U.S. patent application number 10/562852 was filed with the patent office on 2007-01-25 for peptides antibodies directed thereagainst and methods using same for diagnosing and treating amyloid-associated diseases.
Invention is credited to Ehud Gazit.
Application Number | 20070021345 10/562852 |
Document ID | / |
Family ID | 33555600 |
Filed Date | 2007-01-25 |
United States Patent
Application |
20070021345 |
Kind Code |
A1 |
Gazit; Ehud |
January 25, 2007 |
Peptides antibodies directed thereagainst and methods using same
for diagnosing and treating amyloid-associated diseases
Abstract
Peptides having at least 2 amino acids and no more than 15 amino
acids are provided. The peptides comprise amino acid sequence X-Y
or Y-X, wherein X is an aromatic amino acid and Y is any amino acid
other than glycine. Also provided are pharmaceutical compositions
and kits including such peptides as well as methods using same for
diagnosing and treating amyloid associated diseases.
Inventors: |
Gazit; Ehud;
(Ramat-HaSharon, IL) |
Correspondence
Address: |
Martin D Moynihan;Prtsi Inc
PO Box 16446
Arlington
VA
22215
US
|
Family ID: |
33555600 |
Appl. No.: |
10/562852 |
Filed: |
June 29, 2004 |
PCT Filed: |
June 29, 2004 |
PCT NO: |
PCT/IL04/00577 |
371 Date: |
April 19, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60483180 |
Jun 30, 2003 |
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60514974 |
Oct 29, 2003 |
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Current U.S.
Class: |
424/130.1 ;
514/17.8; 530/326; 530/327; 530/328; 530/329; 530/330; 530/331;
548/530 |
Current CPC
Class: |
A61P 25/28 20180101;
A61P 25/20 20180101; C07K 5/06156 20130101; C07K 14/4711 20130101;
A61P 3/10 20180101; A61P 25/16 20180101; A61P 43/00 20180101; A61P
25/00 20180101; C07K 5/0819 20130101; C07K 5/101 20130101; C07K
5/1021 20130101; C07K 5/1024 20130101; A61P 25/14 20180101; C07K
5/1008 20130101; A61K 38/00 20130101; C07K 5/0823 20130101; C07K
5/1016 20130101; A61P 25/02 20180101; C07K 5/06078 20130101; C07K
7/06 20130101; C07K 5/06165 20130101; C07K 5/0812 20130101; A61P
35/00 20180101 |
Class at
Publication: |
514/014 ;
514/015; 514/016; 514/017; 514/018; 514/019; 530/326; 530/327;
530/328; 530/329; 530/330; 530/331; 548/530 |
International
Class: |
A61K 38/10 20070101
A61K038/10; A61K 38/08 20070101 A61K038/08; A61K 38/06 20070101
A61K038/06; A61K 38/05 20070101 A61K038/05; A61K 38/04 20070101
A61K038/04 |
Claims
1. A peptide comprising amino acid sequence X-Y or Y-X, wherein X
is an aromatic amino acid and Y is any amino acid other than
glycine, the peptide being at least 2 and no more than 15 amino
acids in length.
2-11. (canceled)
12. The peptide of claim 1, selected from the group consisting of
SEQ ID NOs. 4, 12-19, 27-45, 112-123, 125, 127, 128-149 and
150.
13-39. (canceled)
40. 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
including the amino acid sequence X-Y or Y-X, wherein X is an
aromatic amino acid and Y is any amino acid other than glycine,
said peptide being at least 2 and no more than 15 amino acids in
length.
41-73. (canceled)
72. A pharmaceutical composition for treating or preventing an
amyloid-associated disease comprising as an active ingredient a
peptide including the amino acid sequence X-Y or Y-X, wherein X is
an aromatic amino acid and Y is any amino acid other than glycine,
said peptide being at least 2 and no more than 15 amino acids in
length and a pharmaceutically acceptable carrier or diluent.
73. The pharmaceutical composition of claim 72, wherein Y is a
polar uncharged amino acid selected from the group consisting of
serine, threonine, asparagine, glutamine and natural derivatives
thereof.
74. The pharmaceutical composition of claim 72, wherein Y is a
.beta.-sheet breaker amino acid.
75. The pharmaceutical composition of claim 74, wherein said
.beta.-sheet breaker amino acid is a naturally occurring amino
acid.
76. The pharmaceutical composition of claim 75, wherein said
naturally occurring amino acid is selected from the group
consisting of proline, aspartic acid, glutamic acid, glycine,
lysine and serine.
77. The pharmaceutical composition of claim 74, wherein said
.beta.-sheet breaker amino acid is a synthetic amino acid.
78. The pharmaceutical composition of claim 77, wherein said
synthetic amino acid is a C.alpha.-methylated amino acid.
79. The pharmaceutical composition of claim 78, wherein said
C.alpha.-methylated amino acid is .alpha.-aminoisobutyric acid.
80. (canceled)
81. The pharmaceutical composition of claim 72, wherein said
peptide is selected from the group consisting of SEQ ID NOs. 4,
12-19, 27-45, 112-123, 125 and 127.
82. The pharmaceutical composition of claim 72, wherein said
peptide is at least 4 amino acids in length and includes at least
two serine residues at a C-terminus thereof.
83. The pharmaceutical composition of claim 72, wherein said
peptide is at least 3 amino acids in length and whereas at least
one of said amino acids of said peptide other than X-Y is a polar
uncharged amino acid selected from the group consisting of serine,
threonine, asparagine, glutamine and natural derivatives
thereof.
84. The pharmaceutical composition of claim 72, wherein said
peptide is at least 3 amino acids in length and whereas at least
one of said amino acids of said peptide other than X-Y is a is a
.beta.-sheet breaker amino acid.
85. The pharmaceutical composition of claim 84, wherein said
.beta.-sheet breaker amino acid is a naturally occurring amino
acid.
86. The pharmaceutical composition of claim 85, wherein said
naturally occurring amino acid is selected from the group
consisting of proline, aspartic acid, glutamic acid, glycine,
lysine and serine.
87. The pharmaceutical composition of claim 84, wherein said
.beta.-sheet breaker amino acid is a synthetic amino acid.
88. The pharmaceutical composition of claim 87, wherein said
synthetic amino acid is a C.alpha.-methylated amino acid.
89. The pharmaceutical composition of claim 88, wherein said
C.alpha.-methylated amino acid is .alpha.-aminoisobutyric acid.
90. The pharmaceutical composition of claim 84, wherein said
.alpha.-sheet breaker amino acid is located downstream to said X-Y
in said peptide.
91. The pharmaceutical composition of claim 84, wherein said
.beta.-sheet breaker amino acid is located upstream to said X-Y in
said peptide.
92. The pharmaceutical composition of claim 72, wherein said
peptide is at least 3 amino acids in length and whereas at least
one of said amino acids of said peptide is a positively charged
amino acid and at least one of said amino acids of said peptide is
a negatively charged amino acid.
93. The pharmaceutical composition of claim 92, wherein said
positively charged amino acid is selected from the group consisting
of lysine, arginine, and natural and synthetic derivatives
thereof.
94. The pharmaceutical composition of claim 92, wherein said
negatively charged amino acid is selected from the group consisting
of aspartic acid, glutamic acid and natural and synthetic
derivatives thereof.
95. The pharmaceutical composition of claim 72, wherein at least
one amino acid of said at least 2 and no more than 15 amino acids
of the peptide is a D stereoisomer.
96. The pharmaceutical composition of claim 72, wherein at least
one amino acid of said at least 2 and no more than 15 amino acids
of the peptide is an L stereoisomer.
97. The pharmaceutical composition of claim 72, wherein the peptide
is two amino acids in length and Y is a .beta.-sheet breaker amino
acid.
98. The pharmaceutical composition of claim 97, wherein the peptide
is as set forth in SEQ ID NO: 145.
99. The pharmaceutical composition of claim 72, wherein the peptide
is 3 amino acids in length, whereas Y is an aromatic amino acid and
an amino acid residue attached to said amino acid sequence X-Y or
Y-X is a .beta.-sheet breaker amino acid.
100. The pharmaceutical composition of claim 99, wherein said
.beta.-sheet breaker amino acid is at a C-terminus of the
peptide.
101. The pharmaceutical composition of claim 72, wherein the
peptide is at least 3 amino acids in length and includes a
thiolated amino acid at an N-terminus thereof.
102. A nucleic acid construct comprising a polynucleotide segment
encoding the peptide of claim 1.
103-117. (canceled)
118. An antibody or an antibody fragment comprising an antigen
recognition region capable of binding the peptide of claim 1.
119-140. (canceled)
141. 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 the peptide of claim 1 and a
pharamaeutical acceptable carrier or diluent.
142-147. (canceled)
148. A method of treating or preventing an amyloid-associated
disease in an individual, the method comprising providing to the
individual therapeutically effective amount of an antibody or an
antibody fragment having an antigen recognition region capable of
binding the peptide of claim 1, thereby treating or preventing the
amyloid-associated disease in the individual.
149-154. (canceled)
155. A peptide having the general Formula: ##STR4## wherein: C* is
a chiral carbon having a D configuration. R.sub.1 and R.sub.2 are
each independently selected from the group consisting of hydrogen,
alkyl, cycloalkyl, aryl, carboxy, C-thiocarb; R.sub.3 is selected
from the group consisting of hydroxy, alkoxy, aryloxy, thiohydroxy,
thioalkoxy, thioaryloxy, halo and amine; and R4 is alkyl.
156. The peptide of claim 155, wherein R.sub.4 is methyl.
157. The peptide of claim 155, wherein R.sub.1 and R.sub.2 are each
hydrogen and R.sub.3 is hydroxy.
158. The peptide of claim 155 is a cyclic peptide.
159. 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 the peptide
160-162. (canceled)
Description
FIELD AND BACKGROUND OF THE INVENTION
[0001] 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 and Alzheimer's disease.
[0002] 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.
[0003] 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 or
parallel .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;
Balbach et al. (2002) Biophys J. 83:1205-16].
[0004] 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.
[0005] Furthermore, 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].
[0006] Thus, therapies designed to inhibiting the production of
amyloid polypeptides or inhibiting amyloidosis may be useful for
treating amyloid associated diseases.
[0007] 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). 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.
[0008] 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].
[0009] The following summarizes current attempts to engineer drugs
having the capability of destabilizing amyloid structures.
[0010] 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 (Nature 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 A.beta.
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
92:2959-63].
[0011] 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.
[0012] 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.
[0013] Small molecules--The potential use of small molecules which
bind the amyloid polypeptide, stabilizing the native fold of the
protein has been attempted in the case of the transthyretin (TTR)
protein [Peterson (1998) Proc. Natl. Acad. Sci. USA 95:12965-12960;
Oza (1999) Bioorg. Med. Chem. Lett. 9:1-6]. Thus far, it has been
demonstrated that molecules such as thyroxine and flufenamic acid
are capable of preventing the conformation change, leading to
amyloid formation. However, the use of the compounds in animal
models has not been proved yet and might be compromised due to the
presence in blood or proteins, other than TTR, capable of binding
these ligands.
[0014] 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 of the TTR both in vitro and in vivo [Altland (1999)
Neurogenetics 2:183-188]. However, a complete characterization of
the antioxidant effect is still not available and the
interpretation of results concerning possible therapeutic
strategies remains difficult.
[0015] 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
[0016] According to one aspect of the present invention there is
provided a peptide comprising amino acid sequence X-Y or Y-X,
wherein X is an aromatic amino acid and Y is any amino acid other
than glycine, the peptide being at least 2 and no more than 15
amino acids in length .
[0017] According to another aspect of the present invention there
is provided a peptide comprising an amino acid sequence selected
from the group consisting of SEQ ID) NOs. 4, 12-19, 2745, 112-123,
125 and 127, the peptide being at least 2 and no more than 15 amino
acids in length.
