptide having at least 5 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.
[0028] According to yet additional aspect of the present invention
there is provided a method of treating or preventing an
amyloid-associated disease in an individual, the method comprising
providing to the individual therapeutically effective amount of a
peptide having at least 5 amino acid residues and less than 15
amino acid residues, the peptide including an amino acid sequence
as set forth in SEQ ID NO: 13 and being capable of self aggregating
under physiological conditions.
[0029] According to still additional aspect of the present
invention there is provided a method of treating or preventing an
amyloid-associated disease in an individual, the method comprising
providing to the individual a therapeutically effective amount of a
peptide selected from the group consisting of SEQ ID NOs: 8, 9, 10
and 11, wherein the peptide is an active ingredient of a
pharmaceutical compositions which also includes a physiologically
acceptable carrier.
[0030] According to a further aspect of the present invention there
is provided a method of treating or preventing an
amyloid-associated disease in an individual, the method comprising
providing to the individual a therapeutically effective amount of a
peptide having the amino acid sequence set forth in SEQ ID NO: 13
wherein the peptide is an active ingredient of a pharmaceutical
compositions which also includes a physiologically acceptable
carrier.
[0031] According to yet a further aspect of the present invention
there is provided a pharmaceutical composition for treating or
preventing an amyloid-associated disease comprising as an active
ingredient a peptide having at least 5 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.
[0032] According to still a further aspect of the present invention
there is provided 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, 9, 10 and 11 and a pharmaceutically acceptable carrier or
diluent.
[0033] According to still a further aspect of the present invention
there is provided a pharmaceutical composition for treating or
preventing an amyloid-associated disease comprising as an active
ingredient a peptide having the amino acid sequence set forth in
SEQ ID NO: 13 and a pharmaceutically acceptable carrier or
diluent.
[0034] 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: 8, 9, 10 and 11.
[0035] According to still a further aspect of the present invention
there is provided a nucleic acid construct comprising a
polynucleotide segment encoding a peptide having the amino acid
sequence set forth in SEQ ID NO: 13.
[0036] According to still a further aspect of the present invention
there is provided a nucleic acid construct comprising a
polynucleotide segment encoding a peptide having at least 5 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.
[0037] According to further features in preferred embodiments of
the invention described below, the amino acid sequence is selected
from the group consisting of SEQ ID NO: 4, 12 and 13.
[0038] According to still further features in the described
preferred embodiments the peptide is as set forth in SEQ ID NO:
13.
[0039] According to still further features in the described
preferred embodiments the peptide is as set forth in SEQ ID NO:
12.
[0040] According to still further features in the described
preferred embodiments the peptide further comprising at least two
serine residues at a C-terminus thereof.
[0041] According to still further features in the described
preferred embodiments the peptide is a linear or cyclic
peptide.
[0042] According to still further features in the described
preferred embodiments the peptide is an active ingredient of a
pharmaceutical composition which also includes a physiologically
acceptable carrier.
[0043] According to still further features in the. described
preferred embodiments the peptide is expressed from a nucleic acid
construct.
[0044] According to still further features in the described
preferred embodiments the nucleic acid construct further comprising
a promoter.
[0045] The present invention successfully addresses the
shortcomings of the 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0047] In the drawings:
[0048] FIGS. 1a-c are schematic illustrations of a primary sequence
comparison between human and rodent IAPP and the synthetic peptides
of the present invention. FIG. 1a is a sequence alignment of human
and rodent IAPP. 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. FIG. 1b illustrates the chemical structure of the wild
type IAPP peptide (SEQ ID NO: 1). FIG. 1c illustrates the primary
sequences and SEQ ID NOs of the peptides of the present
invention.
[0049] FIGS. 2a-b are graphs illustrating light absorbance at 405
nm as a function of time during fibril formation thus reflecting
the aggregation kinetics of the peptides of the present invention.
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.
[0050] FIG. 3 is a histogram depicting mean particle size of
assembled IAPP peptide and derivatives as measured by light
scattering. Each column represents the results of 3-5 independent
measurements.
[0051] FIGS. 4a-n are photomicrographs illustrating Congo Red
binding to pre-assembled IAPP peptides. Normal field and polarized
field micrographs are shown respectively for each of the following
aged peptide suspensions: N1A peptide (FIGS. 4a-b), F2A peptide
(FIGS. 4c-d), G3A peptide (FIGS. 4e-f), wild type peptide (FIGS.
4g-h), I5A peptide (FIGS. 4i-j) and L6A (FIGS. 4k-l).
[0052] FIGS. 5a-f are electron micrographs of "aged" IAPP peptide
and derivatives. N1A peptide (FIG. 5a), F2A peptide (FIG. 5b), G3A
peptide (FIG. 5c), wild type peptide (FIG. 5d), I5A peptide (FIG.
5e) and L6A (FIG. 5f). The indicated scale bar represents 100
nm.
[0053] FIGS. 6a-c are schematic illustrations of amyloid binding
with the inhibitory aromatic reagents: Ro 47-1816/001 (FIG. 6a),
Thioflavin T(FIG. 6b) and CR dye (FIG. 6c).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0054] The present invention is of novel peptides, compositions and
methods, which can be used to diagnose and treat amyloid associated
diseases such as type II Diabetes mellitus.
[0055] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0056] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details of construction and the
arrangement of the components set forth in the following
description or illustrated in the drawings. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0057] Numerous therapeutic approaches for prevention of amyloid
fibril formation or disaggreagtion of amyloid material have been
described in the prior art. However, current therapeutic approaches
are limited by cytotoxicity, non-specificity and delivery
barriers.
[0058] While reducing the present invention to practice and while
searching for a novel therapeutic modality to amyloid associated
diseases, in particular type II diabetes mellitus, the present
inventors have identified a sequence characteristic of amyloid
forming peptides which governs fibril formation.
[0059] Previous efforts to define the contributions of specific
residues in the IAPP basic amyloidogenic sequence involved the use
of site-directed deletion mutagenesis [U.S. Pat. No. 6,359,112 and
Konstantinos et al. (2000) J. Mol. Biol. 295:1055-71]. This
approach resulted in the findings that the structural determinants
for IAPP peptide assembly are consecutive glycine and alanine
residues.
[0060] In sharp contrast, the present inventors have uncovered that
the largest contribution to peptide aggregation comes from the
aromatic residue of the peptide rather than from the glycine and
alanine residues.
[0061] This discrepancy in results can be explained by the
different biochemical approaches, which were utilized to elucidate
the minimal amyloidogenic sequence. While Kapurniotu et al.
implemented a deletion mutant analysis, which overlooks the need to
retain tertiary structures [Konstantinos et al. (2000) J. Mol.
Biol. 295:1055-71], the inventors of the present invention used an
alanine scanning approach, wherein each alanine lo substitution
examines the contribution of an individual amino acid side chain to
the fimctionality of the protein, without extensively changing its
hydrophobicity or tendency to form .beta.-sheet structures (see
Example 1 of the Examples section).
[0062] The present findings enable for the first time, to generate
highly efficient diagnostic and therapeutic peptides which can be
utilized to treat or diagnose diseases characterized by amyloid
plaque formation.
[0063] Thus, according to one aspect of the present invention there
is provided a peptide which preferably includes an amino acid
sequence as set forth in SEQ ID NO: 7.
[0064] The sequence set forth in 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.
[0065] 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 finctional aromatic portions thereof. Examples of
aromatic residues which can be used by the present invention are
provided in Table 2 below.
[0066] 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.
[0067] As used herein, the phrase "self-aggregation" refers to the
capability of a peptide to form aggregates (e.g. fibrils) in an
aqueous solution. 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.
[0068] Since aggregation kinetics and aggregate structures are
largely determined by the specific residue composition and possibly
the length of the peptides generated, the present invention
encompasses both longer peptides (e.g., 10-50 amino acids) which
include the sequences set forth in SEQ ID NOs: 4, 12 or 13, or
shorter peptides (5-10 amino acid residues) including any of these
sequences. Due to their self-aggregating nature these peptides can
be used as potent diagnostic reagents.
[0069] Alternatively, the peptides of the present invention include
sequences set forth in SEQ ID NOs: 8, 9, 10 and b 11.
[0070] For example, a peptide encompassed by SEQ ID NO: 9, 10 or 11
can be utilized for therapy since as is shown in the Examples
section which follows, such a peptide displays no aggregation (SEQ
ID NO: 9) or slow aggregation kinetics as compared to the wild type
peptide (SEQ ID NOs: 9 and 10). It is conceivable that since
amyloid formation is in any case a very slow process the peptides
of the present invention will completely inhibit or significantly
delay amyloidosis under physiological conditions.
[0071] The term "peptide" as used herein 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. Such
modifications include, but are not limited to N terminus
modification, C terminus modification, peptide bond modification,
including, but not limited to, CH2--NH, CH2--S, CH2--S.dbd.O,
O.dbd.C--NH, CH2--O, CH2--CH2, S.dbd.C--NH, CH.dbd.CH or CF.dbd.CH,
backbone modifications, and residue modification. Methods for
preparing peptidomimetic compounds are well known in the art and
are specified, for example, in Quantitative Drug Design, C. A.
Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which
is incorporated by reference as if fully set forth herein. Further
details in this respect are provided hereinunder.
[0072] Peptide bonds (--CO--NH--) within the peptide may be
substituted, for example, by N-methylated bonds (--N(CH3)--CO--),
ester bonds (--C(R)H--C--O-- O--C(R)--N--), ketomethylen bonds
(--CO--CH2--), .alpha.-aza bonds (--NH--N(R)--CO--), wherein R is
any alkyl, e.g., methyl, carba bonds (--CH2--NH--), hydroxyethylene
bonds (--CH(OH)--CH2--), thioamide bonds (--CS--NH--), olefinic
double bonds (--CH.dbd.CH--), retro amide bonds (--NH--CO--),
peptide derivatives (--N(R)--CH2--CO--), wherein R is the "normal"
side chain, naturally presented on the carbon atom.
[0073] These modifications can occur at any of the bonds along the
peptide chain and even at several (2-3) at the same time.
[0074] 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.
[0075] In addition to the above, the peptides of the 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).
[0076] As used herein in the specification and in the claims
section below the term "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. Furthermore, the term "amino acid"
includes both D- and L-amino acids.