[0018] According to yet another aspect of the present invention
there is provided a peptide comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs. 4, 12-19, 27-45,
112-123, 125 and 127, the peptide being at least 2 and no more than
15 amino acids in length.
[0019] According to still another aspect of the present invention
there is provided a peptide selected from the group consisting of
SEQ ID NOs. 4, 12-19, 27-45, 112-123, 125 and 127.
[0020] 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 including the amino acid sequence X-Y or Y-X, wherein X is
an aromatic amino acid and Y is any amino acid other than glycine,
the peptide being at least 2 and no more than 15 amino acids in
length.
[0021] According to further features in preferred embodiments of
the invention described below, the peptide is an active ingredient
of a pharmaceutical composition which also includes a
physiologically acceptable carrier.
[0022] According to still further features in the described
preferred embodiments the peptide is expressed from a nucleic acid
construct.
[0023] According to yet an additional 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 including the amino acid sequence
X-Y or Y-X, wherein X is an aromatic amino acid and Y is any amino
acid other than glycine, the peptide being at least 2 and no more
than 15 amino acids in length and a pharmaceutically acceptable
carrier or diluent.
[0024] According to still further features in the described
preferred embodiments at least one amino acid of the at least 2 and
no more than 15 amino acids of the peptide is a D stereoisomer.
[0025] According to still further features in the described
preferred embodiments at least one amino acid of the at least 2 and
no more than 15 amino acids of the peptide is an L
stereoisomer.
[0026] According to still further features in the described
preferred embodiments the peptide is two amino acids in length and
Y is a .beta.-sheet breaker amino acid.
[0027] According to still further features in the described
preferred embodiments the peptide is as set forth in SEQ ID NO:
145.
[0028] According to still further features in the described
preferred embodiments the peptide is 3 amino acids in length,
whereas Y is an aromatic amino acid and an amino acid residue
attached to the amino acid sequence X-Y or Y-X is a .beta.-sheet
breaker amino acid.
[0029] According to still further features in the described
preferred embodiments the .beta.-sheet breaker amino acid is at a
C-terminus of the peptide.
[0030] According to still further features in the described
preferred embodiments the peptide is at least 3 amino acids in
length and includes a thiolated amino acid at an N-terminus
thereof.
[0031] According to still an additional aspect of the present
invention there is provided a nucleic acid construct comprising a
polynucleotide segment encoding a peptide including the amino acid
sequence X-Y or Y-X, wherein X is an aromatic amino acid and Y is
any amino acid other than glycine, the peptide being at least 2 and
no more than 15 amino acids in length.
[0032] According to still further features in the described
preferred embodiments the nucleic acid construct further comprises
a promoter.
[0033] According to a further aspect of the present invention there
is provided an antibody or an antibody fragment comprising an
antigen recognition region capable of binding a peptide including
the amino acid sequence X-Y or Y-X, wherein X is an aromatic amino
acid and Y is any amino acid other than glycine, the peptide being
at least 2 and no more than 15 amino acids in length.
[0034] According to still further features in the described
preferred embodiments Y is a polar uncharged amino acid selected
from the group consisting of serine, threonine, asparagine,
glutamine and natural derivatives thereof.
[0035] According to still further features in the described
preferred embodiments Y is a .beta.-sheet breaker amino acid.
[0036] According to still further features in the described
preferred embodiments the .beta.-sheet breaker amino acid is a
naturally occurring amino acid.
[0037] According to still further features in the described
preferred embodiments the naturally occurring amino acid is
selected from the group consisting of proline, aspartic acid,
glutamic acid, glycine, lysine and serine.
[0038] According to still further features in the described
preferred embodiments the .beta.-sheet breaker amino acid is a
synthetic amino acid.
[0039] According to still further features in the described
preferred embodiments the synthetic amino acid is a
C.alpha.-methylated amino acid.
[0040] According to still further features in the described
preferred embodiments the C.alpha.-ethylated amino acid is
.alpha.-aminoisobutyric acid.
[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 selected from the group
consisting of SEQ ID NOs. 4, 12-19, 27-45, 112-123, 125 and
127.
[0043] According to still further features in the described
preferred embodiments the peptide is at least 4 amino acids in
length and includes at least two serine residues at a C-terminus
thereof.
[0044] According to still further features in the described
preferred embodiments the peptide is at least 3 amino acids in
length and whereas at least one of the amino acids of the peptide
other than X-Y is a polar uncharged amino acid selected from the
group consisting of serine, threonine, asparagine, glutamine and
natural derivatives thereof.
[0045] According to still further features in the described
preferred embodiments the peptide is at least 3 amino acids in
length and whereas at least one of the amino acids of the peptide
other than X-Y is a is a .beta.-sheet breaker amino acid.
[0046] According to still further features in the described
preferred embodiments the .beta.-sheet breaker amino acid is a
naturally occurring amino acid.
[0047] According to still further features in the described
preferred embodiments the naturally occurring amino acid is
selected from the group consisting of proline, aspartic acid,
glutamic acid, glycine, lysine and serine.
[0048] According to still further features in the described
preferred embodiments the .beta.-sheet breaker amino acid is a
synthetic amino acid.
[0049] According to still further features in the described
preferred embodiments the synthetic amino acid is a Ca-methylated
amino acid.
[0050] According to still further features in the described
preferred embodiments the C.alpha.-methylated amino acid is
.alpha.-saminoisobutyric acid.
[0051] According to still further features in the described
preferred embodiments the .beta.-sheet breaker amino acid is
located downstream to the X-Y in the peptide.
[0052] According to still further features in the described
preferred embodiments the .beta.-sheet breaker amino acid is
located upstream to the X-Y in the peptide.
[0053] According to still further features in the described
preferred embodiments the peptide is at least 3 amino acids in
length and whereas at least one of the amino acids of the peptide
is a positively charged amino acid and at least one of the amino
acid residues of the peptide is a negatively charged amino
acid.
[0054] According to still further features in the described
preferred embodiments the positively charged amino acid is selected
from the group consisting of lysine, arginine, and natural and
synthetic derivatives thereof.
[0055] According to still further features in the described
preferred embodiments the negatively charged amino acid is selected
from the group consisting of aspartic acid, glutamic acid and
natural and synthetic derivatives thereof.
[0056] 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 an antibody or an antibody fragment having an antigen
recognition region capable of binding a peptide including the amino
acid sequence X-Y or Y-X, wherein X is an aromatic amino acid and Y
is any amino acid other than glycine, the peptide being at least 2
and no more than 15 amino acids in length and a pharmaceutical
acceptable carrier or diluent.
[0057] According to stilt 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 therapeutically effective amount of an
antibody or an antibody fragment having an antigen recognition
region capable of binding a peptide including the amino acid
sequence X-Y or Y-X, wherein X is an aromatic amino acid and Y is
any amino acid other than glycine, the peptide being at least 2 and
no more than 15 amino acids in length.
[0058] According to still a further aspect of the present invention
there is provided a peptide having the general Formula:
##STR1##
[0059] wherein:
[0060] C* is a chiral carbon having a D configuration.
[0061] R.sub.1 and R.sub.2 are each independently selected from the
group consisting of hydrogen, alkyl, cycloalkyl, aryl, carboxy,
C-thiocarb;
[0062] R.sub.3 is selected from the group consisting of hydroxy,
alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, halo and
amine; and
[0063] R.sub.4 is alkyl.
[0064] According to still 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 general Formula: ##STR2##
[0065] wherein:
[0066] C* is a chiral carbon having a D configuration.
[0067] R.sub.1 and R.sub.2 are each independently selected from the
group consisting of hydrogen, alkyl, cycloalkyl, aryl, carboxy,
C-thiocarb;
[0068] R.sub.3 is selected from the group consisting of hydroxy,
alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, halo and
amine; and
[0069] R.sub.4 is alkyl.
[0070] According to still further features in the described
preferred embodiments R.sub.4 is methyl.
[0071] According to still further features in the described
preferred embodiments R.sub.1 and R.sub.2 are each hydrogen and
R.sub.3 is hydroxy.
[0072] According to still further features in the described
preferred embodiments the peptide is a cyclic peptide.
[0073] 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.
[0074] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the patent specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] 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.
[0076] In the drawings:
[0077] 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.
[0078] FIGS. 2a-c are schematic illustrations of amyloid binding
with the inhibitory aromatic reagents: Ro 47-1816/001 (FIG. 2a),
Thioflavin T (FIG. 2b) and CR dye (FIG. 2c).
[0079] FIGS. 3a-c are schematic illustrations of a primary sequence
comparison between human and rodent IAPP and the synthetic peptides
of the present invention. FIG. 3a 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. 3b illustrates the chemical structure of the wild
type IAPP peptide (SEQ ID NO: 1). FIG. 3c illustrates the primary
sequences and SEQ ID NOs. of the peptides derived from the basic
amyloidogenic unit.
[0080] 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.
[0081] FIG. 5 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.
[0082] FIGS. 6a-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. 6a-b), F2A peptide
(FIGS. 6c-d), G3A peptide (FIGS. 6e-f), wild type peptide (FIGS.
6g-h), I5A peptide (FIGS. 6i-j) and L6A (FIGS. 6k-1). Buffer with
Congo red reagent was used as a negative control visualized with
and without polarized light as shown in FIGS. 6m and 6n,
respectively.
[0083] FIGS. 7a-f are electron micrographs of "aged" IAPP peptide
and derivatives. NIA peptide (FIG. 7a), F2A peptide (FIG. 7b), G3A
peptide (FIG. 7c), wild type peptide (FIG. 7d), I5A peptide (FIG.
7e) and L6A (FIG. 7f). The indicated scale bar represents 100
nm.
[0084] FIG. 8a is a nucleic acid sequence alignment of wild type
hIAPP and a corresponding sequence modified according to a
bacterial codon usage. Modified bases are underlined.
[0085] 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).sub.6 tag are fused
C-terminally to the malE tag vector sequence. A factor Xa cleavage
site for removal of the MBP tag is indicated.
[0086] 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 .mu.g
protein were loaded in each of Lanes 1-3 whereas 5 .mu.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. Lane 4--purified MBP. Lane 5--purified MBP-IAPP. An arrow
marks the MBP-IAPP.
[0087] FIGS. 10a-b are a dot-blot image (FIG. 10a) and
densitometric quantitation thereof (FIG. 10b) depicting putative
amyloidogenic sequences in hIAPP.
[0088] FIG. 11 is a graphic illustration depicting light absorbance
at 405 nm as a fuiction of time during fibril formation thus
reflecting the aggregation kinetics of IAPP-derived peptides (SEQ
ID NOs. 14-19).
[0089] 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 (FIGS. 12a), NFLVHSS (FIG. 12b),
FLVHSS (FIG. 12c), NFLVH (FIG. 12d), FLVHS (FIG. 12e) and FLVH
(FIG. 12f).
[0090] FIGS. 13a-f are electron micrographs of "aged" IAPP
peptides. NFLVHSSNN peptide (FIGS. 13a), NFLVHSS (FIG. 13b), FLVHSS
(FIG. 13c), NFLVH (FIG. 13d), FLVHS (FIG. 13e) and FLVH (FIG. 13f).
The indicated scale bar represents 100 nm.
[0091] FIGS. 14a-f are graphs showing secondary structures in the
insoluble IAPP aggregates as determined by Fourier transformed
infrared spectroscopy. NFLVHSSNN peptide (FIGS. 14a), NFLVHSS (FIG.
14b), FLVHSS (FIG. 14c), NFLVH (FIG. 14d), FLVHS (FIG. 14e) and
FLVH (FIG. 14f).
[0092] FIG. 15 is a chemical structure of a previously reported
amyloidogenic peptide fragment of Medin [Haggqvist (1999) Proc.
Natl. Acad. Sci. U S A 96:8669-8674).
[0093] 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. FIG. 16a
illustrates a short-term kinetic assay. FIG. 16b illustrates a
long-term kinetic assay.
[0094] FIGS. 17a-f are electron micrographs of "aged" Medin-derived
peptides. NFGSVQFA--FIGS. 17a, NFGSVQ--FIG. 17b, NFGSV--FIG. 17c,
FGSVQ--FIG. 17d, GSVQ--FIG. 17e and FGSV--FIG. 17f. The indicated
scale bar represents 100 nm.
[0095] 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--FIGS. 18a, NFGSVQ--FIG. 18b, NFGSV--FIG.
18c, FGSVQ--FIG. 18d, GSVQ--FIG. 18e and FGSV--FIG. 18f.
[0096] FIGS. 19a-c depict the effect of an alanine mutation on the
amyloidogenic features of the hexapeptide amyloidogenic fragment of
Medin. FIG. 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; FIG. 19b is
an electron micrograph of "aged" Medin--derived alanine mutant, The
scale bar represents 100 nm; FIG. 19c--is a photomicrograph
illustrating Congo Red binding to pre-assembled Medin-derived
peptide mutant.
[0097] FIGS. 20a-b are the amino acid sequence of human Calcitonin
(FIG. 20a) and chemical structure of an amyloidogenic peptide
fragment of human Calcitonin (FIG. 20b). Underlined are residues 17
and 18 which are important to the oligomerization state and
hormonal activity of Calcitonin [Kazantzis (2001) Eur. J. Biochem.
269:780-791].
[0098] FIGS. 21a-d are electron micrographs of "aged"
Calcitonin-derived peptides. DFNKF--FIG. 21a, DFNK--FIG. 21b,
FNKF--FIG. 21c and DFN--FIG. 21d. The indicated scale bar
represents 100 nm.
[0099] 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--FIG. 22a, DFNK--FIG. 22b, FNKF--FIG. 22c and
DFN--FIG. 22d.
[0100] FIG. 23 is a graphic illustration showing secondary
structures in the insoluble Calcitonin aggregates as determined by
Fourier transformed infrared spectroscopy.
[0101] FIGS. 24a-c depict the effect an alanine mutation on the
amyloidogenic features of the pentapeptide amyloidogenic fragment
of Calcitonin. FIG. 24a is an electron micrograph of "aged"
Calcitonin-derived alanine mutant. The scale bar represents 100 nm;
FIG. 24b--is a photomicrograph illustrating Congo Red binding to
pre-assembled Calcitonin-derived peptide mutant; FIG. 24c is a
graph showing secondary structures in the mutant peptide as
determined by Fourier transformed infrared spectroscopy.
[0102] FIG. 25 is an electron micrograph depicting self-assembly of
"aged" Lactotransferrin-derived peptide. The scale bar represents
100 nm.
[0103] FIG. 26 is an electron micrograph depicting self-assembly of
"aged" Serum amyloid A protein-derived peptide. The scale bar
represents 100 nm.
[0104] FIG. 27 is an electron micrograph depicting self-assembly of
"aged" BriL-derived peptide. The scale bar represents 100 nm.
[0105] FIG. 28 is an electron micrograph depicting self-assembly of
"aged" Gelsolin-derived peptide. The scale bar represents 100
nm.
[0106] FIG. 29 is an electron micrograph depicting self-assembly of
"aged" Serum amyloid P-derived peptide. The scale bar represents
100 nm.
[0107] FIG. 30 is an electron micrograph depicting self-assembly of
"aged" Immunoglobulin light chain-derived peptide. The scale bar
represents 100 nm.
[0108] FIG. 31 is an electron micrograph depicting self-assembly of
"aged" Cystatin C-derived peptide. The scale bar represents 100
nm.
[0109] FIG. 32 is an electron micrograph depicting self-assembly of
"aged" Transthyretin-derived peptide. The scale bar represents 100
nm.
[0110] FIG. 33 is an electron micrograph depicting self-assembly of
"aged" Lysozyme-derived peptide. The scale bar represents 100
nm.
[0111] FIG. 34 is an electron micrograph depicting self-assembly of
"aged" Fibrinogen-derived peptide. The scale bar represents 100
nm.
[0112] FIG. 35 is an electron micrograph depicting self-assembly of
"aged" Insulin-derived peptide. The scale bar represents 100
nm.
[0113] FIG. 36 is an electron micrograph depicting self-assembly of
"aged" Prolactin-derived peptide. The scale bar represents 100
nm.
[0114] FIG. 37 is an electron micrograph depicting self-assembly of
"aged" Beta 2 microglobulin-derived peptide. The scale bar
represents 100 nm.
[0115] FIG. 38 is a graphic representation of the effect of an
inhibitory peptide on IAPP self-assembly. Squares--wild type (wt)
IAPP peptide; triangles--wt-IAPP+inhibitory peptide; circles--no
peptides.
[0116] 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).
[0117] FIG. 40 is a histogram representation illustrating turbidity
of IAPP analogues following seven day aging.
[0118] FIG. 41a-f are electron rnicrographs of "aged" IAPP
analogues. NFGAILSS --FIG. 41a; NFGAILSS--FIG. 41b; NIGAILSS--FIG.
41c; NLGAILSS--FIG. 41d; NVGAILSS--FIG. 41e and NAGAILSS--FIG. 41f.
The indicated scale bar represents 100 nm.
[0119] FIGS. 42a-c illustrate the binding of IAPP--NFGAILSS to
analogues of the minimal amyloidogenic sequence SNNXGAILSS (X=any
natural amino acid but cysteine). FIG. 42a shows short exposure of
the bound peptide-array. FIG. 42b shows long exposure of the bound
peptide-array. FIG. 42c shows quantitation of the short exposure
(FIG. 42a) using densitometry and arbitrary units.
[0120] FIG. 43a is a Ramachandran plot showing the sterically
allowed regions for all residues (yellow for fully allowed, orange
for partially allowed), for L-Proline (blue) and for the achiral
Aib residue (magenta).
[0121] FIGS. 43b-c are schematic illustrations showing the chemical
structure of the longer wild-type IAPP peptide (ANFLVH, SEQ ID NO:
124, FIG. 43b) and the Aib modified structure thereof peptide
(Aib-NF-Aib-VH, SEQ ID NO: 125, FIG. 43c). Functional groups
suitable for modicifcation are marked in blue (FIG. 43b) while
modified groups are marked in red (FIG. 43c).
[0122] FIGS. 44a-d are electron micrographs of "aged" IAPP
analogues. FIG. 44a--ANFLVH; FIG. 44b--ANFLV; FIG.
44c--Aib-NF-Aib-VH; and FIG. 44d--Aib-NF-Aib-V. The indicated scale
bar represents 100 nm.
[0123] FIGS. 45a-d are photomicrographs illustrating Congo Red
binding to pre-assembled wild type and Aib modified IAPP peptides.
Polarized field micrographs are shown for each of the following
aged (i.e., 11 days) peptide suspensions. FIG. 45a--ANFLVH; FIG.
45b--ANFLV; FIG. 45c--Aib-NF-Aib-VH; FIG. 45d--Aib-NF-Aib-V.
[0124] FIGS. 46a-b are graphs showing secondary structures in the
insoluble wild type and Aib modified hIAPP aggregates as determined
by Fourier transformed infrared spectroscopy (FT-IR). FIG.
46a--wild-type peptide ANFLVH and the corresponding Aib modified
peptide as designated by arrows. FIG. 46b--wild-type ANFLVH and the
corresponding Aib modified peptide as designated by errows.
[0125] FIG. 47 is a graph showing the inhibitory effect of Aib
modified peptides on amyloid fibril formation. Wild type IAPP was
incubated alone or with the various peptides of the present
invention. Fibril formation as a function of time was determined
using ThT fluorescence.
[0126] FIG. 48 is a histogram showing the inhibitory effect of
short aromatic sequences (SEQ ID NOs. 112-123) on IAPP
self-assembly.
[0127] FIGS. 49a-d are graphs depicting iterative cycles of
selection of IAPP fibrilization inhibitors. Fibrilization was
monitored by ThT fluorescence assay. Fluorescence values of IAPP
alone (4 .mu.M) or in the presence of assayed compounds (40 AM)
were tested. Measurements were taken once IAPP fluorescence reached
a plateau. IAPP fluorescence was arbitrary set as 100. FIG. 49a
shows the results of the first round of selection of IAPP
fibrilization inhibitors. EGI=D-Phe-D-Phe-D-Pro;
EG2=Aib-D-Phe-D-Asn-Aib; EG3=D-Phe-D-Asn-D-Pro; EG4
-Aib-Asn-Phe-Aib; EG5=Gln-Lys-Leu-Val-Phe-Phe; EG6=Tyr-Tyr;
EG7=Tyr-Tyr-NH2; EG8=Aib-Phe-Phe. FIG. 49b shows the results of the
second round of selection of IAPP fibrilization inhibitors.
EG13=Asn-Tyr-Aib; EG14=Asn-Tyr-Pro; EG15=D-Pro-D-Tyr-D-Asn;
EG16=D-Tyr-Aib; EG17=D-Pro-D-Tyr; EG18=D-Tyr-D-Pro. FIG. 49c shows
the results of the third round of selection of IAPP fibrilization
inhibitors. d-F-P=D-Phe-Pro; P-d-F=Pro-D-Phe; EG19=Asn-Tyr-Tyr-Pro;
EG20=Tyr-Tyr-Aib; EG21=Aib-Tyr-Tyr; EG22=Aib-Tyr-Tyr-Aib;
EG23=D-Asn-Tyr-Tyr-D-Pro. FIG. 49d shows the results of the forth
round of selection of IAPP fibrilization inhibitors.
EG24=Pro-Tyr-Tyr; EG25=Tyr-Tyr-Pro; EG26=Pro-Tyr-Tyr-Pro;
EG27=D-Tyr-D-Tyr; EG28=D-Pro-Aib; EG29=D-Phe-D-Pro; EG30=D-Trp-Aib;
EG31=D-Trp-D-Pro.
[0128] FIG. 50 is a graph depicting Inhibition of A.beta.(140)
fibril formation by D-Trp-Aib. A.beta.1-40 stock solution was
diluted to a final concentration of 5 .mu.M in 100 mM NaCI, 10 mM
sodium phosphate buffer (pH 7.4) with 10 .mu.M D-Trp-Aib
(triangles) or without any addition (squares). Fluorescence values
were measured after addition of 0.3 .mu.M Tht to each sample. The
results represent the mean of two independent measurements.
[0129] FIGS. 51a-c are photomicrographs depicting the inhibitory
effect of D-Trp-Aib on the fibrilization of A.beta. as visualized
by TEM. FIG. 51a shows A.beta.alone. FIGS. 51b-c shows two
different field of A.beta. incubated in the presence of the
inhibitor.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0130] 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.
[0131] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0132] 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.
[0133] 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.
[0134] While reducing the present invention to practice and while
searching for a novel therapeutic modality to amyloid associated
diseases, such as Type 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].
[0135] 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. The involvement of
aromatic residues in the process of amyloid formation is in-line
with the well-established role of .pi.-stacking interactions in
molecular recognition and self-assembly [Gillard et al (1997) Chem.
Eur. J. 3: 1933-40; Claessens and Stoddart, (1997) J. Phys. Org.
Chem. 10: 254-72; Shetty et al (1996) J. Am. Chem. Soc. 118:
1019-27; McGuaghey et al (1998) .pi.-stacking interactions: Alive
and well in proteins. J. Biol. Chem. 273, 15458-15463; Sun and
Bernstein (1996) J. Phys. Chem. 100: 13348-66]. .pi.-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 .pi.-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 of the tertiary structure of
proteins, host-guest interactions, and porphyrin aggregation in
solution [for further review on the possible role of .pi.-stacking
interaction in the self-assembly of amyloid fibrils see Gazit
(2002) FASEB J. 16:77-83].
[0136] The present inventor demonstrated the ability of short
aromatic peptide sequences, as short as di-peptides (see Example
45-47), to mediate molecular recognition, enabling for the first
time, to generate highly efficient diagnostic, prophylactic and
therapeutic peptides which can be utilized to treat or diagnose
diseases characterized by amyloid plaque formation.