[0077] 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. TABLE-US-00001 TABLE
1 Three-Letter One-letter Amino Acid Abbreviation Symbol alanine
Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine
Cys C Glutamine Gln Q Glutamic Acid Glu E glycine Gly G Histidine
His H isoleucine Iie I leucine Leu L Lysine Lys K Methionine Met M
phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T
tryptophan Trp W tyrosine Tyr Y Valine Val V Any amino acid as
above Xaa X
[0078] TABLE-US-00002 TABLE 2 Non-conventional amino acid Code
.alpha.-aminobutyric acid Abu .alpha.-amino-.alpha.-methylbutyrate
Mgabu aminocyclopropane-carboxylate Cpro aminoisobutyric acid Aib
aminonorbornyl-carboxylate Norb cyclohexylalanine Chexa
cyclopentylalanine Cpen D-alanine Dal D-arginine Darg D-aspartic
acid Dasp D-cysteine Dcys D-glutamine Dgln D-glutamic acid Dglu
D-histidine Dhis D-isoleucine Dile D-leucine Dleu D-lysine Dlys
D-methionine Dmet D-ornithine Dorn D-phenylalanine Dphe D-proline
Dpro D-serine Dser D-threonine Dthr D-tryptophan Dtrp D-tyrosine
Dtyr D-valine Dval D-.alpha.-methylalanine Dmala
D-.alpha.-methylarginine Dmarg D-.alpha.-methylasparagine Dmasn
D-.alpha.-methylaspartate Dmasp D-.alpha.-methylcysteine Dmcys
D-.alpha.-methylglutamine Dmgln D-.alpha.-methylhistidine Dmhis
D-.alpha.-methylisoleucine Dmile D-.alpha.-methylleucine Dmleu
D-.alpha.-methyllysine Dmlys D-.alpha.-methylmethionine Dmmet
D-.alpha.-methylornithine Dmorn D-.alpha.-methylphenylalanine Dmphe
D-.alpha.-methylproline Dmpro D-.alpha.-methylserine Dmser
D-.alpha.-methylthreonine Dmthr D-.alpha.-methyltryptophan Dmtrp
D-.alpha.-methyltyrosine Dmty D-.alpha.-methylvaline Dmval
D-.alpha.-methylalnine Dnmala D-.alpha.-methylarginine Dnmarg
D-.alpha.-methylasparagine Dnmasn D-.alpha.-methylasparatate Dnmasp
D-.alpha.-methylcysteine Dnmcys D-N-methylleucine Dnmleu
D-N-methyllysine Dnmlys N-methylcyclohexylalanine Nmchexa
D-N-methylornithine Dnmorn N-methylglycine Nala
N-methylaminoisobutyrate Nmaib N-(1-methylpropyl)glycine Nile
N-(2-methylpropyl)glycine Nile N-(2-methylpropyl)glycine Nleu
D-N-methyltryptophan Dnmtrp D-N-methyltyrosine Dnmtyr
D-N-methylvaline Dnmval .gamma.-aminobutyric acid Gabu
L-t-butylglycine Tbug L-ethylglycine Etg L-homophenylalanine Hphe
L-.alpha.-methylarginine Marg L-.alpha.-methylaspartate Masp
L-.alpha.-methylcysteine Mcys L-.alpha.-methylglutamine Mgln
L-.alpha.-methylhistidine Mhis L-.alpha.-methylisoleucine Mile
D-N-methylglutamine Dnmgln D-N-methylglutamate Dnmglu
D-N-methylhistidine Dnmhis D-N-methylisoleucine Dnmile
D-N-methylleucine Dnmleu D-N-methyllysine Dnmlys
N-methylcyclohexylalanine Nmchexa D-N-methylornithine Dnmorn
N-methylglycine Nala N-methylaminoisobutyrate Nmaib
N-(1-methylpropyl)glycine Nile N-(2-methylpropyl)glycine Nleu
D-N-methyltryptophan Dnmtrp D-N-methyltyrosine Dnmtyr
D-N-methylvaline Dnmval .gamma.-aminobutyric acid Gabu
L-t-butylglycine Tbug L-ethylglycine Etg L-homophenylalanine Hphe
L-.alpha.-methylarginine Marg L-.alpha.-methylaspartate Masp
L-.alpha.-methylcysteine Mcys L-.alpha.-methylglutamine Mgln
L-.alpha.-methylhistidine Mhis L-.alpha.-methylisoleucine Mile
L-.alpha.-methylleucine Mleu L-.alpha.-methylmethionine Mmet
L-.alpha.-methylnorvaline Mnva L-.alpha.-methylphenylalanine Mphe
L-.alpha.-methylserine mser L-.alpha.-methylvaline Mtrp
L-.alpha.-methylleucine Mval Nnbhm N-(N-(2,2-diphenylethyl)
carbamylmethyl-glycine Nnbhm 1-carboxy-1-(2,2-diphenyl
ethylamino)cyclopropane Nmbc L-N-methylalanine Nmala
L-N-methylarginine Nmarg L-N-methylasparagine Nmasn
L-N-methylaspartic acid Nmasp L-N-methylcysteine Nmcys
L-N-methylglutamine Nmgin L-N-methylglutamic acid Nmglu
L-N-methylhistidine Nmhis L-N-methylisolleucine Nmile
L-N-methylleucine Nmleu L-N-methyllysine Nmlys L-N-methylmethionine
Nmmet L-N-methylnorleucine Nmnle L-N-methylnorvaline Nmnva
L-N-methylornithine Nmorn L-N-methylphenylalanine Nmphe
L-N-methylproline Nmpro L-N-methylserine Nmser L-N-methylthreonine
Nmthr L-N-methyltryptophan Nmtrp L-N-methyltyrosine Nmtyr
L-N-methylvaline Nmval L-N-methylethylglycine Nmetg
L-N-methyl-t-butylglycine Nmtbug L-norleucine Nle L-norvaline Nva
.alpha.-methyl-aminoisobutyrate Maib
.alpha.-methyl-.gamma.-aminobutyrate Mgabu
.alpha.-methylcyclohexylalanine Mchexa
.alpha.-methylcyclopentylalanine Mcpen
.alpha.-methyl-.alpha.-napthylalanine Manap
.alpha.-methylpenicillamine Mpen N-(4-aminobutyl)glycine Nglu
N-(2-aminoethyl)glycine Naeg N-(3-aminopropyl)glycine Norn
N-amino-.alpha.-methylbutyrate Nmaabu .alpha.-napthylalanine Anap
N-benzylglycine Nphe N-(2-carbamylethyl)glycine Ngln
N-(carbamylmethyl)glycine Nasn N-(2-carboxyethyl)glycine Nglu
N-(carboxymethyl)glycine Nasp N-cyclobutylglycine Ncbut
N-cycloheptylglycine Nchep N-cyclohexylglycine Nchex
N-cyclodecylglycine Ncdec N-cyclododeclglycine Ncdod
N-cyclooctylglycine Ncoct N-cyclopropylglycine Ncpro