[0137] Thus, according to one aspect of the present invention there
is provided a peptide which includes the amino acid sequence X-Y or
Y-X, wherein X is an aromatic amino acid and Y is any amino acid
other than glycine. Examples of peptides that include this sequence
are set forth in SEQ ID Nos. 4, 12-19, 2745, 112-123, 125 and 127.
As is shown by the results presented in Examples 36-39 of the
Examples section which follows, the present inventor have uncovered
that contrary to the teachings of the prior art, it is aromaticity
rather than hydrophobicity which dictates amyloid self-assembly.
Thus, the aromatic amino acid of the peptides of the present
invention is pivotal to the formation of amyloid fibrils.
[0138] 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. Examples of
aromatic residues which can form a part of the peptides of present
invention are provided in Table 2 below.
[0139] 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.
[0140] 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.
[0141] Since aggregation kinetics and aggregate structures are
largely determined by the specific residue composition and possibly
the length of the peptides generated (see FIG. 1), the present
invention encompasses both longer peptides (e.g., 10-50 amino
acids), which include the sequences set forth in SEQ ID NOs:
4.12-19, 27-45, 112-123, 125, 127, 128-147 or 148, or preferably
shorter peptides (e.g., 2-15 amino acids, preferably at least 2, at
least 3, at least 4, at least 5, at least 6, at least 8, at
least-10, say 12 amino acids, preferably no more than 15 amino
acids) including any of these sequences.
[0142] In order to enhance the rate of amyloid formation, the
peptides of the present invention preferably 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).
[0143] According to one embodiment of this aspect of the present
invention, amino acid residue Y is the polar and uncharged amino
acid.
[0144] According to another embodiment of this aspect of the
present invention, the peptide includes at least 3 amino acids, the
X-Y/Y-X amino acid sequence described hereinabove and an additional
polar and uncharged amino acid positioned either upstream
(N-Terminal end) or downstream (C-Terminal end) of the X-Y/Y-X
sequence.
[0145] The peptides of the present invention, can be at least 3
amino acid in length and may 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 of the Examples section, it is likely
that electrostatic interactions between opposing charges may direct
the formation of ordered antiparallel structure.
[0146] Yet additionally, the peptide of the present invention can
be 4 amino acids in length and include two serine residues at the
C-terminal end of the X-Y/Y-X sequence.
[0147] Still additionally, the peptide of the present invention can
be at least 3 amino acids in length and include a thiolated amino
acid residue (i.e., including a sulfur ion), preferably at an
N-terminal end thereof (e.g., SEQ ID NOs: 149 and 150,
D-Cys-D-Trp-Aib and L-Cys-D-Trp-Aib, respectively as well as their
acytelated and amidated forms). Such a peptide configuration is
highly valuable since it provides reducing properties to the
peptide and as such can serve both as a reducing agent and as an
antioxidant both may be critical for neuroprotection (Offen et al.
(2004) J Neurochem. 89:1241-51); and as an amyloid inhibitor.
Examples of thiolated amino acids include, but are not limited to,
the naturally occurring amino acids cysteine and methionine and
synthetic amino acids such as Tyr (SO.sub.3H).
[0148] Since the present inventor has identified the sequence
characteristics governing fibril formation, the teachings of the
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.
[0149] For example, a peptide encompassed by SEQ ID NO: 9, 10, 11,
17, 19, 25 or 30 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 a very slow process, these peptide
sequences will completely inhibit or significantly delay
amyloidosis under physiological conditions.
[0150] 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=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.
[0151] 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=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.
[0152] These modifications can occur at any of the bonds along the
peptide chain and even at several (2-3) at the same time.
[0153] Natural aromatic amino acids, Trp, Tyr and Phe, may be
substituted for synthetic non-natural acid such as Phenylglycine,
Tic, naphtylalanine (Nal), phenylisoserine, threoninol,
ring-methylated derivatives of Phe, halogenated derivatives of Phe
or o-methyl-Tyr.
[0154] 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).
[0155] 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.
[0156] Tables 1 and 2 below list naturally occurring amino acids
(Table 1) and non-conventional or modified amino acids (e.g.,
synthetic, Table 2) which can be used with the present invention.
TABLE-US-00001 TABLE 1 Three-Letter Amino Acid Abbreviation
One-letter 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
[0157] 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) Nnbhm
carbamylmethyl-glycine 1-carboxy-1-(2,2-diphenyl Nmbc
ethylamino)cyclopropane 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) Nnbhe carbamylmethyl(1)glycine
[0158] 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.
[0159] For therapeutic application, the peptides of the present
invention preferably include at least one .beta.-sheet breaker
amino acid residue, which is positioned in the peptide sequence as
described below. Peptides which include such .beta.-sheet breaker
amino acids retain recognition of amyloid polypeptides but prevent
aggregation thereof (see Examples 40-45 of the Examples section
which follows). According to one preferred embodiment of this
aspect of the present invention, the .beta.-sheet breaker amino
acid is a naturally occurring amino acid such as proline (e.g., SEQ
ID NOs. 45, 112, 119, 120, 122, 123, 128, 130, 134, 138, 139, 140,
141, 143, 144, 146, 147 and 148, see background section) 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 of the amyloid
fibril. Other .beta.-sheet breaker amino acid residues include, but
are not limited to aspartic acid, glutamic acid, glycine, lysine
and serine (according to Chou and Fasman (1978) Annu. Rev. Biochem.
47, 258).
[0160] According to another preferred embodiment of this aspect of
the present invention, the .beta.-sheet breaker amino acid residue
is a synthetic amino acid such as a C.alpha.-amethylated amino
acid, which conformational constrains are restricted [Balaram,
(1999) J. Pept. Res. 54, 195-199]. Unlike natural amino acids,
C.alpha.-methylated amino acids have a hydrogen atom attached to
the C.sub..alpha., which affects widely their sterical properties
regarding the .phi. and .psi. angels of the amide bond. Thus, while
alanine has a wide range of allowed .phi. and .psi. conformations,
.alpha.-aminoisobutyric acid (Aib, see Table 2, above) has limited
.phi. and .psi. conformations. Hence, peptides of the present
invention which are substituted with at least one Aib residue are
capable of binding amyloid polypeptides but prevent aggregation
thereof (see Examples 40-44). Such peptides are set forth in SEQ ID
NOs: 113, 114, 117, 118, 121, 135, 136, 137,143, 145, 149, 129 and
131.
[0161] The .beta.-sheet breaker amino acid of this aspect of the
present invention can be located at position Y of the X-Y/Y-X amino
acid sequence of the peptide (see for Example SEQ ID NOs: 123, 143,
144, 145, 146, 147, 148). Alternatively, the peptides of this
aspect of the present invention can be at least 3 amino acids and
include the breaker amino acid in any position other than the
X-Y/Y-X amino acid sequence (see for example SEQ ID NO: 117).
[0162] The .beta.-sheet breaker amino acid may be positioned
upstream of the aromatic residue (see SEQ ID NO: 122) or downstream
thereto (see SEQ ID NO: 123).
[0163] According to one preferred embodiment of this aspect of the
present invention the peptide is three amino acids in length,
wherein Y is an aromatic amino acid and an amino acid residure
attached to the amino acid sequence X-Y or Y-X is a .beta.-sheet
breaker amino acid, which is preferably attached at the C-terminus
of the peptide (e.g., SEQ ID NOs: 135 and 140).
[0164] According to another preferred embodiment of this aspect of
the present invention the peptide is two amino acids in length and
Y is a .beta.-sheet breaker amino acid (e.g., SEQ ID NOs: 121,
143-148).
[0165] According to a most preferred embodiment of this aspect of
the present invention the peptide is a dipeptide having the
following general formula: ##STR3##
[0166] wherein:
[0167] C* is a chiral carbon having a D configuration (also
referred to in the art as R-configuration).
[0168] R.sub.1 and R.sub.2 are each independently selected from the
group consisting of hydrogen, alkyl, cycloalkyl, aryl, carboxy,
thiocarboxy, C-carboxylate and C-thiocarboxylate;
[0169] R.sub.3 is selected from the group consisting of hydroxy,
alkoxy, aryloxy, thiobydroxy, thioalkoxy, thioaryloxy, halo and
amine; and
[0170] R.sub.4 is alkyl.
[0171] As used herein, the term "alkyl" refers to a saturated
aliphatic hydrocarbon including straight chain and branched chain
groups. Preferably, the alkyl group has 1 to 20 carbon atoms.
Whenever a numerical range; e.g., "1-20", is stated herein, it
implies that the group, in this case the alkyl group, may contain 1
carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and
including 20 carbon atoms. More preferably, the alkyl is a medium
size alkyl having 1 to 10 carbon atoms. Most preferably, unless
otherwise indicated, the alkyl is a lower alkyl having 1 to 4
carbon atoms. The alkyl group may be substituted or unsubstituted.
When substituted, the substituent group can be, for example, halo,
hydroxy, cyano, nitro and amino.
[0172] A "cycloalkyl" group refers to an all-carbon monocyclic or
fused ring (i.e., rings which share an adjacent pair of carbon
atoms) group wherein one of more of the rings does not have a
completely conjugated pi-electron system. Examples, without
limitation, of cycloalkyl groups are cyclopropane, cyclobutane,
cyclopentane, cyclopentene, cyclohexane, cyclohexadiene,
cycloheptane, cycloheptatriene, and adamantane. A cycloalkyl group
may be substituted or unsubstituted. When substituted, the
substituent group can be, for example, alkyl, halo, hydroxy, cyano,
nitro and amino.
[0173] An "aryl" group refers to an all-carbon monocyclic or
fused-ring polycyclic (i.e., rings which share adjacent pairs of
carbon atoms) groups having a completely conjugated pi-electron
system. Examples, without limitation, of aryl groups are phenyl,
naphthalenyl and anthracenyl. The aryl group may be substituted or
unsubstituted. When substituted, the substituent group can be, for
example, alkyl, cycloalkyl, halo, hydroxy, alkoxy, thiohydroxy,
thioalkoxy, cyano, nitro and amino.
[0174] A "hydroxy" group refers to an--OH group.
[0175] An "alkoxy" group refers to both an--O-alkyl and
an--O-cycloalkyl group, as defined herein.
[0176] An "aryloxy" group refers to an --O-aryl group, as defined
herein.
[0177] A "thiohydroxy" group refers to a --SH group.
[0178] A "thioalkoxy" group refers to both an --S-alkyl group, and
an --S-cycloalkyl group, as defined herein.
[0179] A "thioaryloxy" group refers to an --S-aryl group, as
defined herein.
[0180] A "carboxy" group refers to a --C(.dbd.O)--R' group, where
R' is hydrogen, halo, alkyl, cycloalkyl or aryl, as defined
herein.
[0181] A "thiocarboxy" group refers to a --C(.dbd.S)--R' group,
where R' is as defined herein for R'.
[0182] A "C-carboxylate" group refers to a --C(.dbd.O)--O--R'
groups, where R' is as defined herein.
[0183] A "C-thiocarboxylate" group refers to a --C(.dbd.S)--O--R'
groups, where R' is as defined herein.
[0184] A "halo" group refers to fluorine, chlorine, bromie or
iodine.
[0185] An "amine" group refers to an --NR'R'' group where R' is as
defined herein and R'' is as defined for R'.
[0186] A "nitro" group refers to an --NO.sub.2 group.
[0187] A "cyano" group refers to a --C.ident.N group.
[0188] Preferably, R.sub.4 is methyl, such that the compound above
is D-tryptophane-alpha-aminobutyric acid (also referred to herein
as D-Trp-aib or D-tryptophane-alpha-methyl-alanine), or a
derivative thereof.