N-cycloundecylglycine Ncund N-(2,2-diphenylethyl)glycine Nbhm
N-(3,3-diphenylpropyl)glycine Nbhe N-(3-indolylyethyl) glycine
Nhtrp N-methyl-.gamma.-aminobutyrate Nmgabu D-N-methylmethionine
Dnmmet N-methylcyclopentylalanine Nmcpen D-N-methylphenylalanine
Dnmphe D-N-methylproline Dnmpro D-N-methylserine Dnmser
D-N-methylserine Dnmser D-N-methylthreonine Dnmthr
N-(1-methylethyl)glycine Nva N-methyla-napthylalanine Nmanap
N-methylpenicillamine Nmpen N-(p-hydroxyphenyl)glycine Nhtyr
N-(thiomethyl)glycine Ncys penicillamine Pen
L-.alpha.-methylalanine Mala L-.alpha.-methylasparagine Masn
L-.alpha.-methyl-t-butylglycine Mtbug L-methylethylglycine Metg
L-.alpha.-methylglutamate Mglu L-.alpha.-methylhomo phenylalanine
Mhphe N-(2-methylthioethyl)glycine Nmet
N-(3-guanidinopropyl)glycine Narg N-(1-hydroxyethyl)glycine Nthr
N-(hydroxyethyl)glycine Nser N-(imidazolylethyl)glycine Nhis
N-(3-indolylyethyl)glycine Nhtrp N-methyl-.gamma.-aminobutyrate
Nmgabu D-N-methylmethionine Dnmmet N-methylcyclopentylalanine
Nmcpen D-N-methylphenylalanine Dnmphe D-N-methylproline Dnmpro
D-N-methylserine Dnmser D-N-methylthreonine Dnmthr
N-(1-methylethyl)glycine Nval N-methyla-napthylalanine Nmanap
N-methylpenicillamine Nmpen N-(p-hydroxyphenyl)glycine Nhtyr
N-(thiomethyl)glycine Ncys penicillamine Pen
L-.alpha.-methylalanine Mala L-.alpha.-methylasparagine Masn
L-.alpha.-methyl-t-butylglycine Mtbug L-methylethylglycine Metg
L-.alpha.-methylglutamate Mglu L-.alpha.-methylhomophenylalanine
Mhphe N-(2-methylthioethyl)glycine Nmet L-.alpha.-methyllysine Mlys
L-.alpha.-methylnorleucine Mnle L-.alpha.-methylornithine Morn
L-.alpha.-methylproline Mpro L-.alpha.-methylthreonine Mthr
L-.alpha.-methyltyrosine Mtyr L-N-methylhomophenylalanine Nmhphe
N-(N-(3,3-diphenylpropyl) carbamylmethyl(1)glycine Nnbhe
[0079] Since the present peptides are preferably utilized in
therapeutics or diagnostics which require the peptides to be in
soluble form, the peptides of the 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.
[0080] The peptides of the present invention are preferably
utilized in a linear form, although it will be appreciated that in
cases where cyclicization does not severely interfere with peptide
characteristics, cyclic forms of the peptide can also be
utilized.
[0081] 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).
[0082] Thus, the present invention provides conclusive data as to
the identity of the structural determinant of amyloid peptides,
which directs fibril assembly.
[0083] As such, the present invention enables design of a range of
peptide sequences, which can be utilized for prevention/treatment
or diagnosis of amyloidosis.
[0084] The results presented herein are further substantiated by
the observation that the consensus aromatic sequence of the present
invention (SEQ ID NO: 7) is shared by numerous amyloid related
proteins (see Table 3), and the fact that small aromatic molecules,
such as Ro 47-1816/001 [Kuner et al. (2000) J. Biol. Chem.
275:1673-8, see FIG. 6a] and
3-p-toluoyl-2-[4'-(3-diethylaminopropoxy)-phnyl]-benzofuran [Twyman
(1999) Tetrahedron Letters 40:9383-9384] have been demonstrated
effective in inhibiting the polymerization of the beta polypeptide
of Alzheimer's disease [Findeis et al. (2000) Biochem. Biophys.
Acta 1503:76-84], while amyloid specific dyes such as Congo-Red
(FIG. 6b) and thioflavin T (FIG. 6c), which contain aromatic
elements are generic amyloid formation inhibitors.
[0085] As is mentioned hereinabove, one specific use for the
peptides of the present invention is prevention or treatment of
diseases associated with amyloid plaque formation.
[0086] Thus, according to another aspect of the present invention,
there is provided a method of treating an amyloid-associated
disease in an individual. Preferred individual subjects according
to the present invention are mammals such as canines, felines,
ovines, porcines, equines, bovines, humans and the like.
[0087] The term "treating" refers to reducing or preventing amyloid
plaque formation, or substantially decreasing plaque occurrence in
the affected tissue.
[0088] Amyloid-associated diseases treated according to the present
invention include, but are not limited to, type II diabetes
mellitus, Alzheimer's disease (AD), early onset Alzheimer's
disease, late onset Alzheimer's disease, presymptomatic Alzheimer's
disease, SAA amyloidosis, hereditary Icelandic syndrome, multiple
myeloma, and prion diseases including scrapie of sheep and goats
and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith and
Wells (1991) Curr 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 (FFI) [Gajdusek (1977) Science 197: 943-960;
Medori, Tritschler et al. (1992) N Engl J Med 326: 444-449].
[0089] The method includes providing to the individual a
therapeutically effective amount of the peptide of the present
invention.
[0090] It will be appreciated that when utilized for treatment of
amyloid diseases, the peptide of the present invention includes an
amino acid sequence suitable for preventing fibril formation,
reducing fibril formation, or disaggregating formed aggregates by
competitive destabilization of the preformed aggregate. For
example, SEQ ID NO: 9 can be utilized for treatment of amyloid
diseases, particularly type II diabetes mellitus since as shown in
the Examples section which follows, such a sequence exhibits no
amyloid fibril formation in an aqueous solution.
[0091] Alternatively, 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 of the full length IAPP.
[0092] The peptide (i.e., active ingredient) 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.
[0093] As used herein 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.
[0094] Herein the term "active ingredient" refers to the
preparation accountable for the biological effect.
[0095] Hereinafter, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" which may be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the 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).
[0096] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0097] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0098] 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.
[0099] Alternately, one may administer a preparation in a local
rather than systemic manner, for example, via injection of the
preparation directly into a specific region of a patient's
body.
[0100] Pharmaceutical compositions of the 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.
[0101] Pharmaceutical 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. Proper formulation is dependent upon the route of
administration chosen.
[0102] For injection, 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. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0103] For oral administration, 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 polyvinylpyrrolidone (PVP). If desired,
disintegrating agents may be added, such as cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0104] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
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.
[0105] Pharmaceutical 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. In soft capsules, the active ingredients may be
dissolved. or suspended in suitable liquids, such as fatty oils,
liquid paraffm, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0106] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0107] For administration by nasal inhalation, 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. In the case of a
pressurized aerosol, 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 of the compound and a suitable powder base such as
lactose or starch.
[0108] The preparations described herein may be formulated for
parenteral administration, e.g., by bolus injection or continuos
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.
[0109] 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. Optionally, 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.
[0110] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0111] 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.
[0112] Pharmaceutical 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.
[0113] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art.
[0114] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro assays. For example, a dose can be
formulated in animal models and such information can be used to
more accurately determine useful doses in humans.
[0115] Toxicity and therapeutic efficacy of the 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 of the patient's condition. [See
e.g., Fingl, et al., (1975) "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1].
[0116] Depending on the severity and responsiveness of the
condition to be treated, 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 of
the disease state is achieved.
[0117] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0118] 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.
[0119] 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 of the form of the
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.
[0120] It will be appreciated that the peptides 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).
Alternatively, 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).
[0121] To enable cellular expression of the peptides of the present
invention, the nucleic acid construct of the present invention
further includes at least one cis acting regulatory element. As
used herein, the phrase "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.
[0122] Any available promoter can be used by the present
methodology. In a preferred embodiment of the present invention,
the promoter utilized by the nucleic acid construct of the present
invention is active in the specific cell population transformed.
Examples of 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. (1983) Cell
33729-740], neuron-specific promoters such as the neurofilament
promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA
86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985)
Science 230:912-916] or mammary gland-specific promoters such as
the milk whey promoter (U.S. Pat. No. 4,873,316 and European
Application Publication No. 264,166). 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.
[0123] The constructs of the present methodology preferably further
include an appropriate selectable marker and/or an origin of
replication. Preferably, 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.
[0124] Examples of suitable constructs include, but are not limited
to pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay,
pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available
from Invitrogen Co. (www.invitrogen.com). Examples of retroviral
vector and packaging systems are those sold by Clontech, San Diego,
Calif., including Retro-X vectors pLNCX and pLXSN, which permit
cloning into multiple cloning sites and the trasgene is transcribed
from CMV promoter. Vectors derived from Mo-MuLV are also included
such as pBabe, where the transgene will be transcribed from the
5'LTR promoter.
[0125] Because of the self-aggregating nature of the peptides of
the present invention it is conceivable that such peptides 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.
[0126] Thus, according to yet another aspect of the present
invention there is provided a method of detecting a presence or an
absence of an amyloid fibril in a biological sample.
[0127] 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 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.
[0128] 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. Methods of obtaining tissue biopsies and
body fluids from mammals are well known in the art.
[0129] 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 decribed 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.
[0130] 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.
[0131] To facilitate complex detection, 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. As used herein the term "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.
Alternatively, 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. Such
epitope tags include a Myc tag, a Flag tag, a His tag, a leucine
tag, an IgG tag, a streptavidin tag and the like.
[0132] Alternatively, aggregate detection can be effected by an
antibody designed and configured to specifically react with the
peptides of the present invention.
[0133] For example, for an antibody specifically recognizing the
peptides of the present invention one may use the amino acid
sequence epitope of SEQ ID NO:13. 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 [Orlandi 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. These include but are not limited
to, the hybridoma technique, the human B-cell hybridoma technique,
and the Epstein-Bar-Virus (EBV)-hybridoma technique [Kohler G., et
al. (1975) Nature 256:495-497, Kozbor D., lo et al. (1985) J.
Immunol. Methods 81:31-42, Cote R. J. et al. (1983) Proc. Natl.
Acad. Sci. 80:2026-2030, Cole S. P. et al. (1984) Mol. Cell. Biol.
62:109-120].
[0134] Antibody fragments may also be generated. For example, such
fragments include F(ab')2 fragments which may. be produced by
pepsin digestion of the antibody molecule and the Fab fragments
which can be generated by reducing the disulfide bridges of the
F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity [Huse W. D. et al.
(1989) Science 254:1275-1281].
[0135] Thus, 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.
[0136] 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. For example, the
present invention may be used for high throughput screening of test
compounds. Typically, 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].
[0137] It will be appreciated that the peptides of the present
invention may also be used as potent detectors of amyloid deposits
in-vivo. A designed peptides 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.