[0189] It will be appreciated that unmodified di-peptides, peptides
of L-configuration, peptides which are of a reversed configuration
(i.e., C-to-N sequence of tryptophane (D/L) and alpha-methyl
alanine), or alternatively, macromolecules (e.g., peptides,
immobilized peptides) which encompass the above-described peptide
sequence, are known (see e.g., WO 02/094857, WO 02/094857, EP Pat.
No. 966,975, U.S. Pat. Nos. 6,255,286, 6,251,625, 6,162,828 and
5,304,470). However, such molecules are chemically and biologically
different than the above described peptide, which unique activity
is strictly dependent on its structure.
[0190] 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 of the peptide can also be
utilized.
[0191] 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).
[0192] For example, a peptide according to the teachings of the
present invention can include at least two cysteine residues
flanking the core peptide sequence. In this case, cyclization can
be generated via formation of S--S bonds between the two Cys
residues. Side-chain to side chain cyclization can also be
generated via formation of an interaction bond of the formula
--(--CH2-)n-S--CH2-C--, wherein n=1 or 2, which is possible, for
example, through incorporation of Cys or homoCys and reaction of
its free SH group with, e.g., bromoacetylated Lys, Om, Dab or Dap.
Furtherrnore, cyclization can be obtained, for example, through
amide bond formation, e.g., by incorporating Glu, Asp, Lys, Orn,
di-amino butyric (Dab) acid, di-aminopropionic (Dap) acid at
various positions in the chain (--CO--NH or --NH--CO bonds).
Backbone to backbone cyclization can also be obtained through
incorporation of modified amino acids of the formulas
H--N((CH2)n-COOH)--C(R)H--COOH or H--N((CH2)n-COOH)--C (R)H--NH2,
wherein n=1-4, and further wherein R is any natural or non-natural
side chain of an amino acid.
[0193] Thus, the present invention provides conclusive data as to
the identity of the structural determinant of amyloid peptides,
which directs fibril assembly.
[0194] As such, the present invention enables design of a range of
peptide sequences, which can be utilized for prevention/treatment
or diagnosis of amyloidosis.
[0195] As is described in Examples 6-35, the present inventor
identified the consensus aromatic sequence of the present invention
(SEQ ID NO: 7) in numerous amyloid related proteins , thereby
conclusively showing that the present invention enables accurate
identification of amyloidogenic fragments in essentially all
amyloidogenic proteins.
[0196] Furthermore, the fact that small aromatic molecules, such as
Ro 47-1816/001 [Kuner et al. (2000) J. Biol. Chem. 275:1673-8, see
FIG. 2a] 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. 2b) and thioflavin T (FIG. 2c), which contain aromatic
elements are generic amyloid formation inhibitors, substantiate the
recognition motif of the present invention as sufficient for
amyloid self-assembly.
[0197] The availability of the 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).
[0198] The term "antibody" refers to intact antibody molecules as
well as functional fragments thereof, such as Fab, F(ab').sub.2,
and Fv that are capable of binding to macrophages. These 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').sub.2, the fragment of the antibody that can be
obtained by treating whole antibody with the enzyme pepsin without
subsequent reduction; F(ab').sub.2 is a dimer of two Fab' fragments
held together by two disulfide bonds; (iv) Fv, defined as a
genetically engineered fragment containing the variable region of
the light chain and the variable region of the heavy chain
expressed as two chains; and (v) Single chain antibody ("SCA"), a
genetically engineered molecule containing the variable region of
the light chain and the variable region of the heavy chain, linked
by a suitable polypeptide linker as a genetically fused single
chain molecule.
[0199] Methods of making these fragments are known in the art. (See
for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1988, incorporated herein by
reference).
[0200] Methods of generating antibodies (i.e., monoclonal and
polyclonal) are well known in the art. 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., 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].
[0201] 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.
[0202] Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. For example,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.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. Alternatively, an enzymatic
cleavage using pepsin produces two monovalent Fab' fragments and an
Fc fragment directly. These methods are described, for example, by
Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references
contained therein, which patents are hereby incorporated by
reference in their entirety. See also Porter, R. R., Biochem. J.,
73: 119-126, 1959. Other methods of cleaving antibodies, such as
separation of heavy chains to form monovalent light-heavy chain
fragments, further cleavage of fragments, or other enzymatic,
chemical, or genetic techniques may also be used, so long as the
fragments bind to the antigen that is recognized by the intact
antibody.
[0203] Fv fragments comprise an association of V.sub.H and V.sub.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 V.sub.H and V.sub.L chains
connected by a peptide linker. These single-chain antigen binding
proteins (sFv) are prepared by constructing a structural gene
comprising DNA sequences encoding the V.sub.H and V.sub.L domains
connected by an oligonucleotide. 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
Wdomains. Methods for producing sFvs are described, for example, by
Whitlow and Filpula, Methods, 2: 97-105, 1991; Bird et al., Science
242:423-426, 1988; Pack et al., Bio/Technology 11:1271-77, 1993;
and Ladner et al., U.S. Pat. No. 4,946,778, which is hereby
incorporated by reference in its entirety.
[0204] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). 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.
[0205] For human applications, the antibodies of the 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) of the 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. In some instances, 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. In general, 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)].
[0206] Methods for humanizing non-human antibodies are well known
in the art. Generally, 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 332:323-327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such 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. In practice,
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.
[0207] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J.
immunol., 147(1):86-95 (1991)]. Similarly, human can be made by
introducing of human immunoglobulin loci into transgenic animals,
e.g., mice in which the endogenous immunoglobulin genes have been
partially or completely inactivated. Upon challenge, human antibody
production is observed, which closely resembles that seen in humans
in all respects, including gene rearrangement, assembly, and
antibody repertoire. This approach is described, for example, in
U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;
5,633,425; 5,661,016, and in the following scientific publications:
Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg et al.,
Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994);
Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger,
Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intem.
Rev. Immunol. 13 65-93 (1995).
[0208] 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.
[0209] Thus, according to yet 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.
[0210] The term "treating" refers to reducing or preventing amyloid
plaque formation, or substantially decreasing plaque occurrence in
the affected tissue. The phrase "amyloid plaque" refers to
fibrillar amyloid as well as aggregated but not fibrillar amyloid,
hereinafter "protofibrillar amyloid", which may be pathogenic as
well. For example, an aggregated but not necessarily fibrillar form
of IAPP was found to be toxic in culture. As shown by Anaguiano and
co-workers [(2002) Biochemistry 41:11338-43] protofibrillar IAPP,
like protofibrillar .alpha.-synucelin, which is implicated in
Parkinson's disease pathogenesis, permeabilized synthetic vesicles
by a pore-like mechanism. The formation of the IAPP amyloid pore
was temporally correlated to the formation of early IAPP oligomers
and disappearance thereof to the appearance of amyloid fibrils.
These results suggest that protofibrillar IAPP may be critical to
type II diabetes mellitus as other protofibrillar proteins are
critical to the development of Alzheimer's and Parkinson's
diseases.
[0211] 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, Perkinson's disease, SAA amyloidosis, hereditary Icelandic
syndrome, multiple myeloma, medullary carcinoma, aortic medical
amyloid, Insulin injection amyloidosis, prion-systematic
arnyloidosis, 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. Med. Chem. 9:1667-1675] 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].
[0212] 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.
[0213] 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 NOs: 45, 112-123, 125, 127, 128-149 and 150 can be
utilized for treatment of amyloid diseases, particularly type II
diabetes mellitus since as shown in Example 35 and in Example 45 of
the Examples section which follows, such sequences interfere with
IAPP self-assembly as demonstrated by the decreased ability of the
amyloidogenic peptide to bind thioflavin T in the presence of
inhibitory peptides.
[0214] Alternatively, 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 of the full length IAPP.
[0215] Alternatively, self-aggregating peptides such as those set
forth in SEQ ID NOs. 17, 19 and 28-30, can be used as potent
inhibitors of amyloid fibrilization, since such peptides can form
heteromolecular complexes which are not as ordered as the
homomolecular assemblies formed by amyloid fragments.
[0216] It will be appreciated that since one of the main obstacles
in using short peptide fragments in therapy is their proteolytic
degradation by stereospecific cellular proteases, the peptides of
the present invention are preferably synthesized from D-isomers of
natural amino acids [i.e., inverso peptide analogues, Tjernberg
(1997) J. Biol. Chem. 272:12601-5, Gazit (2002) Curr. Med. Chem.
9:1667-1675].
[0217] Additionally, 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).
[0218] As used herein a "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.
[0219] Retro-inverso modification of naturally occurring
polypeptides involves the synthetic assembly of amino acids with
.alpha.-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.
[0220] Additionally, since one of the main issues in amyloid fibril
formation is the transition of the amyloid polypeptide from the
native form to stacked .beta.-strand structure, inhibitory peptides
preferably include N-methylated amino acids which constrain
peptide-backbone due to steric effects [Kapurniotu (2002)
315:339-350]. For example, aminoisobutyric acid (Aib or methyl
alanine) is known to stabilize an .alpha.helical structure in short
natural peptides. Furthermore, the N-methylation also affects the
intermolecular NH to CO H-bonding ability, thus suppressing the
formation of multiplayer .beta.-strands, which are stabilized by
H-bonding interactions.
[0221] It will be further appreciated that addition of organic
groups such as a cholyl groups to the N-terminal or C-terminal of
the peptides of the present invention is preferred since it was
shown to improve potency and bioavailability (e.g., crossing the
blood brain barrier in the case of neurodegenerative diseases) of
therapeutic peptides [Findeis (1999) Biochemistry 38:6791-6800].
Furthermore, introducing a charged amino acid to the recognition
motif, may result in electrostatic repulsion which inhibits further
growth of the amyloid fibrils [Lowe (2001) J. Mol. Biol.
40:7882-7889].
[0222] As mentioned hereinabove, the antibodies of the present
invention may also be used to treat amyloid-associated
diseases.
[0223] 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.
[0224] 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.
[0225] Herein the term "active ingredient" refers to the peptide or
antibody preparation, which is accountable for the biological
effect.
[0226] 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).
[0227] 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.
[0228] 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.
[0229] 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.
[0230] 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.
[0231] 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.
[0232] 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.
[0233] 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.
[0234] 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.
[0235] 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.
[0236] 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 paraffin, 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.
[0237] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0238] 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.
[0239] The preparations 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.
[0240] 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.
[0241] 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.
[0242] 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.
[0243] 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.
[0244] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art.
[0245] 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.
[0246] 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].
[0247] 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.
[0248] 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.
[0249] 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.
[0250] 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.
[0251] It will be appreciated that 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). 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).
[0252] To enable cellular expression of the peptides or antibodies
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.
[0253] 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.
[0254] 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.
[0255] Currently preferred 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. Useful lipids for
lipid-mediated transfer of the gene are, for example, DOTMA, DOPE,
and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65
(1996)]. The most preferred constructs for use in gene therapy are
viruses, most preferably adenoviruses, AAV, lentiviruses, or
retroviruses. 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. In addition, such a
construct typically includes a signal sequence for secretion of the
peptide or antibody from a host cell in which it is placed.
Preferably the signal sequence for this purpose is a mammalian
signal sequence. Optionally, the construct may also include a
signal that directs polyadenylation, as well as one or more
restriction sites and a translation termination sequence. By way of
example, such constructs 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.
[0256] 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.
[0257] 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.
[0258] 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.
[0259] 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.
[0260] 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.
[0261] 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.
[0262] 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.
[0263] Alternatively, aggregate detection can be effected by the
antibodies of the present invention.
[0264] 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.
[0265] 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].
[0266] 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 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-ike deposits can be detected
by in vivo imaging techniques known in the art.
[0267] 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.
[0268] 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.
[0269] In addition, other additives such as stabilizers, buffers,
blockers and the like may also be added.
[0270] 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.
[0271] Peptides included in kits or immobilized to substrates may
be conjugated to a detectable label such as described
hereinabove.
[0272] 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.