[0138] The peptides of the present invention can be included in a
diagnostic or therapeutic kit. For example, 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.
[0139] Thus, the peptides can be each mixed in a single container
or placed in individual containers. Preferably, 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.
[0140] In addition, other additives such as stabilizers, buffers,
blockers and the like may also be added.
[0141] The peptides of such 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.
[0142] Peptides included in kits or immobilized to substrates may
be conjugated to a detectable label such as described
hereinabove.
[0143] 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.
[0144] 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.
EXAMPLES
[0145] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0146] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Maryland
(1989); Perbal, "A Practical Guide to Molecular Cloning", John
Wiley & Sons, New York (1988); Watson et al., "Recombinant
DNA", Scientific American Books, New York; Birren et al. (eds)
"Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold
Spring Harbor Laboratory Press, New York (1998); methodologies as
set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531;
5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook",
Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in
Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al.
(eds), "Basic and Clinical Immunology" (8th Edition), Appleton
& Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds),
"Selected Methods in Cellular Immunology", W. H. Freeman and Co.,
New York (1980); available immunoassays are extensively described
in the patent and scientific literature, see, for example, U.S.
Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987;
3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;
4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;
"Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid
Hybridization" Hames, B. D., and Higgins S. J., eds. (1985);
"Transcription and Translation" Hames, B. D., and Higgins S. J.,
eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986);
"Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical
Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in
Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To
Methods And Applications", Academic Press, San Diego, Calif.
(1990); Marshak et al., "Strategies for Protein Purification and
Characterization - A Laboratory Course Manual" CSHL Press (1996);
all of which are incorporated. by reference as if fully set forth
herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
Example 1
Alanine Scan of the hIAPP Basic Amylodogenic Unit--Rational and
Peptide Synthesis
[0147] 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 (IAPP), the
cytotoxicity hereof being directly associated with the development
of the disease. IAPP amyloid formation follows a
nucleation-dependent polymerization process, which proceeds through
conformational transition of soluble IAPP into aggregated
.beta.-sheets. Recently it has been shown that a hexapeptide
(22-27) (NFGAIL, SEQ ID NO: 14) of IAPP, also termed as the "basic
amylodogenic unit" is sufficient for the formation of
.beta.-sheet-containing amyloid fibrils [Konstantinos et al. (2000)
J. Mol. Biol. 295:1055-1071].
[0148] To gain further insight into the specific role of the
residues that compose "the "basic amylodogenic unit", a systematic
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 .beta.-sheet structures. alanine-scan was
preformed in the context of the block that is unique to human IAPP
(FIG. 1a). This block includes two serine residues that follow the
NFGAIL 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. FIG 1b shows a schematic
representation of the chemical structure of the wild-type peptide
while FIG. 1c indicates the amino-acid substitutions in the
different mutant peptides that were generated.
[0149] Methods and Reagents--Peptide synthesis was performed by
PeptidoGenic Research & Co. Inc (Livermore, Calif. USA). The
sequence identity of the peptides was confirmed by ion spray
mass-spectrometry using a Perkin Elmer Sciex API I spectrometer.
The purity of the peptides was confirmed by reverse phase
high-pressure liquid chromatography (RP-HPLC) on a C.sub.18 column,
using a linear gradient of 10 to 70% acetonitrile in water and 0.1%
trifluoroacetic acid (TFA).
Example 2
Kinetics of Aggregation of IAPP Peptide Fragment and Mutant
Derivatives as Monitored by Turbidity Measurements
[0150] To study self-assembly of the IAPP peptide derived
fragments, aggregation and insolubilization kinetics were monitored
using turbidity measurements at 405 nmn.
[0151] Kinetic aggregation assay--Fresh 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 .mu.l of peptides stock solutions were added to 98 .mu.l of 10 mM
Tris pH 7.2, resulting in a 2 mM fmal 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.
[0152] Results--As shown in FIG. 2a, wild-type peptide fragment
(SEQ ID NO: 1) showed an aggregation kinetic profile that was very
similar to those previously reported for non-seeded hIAPP
hexapeptide [Tenidis et al. (2000) J. Mol. Biol 295:1055-71]. Such
a profile is strongly indicative of a nucleation-dependent
polymerization mechanism [Jarrett 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 ID NO: 4)
showed essentially the same profile as that of wild type peptide.
The N1A peptide (SEQ ID NO: 2) mediated higher kinetics of
aggregation, albeit with different kinetic profile as compared to
that of wild-type peptide. Interestingly, the aggregation of N1A
seemed to be less nucleation-dependent. Substitution of the
isoleucine or leucine to alanine (peptides I5A, SEQ ID NO: 5 and
L6A, SEQ ID NO: 6 respectively) reduced the kinetics of aggregation
but did not abolish it completely. Substitution of the
phenylalanine residue to alanine (peptide F2A, SEQ ID NO:3) led to
a total loss of peptide ability to aggregate. The F2A peptide was
completely soluble in the aqueous assay buffer.
[0153] Altogether, kinetic aggregation studies of the amyloidogenic
fragments suggested a major role to the phenylalanine residue in
the process of amyloid formation by the IAPP active fragment.
Example 3
Measurement of Aggregate Mean Particle Size
[0154] While the turbidity assay provided an important estimate
regarding the aggregation potential and kinetics of the various
peptides, it did not provide information about the size of the
actual aggregates formed. It will be appreciated that although the
apparent hydrodynamic diameter of amyloid structures varies due to
irregularity of the amyloid structure, it may still provide a clear
indication about the order of magnitude of the structure formed and
present a quantitative criterion for comparing the structures
formed by the various peptides.
[0155] Therefore, the average size of the aggregates, formed by the
various peptides, was determined using dynamic light scattering
(DLS) experiments.
[0156] 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 .mu.m filter to a final
concentration of 100 .mu.M peptide and 1% DMSO. Particle size
measurement was conducted with a laser-powered ALV-NIBS/HPPS
non-invasive backscattering instrument. Autocorrelation data was
fitted using the ALV-NIBS/HPPS software to derive average apparent
hydrodynamic diameters.
[0157] Results--The average apparent hydrodynamic diameters of the
structures that were formed by the various peptides are presented
in FIG. 3.
[0158] Altogether, the apparent hydrodynamic diameter of the
structures formed by the various peptides seemed to be consistent
with the results obtained by the turbidity assay. As with the
turbidity assay, the wild-type peptide and G3A peptide formed
particles of very similar hydrodynamic diameters. Smaller
structures were observed with the derivative peptides: N1A, I5A and
L6A. Thus, in accordance with the turbidity assay, the DLS
experiments clearly illustrate that no large particles were formed
by the F2A peptide under the indicated experimental conditions.
Example 4
Examination of Amyloidogenic Performance of Wild Type Peptide and
Derivatives Through Congo Red (CR) Binding Assay
[0159] Congo red (CR) staining combined with polarization
microscopy was utilized to test amyloidogenicity of the peptides of
the present invention. Amyloid fibrils in general, and fibrilar
IAPP in particular, bind CR and exhibit gold/green birefringence
under polarized light [Cooper (1974) Lab. Invest. 31:232-8;
Lansbury (1992) Biochemistry 31:6865-70].
[0160] Method and reagents--Peptide solutions incubated in a 10 mM
Tris buffer (pH 7) for four days were dried on a glass microscope
slide. Staining was effected by the addition of 1 mM CR in 10 mM
Tris buffer pH 7.2 followed by a 1 minute incubation. To remove
excess CR, slides were rinsed with double-distilled water and
dried. Saturated CR solutions solubilized in 80% ethanol (v/v) were
used for poorly aggregating peptides. In such cases, staining was
effected without rinsing. Birefringence was determined using a WILD
Makroskop m420 (.times.70) equipped with a polarizing stage.
[0161] Results--Wild type, N1A and G3A peptides bound CR and
exhibited the characteristic green/gold birefringence (see FIGS.
4g, 4a and 4e for normal field and FIGS. 4h, 4b and 4f for
polarized light microscopy, respectively). Peptides I5A and L6A,
bound CR and exhibited rare but characteristic birefringence (FIGS.
4i and 4k for normal field and FIGS. 4j and 4l for polarized light,
respectively). Peptide F2A (NAGAIL) showed no capability of binding
CR (FIG. 4c for normal field and FIG. 4d for polarized light).
Dried buffer solution stained with CR was used as a negative
control (see FIGS. 4m and 4n for normal and polarized light,
respectively). Interestingly, no significant difference in binding
was observed for the negative control and the F2A peptide.
[0162] To substantiate the inability of F2A peptide to form
fibrils, a peptide solution incubated for 14 days was used in the
binding assay. Although some degree of aggregation was visually
observed following two weeks of peptide "aging", CR staining showed
no amyloid structure (results not shown). Under the same conditions
wild-type peptide incubation resulted in significant CR
birefringence.
Example 5
Ultrastructural Analysis of the Fibrillogenic Peptide and
Mutants
[0163] The fibrillogenic potential of the various peptides was
assessed by electron microscopy analysis.
[0164] Method--Peptide solutions (2 mM peptide in 10 mM Tris buffer
pH 7.2), were incubated overnight at room temperature. Fibrils
formation was assessed using 10 .mu.l sample placed on 200-mesh
copper grids, covered with carbon-stabilized formvar film (SPI
Supplies, West Chester Pa.). Following 20-30 seconds of incubation,
excess fluid was removed and the grids were negatively stained with
2% uranyl acetate in water. Samples were viewed in a JEOL 1200EX
electron microscope operating at 80 kV.
[0165] Results--To further characterize the structures formed by
the various peptides, negative staining electron microscopy
analysis was effected. In accordance with previous results,
filamentous structures were observed for all peptides (FIGS. 5a-f)
but F2A which generated amorphous fibrils (FIG. 5b). Frequency of
appearance of fibrils formed by the I5A and L6A peptides (FIGS. 5e
and 5f, respectively) was lower in comparison to that of wild type
(FIG. 5d), NIA, and G3A peptides (FIGS. 5a and 5c, respectively).
Although the EM fields shown for peptides F2A, I5A and L6A, were
rarely observed, the results presented by these images support the
quantitative results presented in the previous sections and thus
provide qualitative analysis of fibril morphology.
[0166] The tangled net-like structures that were observed for the
wild-type, N1A, and 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 I5A and L6A. These longer fibrils may be a
result of a slower kinetics, which allow for a more ordered fibril
organization.
[0167] Taken together, the qualitative results of the electron
microscopy and CR analyses strongly suggest that the phenylalanine
residue in the hexaamyloid peptide is crucial for its amyloidogenic
potential.