[0273] 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
[0274] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0275] 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 1-111
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, CT (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, Calf. (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
[0276] 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, GenBank
Accession No. gi:4557655), the cytotoxicity thereof 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: 1) of IAPP, also termed as
the "basic amyloidogenic unit" is sufficient for the formation of
.beta.-sheet-containing amyloid fibrils [Konstantinos et al. (2000)
J. Mol. Biol. 295:1055-1071].
[0277] To gain further insight into the specific role of the
residues that compose "the "basic amyloidogenic 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. 3a). 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. 3b shows a schematic
representation of the chemical structure of the wild-type peptide
while FIG. 3c indicates the amino-acid substitutions in the
different mutant peptides that were generated.
[0278] Methods and Reagents--Peptide synthesis was performed by
PeptidoGenic Research & Co. Inc (Livermore, Calf. 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
[0279] To study self-assembly of the IAPP peptide derived
fragments, aggregation and insolubilization kinetics were monitored
using turbidity measurements at 405 nm.
[0280] 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 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.
[0281] Results--As shown in FIG. 4a, 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.
[0282] 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
[0283] 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.
[0284] Therefore, the average size of the aggregates, formed by the
various peptides, was determined using dynamic light scattering
(DLS) experiments.
[0285] 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.
[0286] Results--The average apparent hydrodynamic diameters of the
structures that were formed by the various peptides are presented
in FIG. 5.
[0287] 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
[0288] 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].
[0289] 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. Birefiingence was determined using a WILD
Makroskop m420 (x70) equipped with a polarizing stage.
[0290] Results--Wild type, NIA and G3A peptides bound CR and
exhibited the characteristic green/gold birefiingence (see FIGS.
6g, 6a and 6e for normal field and FIGS. 6h, 6b and 6f for
polarized light microscopy, respectively). Peptides I5A and L6A,
bound CR and exhibited rare but characteristic birefringence (FIGS.
6i and 6k for normal field and FIGS. 6j and 6l for polarized light,
respectively). Peptide F2A (NAGAIL) showed no capability of binding
CR (FIG. 6c for normal field and FIG. 6d for polarized light).
Dried buffer solution stained with CR was used as a negative
control (see FIGS. 6m and 6n for normal and polarized light,
respectively). Interestingly, no significant difference in binding
was observed for the negative control and the F2A peptide.
[0291] 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
[0292] The fibrillogenic potential of the various peptides was
assessed by electron microscopy analysis.
[0293] 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 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.
[0294] 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. 7a-f)
but F2A which generated amorphous fibrils (FIG. 7b). Frequency of
appearance of fibrils formed by the I5A and L6A peptides (FIGS. 7e
and 7f, respectively) was lower in comparison to that of wild type
(FIG. 7d), N1A, and G3A peptides (FIGS. 7a and 7c, 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.
[0295] 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.
[0296] 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
Mapping Recognition Domains in the hIAPP Basic Amyloidogenic
Unit--Rational and MBP-IAPP Fusion Protein Synthesis
[0297] To systematically map and compare potential recognition
domains, the ability of hIAPP (GenBank Accession No. gi:4557655) to
interact with an array of 28 membrane-spotted overlapping peptides
that span the entire sequence of HIAPP (i.e., hIAPP.sub.1-10,
hIAPP.sub.2-11 . . . , hIAPP.sub.28-37) was addressed [Mazor (2002)
J. Mol. Biol. 322:1013-24].
[0298] Materials and Experimental Procedures
[0299] 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.
[0300] Engineering synthetic IAPP and MBP-IAPP fusion proteins--A
synthetic DNA sequence of human IAPP modified to include a
bacterial codon usage (SEQ ID NO: 58) was generated by annealing 8
overlapping primers (SEQ ID NOs. 50-57). PCR was effected through
30 cycles of 1 minute at 95.degree. C., one minute at 55.degree.
C., and one minute at 72.degree. C. The annealing product was
ligated and amplified using primers IAPP1 (SEQ ID NO: 50) and IAPP8
(SEQ ID NO: 57). An MBP-IAPP (MBP GenBank Accession No. gi:2654021)
fusion sequence was then constructed using the IAPP synthetic
template, which was amplified using primer YAR2 (SEQ ID NO: 60) and
primer YAR1 (SEQ ID NO. 59), thereby introducing a V8 Ek cleavage
site and a (His).sub.6 tag at the N-terminus of IAPP. 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.sup.9 into pMALc2x (New England Biolabs, USA) [BACH (2001)
J. Mol. Biol. 312:79-93].
[0301] Protein expression and purification--E. coli BL21 cells
transformed with expression plasmid pMALc2x-IAPP encoding MBP-IAPP
under the strong Ptac promoter were grown in 200 ml of LB medium
supplemented with 100 .mu.g/ml ampicillin and 1% (W/V) glucose.
Once reaching an optical density of A.sub.600=0.8, protein
expression was induced with 0.1 or 0.5 mM IPTG at 30.degree. C. for
3 hours (h).
[0302] Cell extracts were prepared in 20 mM Tric-HCI (pH 7.4), 1 mM
EDTA, 200 mM NaCi 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.degree. 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.
Purified MBP-IAPP was stored at 420 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 SDS12% polyacrylamide gels, which were stained with
GelCode Blue (Pierce, USA).
[0303] To study whether the disulfide bond in the MBP-IAPP are
oxidized, purified 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.
Only small fluorescence labeling was detected (on average less than
0.1 probe molecules per protein molecules) and there was no
significant difference between the labeling of MBP and MBP-IAPP,
which suggested that the disulfide bridge in the expressed IAPP
molecules was predominantly oxidized.
[0304] Results
[0305] Expression and purifwation of recombinant MBP-IAPP--Since
previous attempts to express the intact hIAPP in bacteria were
unsuccessful, the protein was expressed as an MBP fusion, which
protected hIAPP from undesirable aggregation S during expression
[Bach (2001) J. Mol. Biol. 312:79-93]. Synthesis of the fusion
protein was effected using a bacterial codon usage as shown in FIG.
8a. The resulting fusion sequence was cloned into pMALc2x-NN as
shown in FIG. 8b and introduced into E. coli BL21(DE3). Growth
conditions, cell extract preparation and protein purification were
effected as described hereinabove. IPTG induction resulted in the
accumulation of high levels of MBP-IAPP in the soluble fraction
with less then 5% of the MBP-IAPP fusion protein was found in the
insoluble fraction of the cell extract (data not shown). Aliquots
from typical purification steps of MBP and MBP-IAPP are shown in
FIG. 9. As shown, the 48 kDa MBP-IAPP accumulated to 25% of the
total soluble protein as calculated by densitometric scanning of
GelCode Blue-stained SDS/Polyacrylamide gels. When induced at
30.degree. C. in a shake flask (A.sub.600=2.0), MBP-IAPP
accumulated as soluble protein in the cytoplasm at a level of about
150 mg/l of cell culture. Despite losses during purification,
MBP-IAPP was purified to near-homogeneity at a yield of 80 mg/l of
cells. For future application and convenient homogeneity
purification of IAPP, in addition to the factor Xa cleavage site
for removal of the MBP tag, an additional His-Tag was also included
(FIG. 8b). The His-Tag could be removed by Ek V8 cleavage at the
N-terminal Lys residue of the IAPP sequence, resulting in the
release of wild type IAPP.
Example 7
Identification of Molecular Recognition Sequences in the hIAPP
Polypeptide
[0306] IAPP Peptide Array Construction--Decamers Corresponding to
Consecutive overlapping sequences of hIAPP.sub.1-37 SEQ ID 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,
Maidstone, England) via the C-terminal amino-acids. N-terminal
acetylation was used for peptide scanning because of higher
stability to peptide degradation, and better representation of the
native recognition motif.
[0307] Peptides Synthesis--Peptide synthesis was effected using
solid-phase synthesis methods performed by Peptron, Inc. (Taejeon,
Korea). Correct identity of the 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.sub.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.
[0308] Binding studies--The cellulose peptide array was initially
blocked with 5% (V/V) non fat milk in Tris buffered saline (TBS, 20
mM Tris pH 7.5, 150 mM NaCI). Thereafter, cellulose membrane was
incubated in the presence of 10 .mu.g/ml MBP-IAPP.sub.1-37 at
4.degree. C. for 12 h in the same blocking buffer. The cellulose
membrane was then washed repeatedly with 0.05% Tween 20 in TBS.
MBP-IAPP.sub.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. Immunoblots were developed using the
Renaissance western blot Chemiluminescence Reagent (NEN, USA)
according to Manufacturer's instructions and signal was quantified
using densitometry. Regeneration of the cellulose membrane for
reuse was carried out by sequential washing with Regeneration
buffer I including 62.5 mM Tris, 2% SDS, 100 Mm 2-mercaptoethanol,
pH 6.7, and Regeneration buffer II including 8 M urea, 1% SDS, 0.1%
2-mercaptoethanol. Efficiency of the washing steps was monitored by
contacting the membrane with the chemiluminescence reagent, as
described.
[0309] Results
[0310] Identification of binding sequences in the IAPP
polypeptide--To identify structural motifs in the IAPP molecule
that mediates the intermolecular recognition between hIAPP
molecules, 28 possible overlapping decamers corresponding to amino
acids 1-10 up to 28-37 of the hIAPP.sub.1-37 molecule were
synthesized on a cellulose membrane matrix. Cellulose
membrane-bound peptides were incubated with MBP-hIAPP.sub.1-37
overnight. Following washing of the cellulose membrane in a
high-salt buffer, immunoblots on the cellulose membrane were
analyzed and binding was quantified by densitometry (FIG. 10b). It
will be appreciated that the measured binding is semiquantitative,
since peptide coupling efficiency during synthesis can vary.
[0311] As shown in FIGS. 10a-b, a number of peptide segments
exhibited binding to MBP-IAPP; An amino acid sequence localized to
the center of the IAPP polypeptide (i.e., hIAPP.sub.7-16 to
hIAPP.sub.12-21) displayed the most prominent binding to
MBP-hIAPP.sub.1-37. Another binding region was identified at the
C-terminal part of IAPP (hIAPP.sub.19-28 to hIAPP.sub.21-30),
although binding in this case was considerably less prominent; A
third binding spot was located to the N-terminal part of IAPP
(hIAPP.sub.2-11), however, no typical distribution around a central
motif was evident in this case, suggesting that this result may be
false. Even after overexposure of the blot (data not shown), no
binding near this peptide (to either hIAPP.sub.1-10 or
hIAPP.sub.3-12) was detected. Furthermore given the close proximity
of the 2-11 region to the disulfide bridge may not allow the
process of fibrillization under physiological conditions.
[0312] To rule out involvement of MBP itself in binding the arrayed
peptides, the peptide coupled cellulose membrane was incubated with
MBP alone and analyzed by immunobloting. No binding was identified
after development of the membrane (not shown).
[0313] These results identified in addition to the previously
defined binding motif of hIAPP [i.e., basic amyloidogenic unit,
hIAPP20-29, Westermark (1990) Proc. Natl. Acad. Sci. 13:5036-40;
Tenidis (2000) J. Mol. Biol. 295:1055-1071; Azriel and Gazit (2001)
J. Biol. Chem. 276:34156-34161], a major central domain of
molecular recognition within hIAPP. The profile of the binding
distribution of the peptide array (FIG. 10b), suggests that NFVLH
(SEQ ID No. 17) may serve as the core recognition motif.
Example 8
Characterization of Aggregation Kinetics of hIAPP Peptide Fragments
as Monitored by Turbidity Measurements
[0314] Binding analysis of the recombinant MBP-hIAPP fusion protein
to the hIAPP peptide array (Example 7), identified a putative
self-assembly domain within the central part of the hIAPP
protein.
[0315] In order to identify the minimal structural motif that is
capable of forming amyloid fibrils, a series of peptides
encompassed within the putative self-assembly domain were tested
for aggregation as monitored using turbidity measurements at 405
nm.
[0316] Table 3 below, illustrates the examined peptides.