Example 6
Amyloid-Related Proteins Share an Aromatic Consensus Sequence
[0168] To substantiate the critical role of the aromatic residue in
IAPP assembly and to expand it to other amyloid-related proteins, a
homology search was conducted. Homology analysis indicated that the
aromatic sequence characteristic is prevalent in numerous other
amyloid-related proteins as shown in Table 3, below. TABLE-US-00003
TABLE 3 Amyloid- Pathological/ related physiological protein
condition Active sequence Reference Islet Type II FGAIL Tenidis et
al J amyloid Diabetes SEQ ID NO: 15 Mol Biol (2000) polypeptide
mellitus 295: 1055-71 This study beta- Alzheimer's QKLVFF Tjenberg
J Biol amyloid disease SEQ ID NO: 16 Chem (1996) 271: peptide LVFFA
8545-8 Findeis SEQ ID NO: 17 Bio-chemistry LPFFD (1999) 38: 6791-
SEQ ID NO: 18 80 Pallitto Bio- chemistry (1999) 38: 3570-8 Soto Nat
Med (1998) 4: 822-26 Lactad- Aortic NFGSVQFV* Haggvist Proc herin
medical SEQ ID NO: 19 Natl Acad Sci amyloid USA (1999) 96: 8669-74
Gelsolin Finnish SFNNGDCCFILD* Maury Biophys Res hereditary SEQ ID
NO: 20 Commun (1992) 183: amyloidosis 227-31 Serum Chronic
SFFSFLGEAFD* Westermark Biochem amyloid A inflammation SEQ ID NO:
21 Biophys Res Commun amyloidosis 182 27-33 PrP Creutzfeldt-
PHGGGWGQ Priola J Biol Chem Jakob SEQ ID NO: 22 (1998) 11980-5
disease Prusiner Cell (CJD) (1998) 93: 337-48 .sup.35P Yeast
PQGGYQQYN* Patino Science prion SEQ ID NO: 23 (1996) 273: 622-6
protein Tuite Cell (2000) 100: 289-92 Aromatic residues are
underlined. An asterisk indicates that the minimal active fragment
may be shorter.
[0169] The significance of aromatic residues in molecular
recognition and self-assembly is consistent with the role of
.pi.-stacking interactions in chemistry. It is suggested that
.pi.-stacking contributes to the enthalpic change (.DELTA.H) in
free energy interaction (.DELTA.G). Furthermore .pi.-stacking has
an entropic role. Accordingly, ordered water molecules are being
released from the aromatic ring by hydrophobic interactions. In
line with this, it is suggested that aromatic interactions may
significantly reduce the energetic barrier for amyloid formation,
thereby accelerating the amyloidosis process. Acceleration in
amyloidosis may be accomplished by the geometrically restricted
assembly of and high affinity between aromatic moieties. As amyloid
fibrils formation is basically a process of molecular recognition
and self-assembly, stacking interactions between aromatic residues
can provide both an energetic contribution as well as
directionality and orientation that is provided by the restricted
geometry of planar aromatic rings stacking.
[0170] Although the invention has been described in conjunction
with specific. embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents, and patent applications mentioned in this
specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent, or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
Sequence CWU 1
1
23 1 8 PRT Artificial sequence Synthetic peptide 1 Asn Phe Gly Ala
Ile Leu Ser Ser 1 5 2 8 PRT Artificial sequence Synthetic peptide 2
Ala Phe Gly Ala Ile Leu Ser Ser 1 5 3 8 PRT Artificial sequence
Synthetic peptide 3 Asn Ala Gly Ala Ile Leu Ser Ser 1 5 4 8 PRT
Artificial sequence Synthetic peptide 4 Asn Phe Ala Ala Ile Leu Ser
Ser 1 5 5 8 PRT Artificial sequence Synthetic peptide 5 Asn Phe Gly
Ala Ala Leu Ser Ser 1 5 6 8 PRT Artificial sequence Synthetic
peptide 6 Asn Phe Gly Ala Ile Ala Ser Ser 1 5 7 5 PRT Artificial
sequence consensus sequence misc_feature (1)..(1) Aromatic amino
acid misc_feature (2)..(2) Non glycine misc_feature (3)..(5) any
amino acid 7 Xaa Xaa Xaa Xaa Xaa 1 5 8 6 PRT Artificial sequence
Synthetic peptide 8 Ala Phe Gly Ala Ile Leu 1 5 9 6 PRT Artificial
sequence Synthetic peptide 9 Asn Ala Gly Ala Ile Leu 1 5 10 6 PRT
Artificial sequence Synthetic peptide 10 Asn Phe Gly Ala Ala Leu 1
5 11 6 PRT Artificial sequence Synthetic peptide 11 Asn Phe Gly Ala
Ile Ala 1 5 12 6 PRT Artificial sequence Synthetic peptide 12 Asn
Phe Ala Ala Ile Leu 1 5 13 5 PRT Artificial sequence Synthetic
peptide 13 Phe Ala Ala Ile Leu 1 5 14 6 PRT Artificial sequence
Synthetic peptide derived from hIAPP 14 Asn Phe Gly Ala Ile Leu 1 5
15 5 PRT Artificial sequence Islet amyloid polypeptide derived,
active site sequence 15 Phe Gly Ala Ile Leu 1 5 16 6 PRT Artificial
sequence Beta-amyloid peptide derived, active site sequence 16 Gln
Lys Leu Val Phe Phe 1 5 17 5 PRT Artificial sequence Beta-amyloid
peptide derived, active site sequence 17 Leu Val Phe Phe Ala 1 5 18
5 PRT Artificial sequence Beta-amyloid peptide derived, active site
sequence 18 Leu Pro Phe Phe Asp 1 5 19 8 PRT Artificial sequence
Lactadherin derived, active site sequence 19 Asn Phe Gly Ser Val
Gln Phe Val 1 5 20 12 PRT Artificial sequence Gelsolin derived,
active site sequence 20 Ser Phe Asn Asn Gly Asp Cys Cys Phe Ile Leu
Asp 1 5 10 21 11 PRT Artificial sequence Serum amyloid A derived,
active site sequence 21 Ser Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp
1 5 10 22 8 PRT Artificial sequence PrP derived, active site
sequence 22 Pro His Gly Gly Gly Trp Gly Gln 1 5 23 9 PRT Artificial
sequence 35P - Yeast prion protein derived, active site sequence 23
Pro Gln Gly Gly Tyr Gln Gln Tyr Asn 1 5
* * * * *