TABLE-US-00003 TABLE 3 HIAPP peptide fragments Peptide (hIAPP
coordinates) SEQ ID NO: sequence 14-22 14 NFLVHSSNN 14-20 15
NFLVHSS 15-20 16 FLVHSS 14-18 17 NFLVH 15-19 18 FLVHS 15-18 19
FLVH
[0317] Materials and Experimental Procedures
[0318] 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 .mu.L of peptide stock solutions were
added to 92 .mu.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 Calif.).
[0319] Results
[0320] 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 25
solution and turbidity, as a hallmark of protein aggregation, was
monitored for two hours. As shown in FIG. 11, the peptides NFLVHSS,
FLVHSS and FLVHS exhibited high turbidity. It will be appreciated
that the lag-time, as was previously reported for amyloid formation
by the NFGAIL short peptide [Tenidis (2000) Supra], is very short
or lacking at all and thus could not be detected under these
experimental conditions, however the aggregation kinetic profiles
were similar to those obtained for the hexapeptide hIAPP.sub.22-27
(NFGAIL). On the other hand, the peptide NFLVHSSNN exhibited very
low turbidity, while NFLVH and FLVH have shown almost no turbidity
at all. Even after significantly longer incubation no significant
turbidity was observed with the latter two peptides. The lack of
amyloid fibrils formation may be due to electrostatic repulsion of
the partially charged histidine residues.
Example 9
Examination of hIAPP peptide Amyloidogenic through Congo Red (CR)
Binding Assay
[0321] 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) J.
Histochem. Cytochem. 10:355-364].
[0322] Materials and Experimental Procedures
[0323] Congo Red Staining and Birefringence--A 10 .mu.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 .mu.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 .mu.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.
[0324] Results
[0325] Congo Red Staining and Birefringence--In order to determine
any possible amyloidal nature of the aggregates formed at the
turbidity assay (see Example 8), a CR birefringence assay was
performed. Peptide fragments were tested for amyloidogenecity by
staining with CR and examination under a light microscope equipped
with cross-polarizers. Consistent with the kinetic assay results,
and as shown in FIGS. 120b-c and 12e, the peptides NFLVHSS, FLVHSS
and FLVHS exhibited a typical birefringence. On the other hand,
peptides NFLVHSSNN, NFLVH and FLVH exhibited very weak
birefringence or no birefringence at all (FIGS. 12a, 12d and 12f).
Peptide NFLVHSSNN exhibited a weaker characteristic birefringence
(FIG. 12a). T he peptide NFLVH exhibited a powerful smear of
birefringence at the edges of the sample (FIG. 12d). The peptide
FLVH exhibited no birefringence (FIG. 12f). In order to test
whether the FLVH peptide did not form amyloid fibrils due to a long
lag-time, a sample of five days aged peptide solution was examined.
The same peptide was also tested in aqueous solution and at very
high concentrations (10 mg/ml), however no Birefringence was
detected in all cases indicating the peptide did not form amyloid
(data not shown).
Example 10
Ultrastructural Analysis of the Fibrillogenic hIAPP Peptides
[0326] The fibrillogenic potential of the various peptides was
assessed by electron microscopy analysis.
[0327] Materials and Experimental Procedures
[0328] Transmission Electron Microscopy--A 10 .mu.L sample of 8
mg/ml peptide solution in 10 mM Tris buffer, pH 7.2 aged for at
least one day was placed on 400-mesh copper grids (SPI supplies,
West Chester Pa.) covered by carbon-stabilized Formvar film.
Following 1 minute, excess fluid was removed, and the grid was then
negatively stained with 2% uranyl acetate in water for another two
minutes. Samples were viewed in a JEOL I200EX electron microscope
operating at 80 kV.
[0329] Results
[0330] To further characterize the structures formed by the various
peptides, negative staining electron microscopy analysis was
effected. In accordance with previous results, all peptide
fragments exhibited fibrillar structures except the FLVH peptide in
which only amorphous aggregates were found (FIGS. 13a-f). NFLVHSSNN
peptide exhibited long thin coiling filaments similar to those
formed by the full-length peptide as described above (FIG. 13a).
Peptides NFLVHSS, FLVHSS, FLVHS exhibited large broad ribbon-like
fibrils as described for the NFGAIL fragment [Tenidis (2000)
Supra., FIGS. 13c-e, respectively]. The fibrils formed by NFLVH
peptide were thin and short and could be considered as
protofilaments rather than filaments. Their appearance was at much
lower frequency, and the EM picture does not represent the general
fields but rather rare events (FIG. 13d). As shown in FIG. 13f, the
FLVH peptide mediated the formation of amorphous aggregates.
Example 11
Secondary Structure Analysis of hlAPP Peptide Fragments
[0331] Fourier transform infrared spectroscopy (FT-IR) was effected
to determine the secondary structure of the hIAPP amyloidogenic
peptide fibrils and the non-fibrillar peptides.
[0332] Materials and Experimental Procedures
[0333] Fourier Transform Infrared Spectroscopy--Infrared spectra
were recorded using a Nicolet Nexus 470 Fr-IR spectrometer with a
DTGS detector. Samples of aged peptide solutions, taken from
turbidity assay, were suspended on a CaF.sub.2 widows (Sigma)-plate
and dried by vacuum. The peptide deposits were resuspended with
double-distilled water and subsequently dried to form thin films.
The resuspension procedure was repeated twice to ensure maximal
hydrogen to deuterium exchange. The measurements were taken using a
4 cm.sup.-1 resolution and 2000 scans averaging. The transmittance
minima values were determined by the OMNIC analysis program
(Nicolet).
[0334] Results
[0335] FT-IR studies--As shown in FIG. 14a-f, all the fibrillar
peptides exhibited FT-IR spectra with a well-defined minimum bands
typical for .beta.-sheet structure around 1620-1640 cm.sup.-1. On
the other hand the spectrum of the tetrapeptide FLVH that has no
appearance for fibrils according to the other methods, is typical
for a random coil structure. The NFLVHSSNN peptide spectrum
exhibited a transmittance minimum at 1621 cm.sup.-1 indicating a
large .beta.-sheet content, as well as minima at 1640 cm.sup.-1 and
1665 suggesting presence of non- .beta.structures. Another minor
minimum was observed at 1688 cm.sup.-1 indicative for anti-parallel
.beta.-sheet (FIG. 14a). The NFLVHSS peptide spectrum exhibited
major minimum band at 1929 cm.sup.-1675 cm.sup.-1. this spectrum is
classical for an anti-parallel .beta.-sheet structure (FIG. 14b). A
similar spectrum was observed for the peptide FLVHS with a major
minimum at 1625 cm.sup.-1. and a minor mininum at 1676 cm.sup.-1
(FIG. 14e). The spectrum of FLVHSS peptide showed also a major
minimum at 1626 cm.sup.-1. The spectrum had also some minor minima
around 1637-1676 cm.sup.-1 but those were shaped more like noise
than signal (FIG. 14c). The spectrum of NFLVH peptide showed a
minimum at 1636 cm.sup.-1 which was also indicative of
.beta.-sheet, however, in comparison with the other spectra, this
band was shifted which could indicate presence of non- .beta.
structures, as well as observed minima at 1654 cm.sup.-1 and 1669
cm.sup.-1 (FIG. 14d). By contrast, the FLVH peptide spectrum
exhibited no minimum at 1620-1640 cm.sup.-1, but showed multiple
minima around 1646-1675 cm.sup.-1 typical to random coil structure
(FIG. 14f).
[0336] To study whether the FLVH tetrapeptide could not form
amyloid fibrils at all or the undetectable fibrils formation was a
result of a slow kinetics, a solution of the peptide at the same
experimental conditions was incubated for two months and the
existence of fibrils was tested. However, no evidence for amyloid
fibril formation was detected using EM microscopy, CR staining, or
FT-IR spectroscopy. These results may suggest that tetrapeptides
are incapable of forming fibrils due to energetic consideration.
That is, the energetic contribution of the stacking of a strand
composed of three peptide bonds is lower than the entropic cost of
oligomerization.
[0337] Taken together, the ultrastructural observations are
consistent with the findings as determined by the turbidity and
Congo red birefiingence assays. All together the experimental data
identified a novel pentapeptide element within the hIAPP peptide,
the FLVHS peptides, which has strong amyloid forming capability.
Interestingly, an NFLVH peptide found in the same central domain of
the hIAPP polypeptide was found to be amyloidogenic however, the
ability thereof to form fibrils was somehow inferior.
Example 12
Identification of the Minimal Amyloidogenic Peptide Fragment of
Medin Background
[0338] 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. It
was suggested that these amyloid play a role in the diminished
elasticity of aortic vessels that is related to old age [Mucchiano
(1992) Supra; Haggqvist (1999) Supra]. While the study clearly
identified a tryptic peptide NFGSVQFV as the medin amyloidogenic
peptide, the minimal sequence of the peptide that is still
amyloidogenic and the molecular determinants that mediate the
amyloid formation process were not determined. Such information is
critical for true understanding of the fibrillization process in
the specific case of Medin but also as a paradigm for the process
of amyloid fibrils formation in general.
[0339] The minimal active fragment of Medin was determined using
functional and structural analyses of truncated analogues derived
from the published octapeptide [Haggqvist (1999) Supra].
[0340] Materials and Experimental procedures
[0341] Peptide synthesis is described in Example 7.
[0342] Table 4 below illustrates the studied peptides.
TABLE-US-00004 TABLE 4 Peptide sequence SEQ ID NO:
NH.sub.2-NFGSVQVF --COOH 20 NH.sub.2-NFGSVQ --COOH 21
NH.sub.2-NFGSV --COOH 22 NH.sub.2- FGSVQ --COOH 23 NH.sub.2- GSVQ
--COOH 24 NH.sub.2- FGSV --COOH 25 NH.sub.2-NAGSVQ --COOH 26
[0343] Results
[0344] In order to get further insights into the structural
elements of Medin that retain the molecular information needed to
mediate a process of molecular recognition and self-assembly, the
ability of short peptide fragments and analogues of Medin to form
amyloid fibrils in vitro was studied. FIG. 15a shows a schematic
representation of the chemical structure of the largest peptide
fragment studied.
Example 13
Kinetics of Aggregation of Medin-derived Peptide Fragments
[0345] Turbidity assay was effected as described in Example 8.
[0346] In order to get first insights regarding the aggregation
potential of the various Medin derived peptides, turbidity assay
was performed. Freshly made stocks of the amyloidogenic octapeptide
and truncated analogues thereof were prepared in DMSO. The peptides
were than diluted to aqueous solution and the turbidity was
monitored by following the absorbance at 405 nm as a function of
time. As shown in FIG. 16a, the NFGSV pentapeptide exhibited the
highest degree of aggregation within minutes of incubation.
Physical examination of the solution indicated that the peptide
formed a gel structure. The kinetics of aggregation of the NFGSVQV
octapeptide was too fast to be measured since turbidity was already
observed immediately with the dilution into aqueous solution (FIGS.
16a-b). Similar fast kinetics were also observed with the GSVQ
tetrapeptide. The truncated NFGSVQ, FGSVQ, and FGSV peptides showed
a gradual increase in turbidity over .about.30 minutes (FIG. 16b)
which was followed by a slight decrease, which could be explained
by sedimentation of large aggregates. Altogether, such kinetics and
turbidity values were similar to those previously observed with
amyloidogenic peptides of similar size (Azriel and Gazit,
2001).
Example 14
Ultrastructural Analysis of Medin-derived Peptide Fragments
[0347] Electron microscopy analysis was effected as described in
Example 10.
[0348] The fibrillization potential of Medin-derived peptide
fragments was effected by electron microscopy (EM) using negative
staining. Stock solutions of the peptide fragments were suspended
and aged for 4 days. Fibrillar structures were clearly seen in
solutions that contained both the NFGSVQFA octapeptide (FIG. 17a)
and the truncated NFGSVQ (FIG. 17b ). In both cases the structures
were similar to those observed with much longer polypeptides, such
the IAPP and the .beta.-amyloid (A.beta.) polypeptides. The shorter
gel-forming NFGSV pentapeptide did not form a typical amyloid
structure but a network of fibrous structures (FIG. 17c). It should
be noted that fibrous networks were recently observed upon the
gelation of the glutathione peptide [Lyon and Atkins, (2001) J. Am.
Chem. Soc. 123:4408-4413]. No typical fibrils could be detected in
solutions that contained the FGSVQ pentapeptide, the GSVQ
tetrapeptide, or the FGSV tetrapeptide in spite of extensive
search. While in the case of the FGSVQ peptide (FIG. 17d) somewhat
fibrillar and ordered structure could be seen, although
significantly different than those formed by typical amyloidogenic
peptide), in the case of the GSVQ and the FGSV peptides, no
fibrillar structures could be found (FIGS. 17e and 17f,
respectively).
Example 15
Examination of Amyloidogenic Performance of Medin-derived Peptides
Through Congo Red (CR) Binding Assay
[0349] CR staining was effected as described in Example 9.
[0350] A CR staining was effected to determine whether the
structures formed by the various Medin-derived peptides show a
typical birefringence. As shown in FIG. 18b, the NFGSVQ hexapeptide
bound CR and exhibited a characteristic bright and strong
green-gold birefringence. The NFGSVQFV octapeptide also exhibited
significant birefringence (FIG. 18a), although less typical than
that observed with the hexapeptide. The gel-forming NFGSV peptide
deposits exhibited very low degree of birefringence (FIG. 18c). The
FGSVQ and FGSV peptide showed no birefringence upon staining with
CR (FIGS. 18d and 18f, respectively). There was clearly no
significant difference between those two peptides and a negative
control (i.e., buffer solution with no peptide) Interestingly,
unexpected high level of birefringence was observed with the GSVQ
tetrapeptide (FIG. 18e), while the morphology of the structures
formed therefrom (FIG. 18e) was clearly different from that of
amyloid fibrils, indicating that these structures may have a
significant degree of order that is reflected in strong
birefringence.
Example 16
The Effect of Phenylalanine Substitution on the Self-assembly of
Medin
[0351] 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 FIG. 19a, a significantly lower
turbidity was observed with the alanine-substituted peptide as
compared to the wild-type hexapeptide. When aged solution of the
NAGSVQ peptide was visualized by EM, no clear fibrillar structures
could be detected (FIG. 19b). This is in complete contrast to the
high abundance fibrillar structures seen with the wild-type peptide
(FIG. 17b). Furthermore, the structures that were visualized did
not show any degree of order as observed with the NFGSV and FGSVQ
peptides as described above, FIGS. 17c-d, but were very similar to
the completely non-fibrillar structures as were observed with the
FGSV tetrapeptide (FIG. 17e). Interestingly, some degree of
birefringence could still be detected (FIG. 19c) with the
alanine-substituted peptide (as was observed with the GSVQ peptide,
FIG. 18e). These results raise further doubts regarding the use of
CR staining as a sole indicator of amyloid formation [Khurana
(2001) J. Biol. Chem. 276:22715-22721].
[0352] Altogether these results show that the truncated fragment of
Medin which is capable of forming amyloid fibrils is the
hexapeptide NFGSVQ (SEQ ID NO: 21), although a shorter pentapeptide
fragment, NFGSV (SEQ ID NO: 22), exhibited a network of fibrous
structures which were not typical of amyloids. The amyloid forming
NFGSVQ hexapeptide is noticeably similar to the minimal
amyloidogenic fragment of the islet amyloid polypeptide (IAPP, see
Examples 1-5). Taken together, the results are consistent with the
assumed role of stacking interactions in the self-assembly
processes that lead to the formation of amyloid fibrils and the
suggested correlation between amyloid fibrils and .beta.-helix
structures.
Example 17
[0353] Identification of the Minimal Amyloidogenic Peptide Fragment
of Human Calcitonin
[0354] 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 30:655-6631.
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.
412:543-551; Arvinte (1993) J. Biol. Chem. 268:6415-6422].
Interestingly, synthetic hCT was found to form amyloid fibrils in
vitro with similar morphology to the deposits found in the thyroid
[Kedar (1976) Supra; Berger (1988) Supra; Arvinte (1993) Supra;
Benvenga (1994) J. Endocrinol. Invest. 17:119-122; Bauer (1994)
Biochemistry 33:12276-12282; Kanaori (1995) Biochemistry
34:1213843; Kamihara (2000) Protein Sci. 9:867-877]. The in vitro
process of amyloid formation is affected by the pH of the medium
[23]. Electron microscopy experiments have revealed that the
fibrils formed by hCT are approximately 80 .ANG. in diameter and up
to several micrometers in length. The fibrils are often associated
with one another and in vitro amyloid formation is affected by the
pH of the medium [Kamihara (2000) Supra.].
[0355] Calcitonin has been used as a drug for various diseases
including Paget's disease and osteoporosis. However, the tendency
of hCT to associate and form amyloid fibrils in aqueous solutions
at physiological pH is a significant limit for its efficient use as
a drug [Austin (1981) Supra; Copp (1970) Supra; Zaidi (2002)
Supra]. Salmon CT [Zaidi (2002) Supra], the clinically used
alternative to hCT, causes immunogenic reaction in treated patients
due to low sequence homology. Therefore, understanding the
mechanism of amyloid formation by hCT and controlling this process
is highly important not only in the context of amyloid formation
mechanism but also as a step toward improved therapeutic use of
Calcitonin.
[0356] 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 a-helical and
.beta.-sheet secondary structure components [Bauer (1994) Supra].
NMR spectroscopy studies have shown that in various structure
promoting solvents like TFE/H.sub.2O, hCT adopts an amphiphilic
.alpha.-helical conformation, predominantly in the residue range
8-22 [Meadows (1991) Biochemistry 30:1247-1254; Motta (1991)
Biochemistry 30:10444-10450]. In DMSO/H.sub.2O, a short
double-stranded antiparallel .beta.-sheet form in the central
region made by residues 16-21 [Motta (1991) Biochemistry
30:2364-71].
[0357] Based on this structural data and the proposed role of
aromatic residues in the process of amyloid formation, the present
inventor has identified a short peptide fragment, which is
sufficient for mediating Calcitonin self-assembly [Reches (2002) J.
Biol. Chem. 277:35475-80].
[0358] The studied peptides--Based on the previously reported
susceptibility of amyloid formation to acidic pH [Kanaori (1995)
Supra], it was suggested that negatively-charged amino-acids, which
undergo protonation at low pH, may play a key role in the process
of amyloid formation. The only negatively-charged amino-acid in hCT
is Asp.sup.15 (FIG. 20a). Furthermore, a critical role for residues
Lys.sup.18 and Phe.sup.9 in the oligomerization state and
bioactivity of hCT was recently shown [Kazantzis (269) Eur. J.
Biochem. 269:780-91]. Together with the occurrence of two
phenylalanine residues in the region focused the structural
analysis of the amyloidogenic determinants in hCT to amino acids
15-19. FIG. 20b shows a schematic representation of the chemical
structure of the longest peptide and Table 5 below, indicates the
various peptide fragments that were used in the study.
TABLE-US-00005 TABLE 5 Amino acid coordinates on hCT Peptide
sequence SEQ ID NO: 15-19 NH.sub.2-DFNKF --COOH 27 16-19 NH.sub.2-
FNKF --COOH 28 15-18 NH.sub.2-DFNK --COOH 29 15-17 NH.sub.2-DFN
--COOH 30 F > A 15-19 NH.sub.2-DANKF --COOH 31
Example 18
Ultrastructural Analysis of Calcitonin-derived Peptide
Fragments
[0359] Electron microscopy analysis was effected as described in
Example 10.
[0360] The fibrillization potential of Calcitonin-derived peptide
fragments was effected by electron microscopy (EM) using negative
staining. Stock solutions of the peptide fragments were suspended
in 0.02M NaCl, 0.01M Tris pH 7.2, aged for 2 days and negatively
stained. Fibrillar structures, similar to those formed by the
full-length polypeptide [Arvinte (1993) Supra; Benvenga (1994)
Supra; Bauer (1994) Supra; Kanaori (1995) Supra; Kamihara (2000)
Supra], were clearly seen with high frequency in solutions that
contained the DFNKF pentapeptide (FIG. 21a). The shorter DFNK
tetrapeptide also formed fibrillar structures (FIG. 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 amorphous aggregates could be found (FIG. 21c). The DFN
tripeptide formed more ordered structures (FIG. 21d) that resembled
the structure formed by gel-forming tripeptide [Lyon (2001) Supra].
To study whether the FNKF tetrapeptide and the DFN tripeptide
peptide cannot form fibrils whatsoever or the observation is a
result of slow kinetics, a solution of the peptides at the same
experimental conditions was incubated for two weeks. Also in this
case no clear fibrillar structures could be detected (data not
shown).
Example 19
Examination of Amyloidogenic Performance of Calcitonin-derived
Peptides through Congo Red (CR) Binding Assay
[0361] CR staining was effected as described in Example 9.
[0362] A CR staining was effected to determine whether the
structures formed by the various hCT-derived peptides show a
typical birefringence. As shown in FIGS. 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 (FIG. 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. The lower level of birefringence of the
DFNK tetrapeptide (FIG. 22b) was consistent with the lower extent
of fibrillization as observed using EM (FIG. 21b). It will be
appreciated, though, that the birefringence observed with the FNKF
tetrapeptide and the DFN tripeptide might represent some degree of
ordered structures [Lyon (2001) Supra].
Example 20
Secondary Structure of the Aggregated hCT-derived Peptides
[0363] FT-IR spectroscopy was effected as described in Example
11.
[0364] Amyloid deposits are characteristic of fibrils rich with
.beta.-pleated sheet structures. To get a quantitative information
regarding the secondary structures that were formed by the various
peptide fragments FT-IR spectroscopy was used. Aged peptide
solutions were dried on CaF2 plates forming thin films as described
in Example 11. As shown in FIG. 23, the DFNKF pentapeptide
exhibited a double minima (at 1639 cm.sup.-1 and 1669 cm.sup.-) an
amide I FT-IR spectrum that is consistent with anti-parallel
.beta.-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 (FIG. 23) was less typical of a
.beta.-sheet structure. While it exhibited a minimum at 1666 cm-1
that may reflect an anti-parallel .beta.-sheet it lacked the
typical minimum around 1620-1640 cm.sup.-1 that is typically
observed with .beta.-sheet structures. The FNKF tetrapeptide
exhibited a FT-IR spectrum that is typical of a non-ordered
structure (FIG. 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.sup.-1 and 1673 cm.sup.-1, FIG. 23) amide I
FT-IR spectrum that is consistent with a mixture of .beta.-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 .beta.-sheet
structural elements.
Example 21
The Effect of Phenylalanine Substitution on the Self-assembly of
Calcitonin-derived Peptides
[0365] In order to get insight into a possible role for the
phenylalanine residues in the process of Calcitonin self-assembly,
the phenylalanine amino acids were replaced with alanine in the
context of the pentapeptide (SEQ ID NO: 31). When aged solution of
the DANKF pentapeptide was visualized by EM, no clear fibrillar
structures could be detected (FIG. 24a). Structures that were
visualized exhibited some degree of order (as compared to the
amorphous aggregates seen with the FNKF tetrapeptide), however, no
green-gold birefringence could be observed (FIG. 24b). The FT-IR
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]. Taken together, the
effect of the phenylalanine to alanine substitution is very similar
to the effect of such a change in the context of a short
amyloid-forming fragment of the islet amyloid polypeptide [Azriel
(2001) Supra].
[0366] Altogether, the ability of an hCT-derived pentapeptide (SEQ
ID NO: 27) to form well-ordered amyloid fibrils was demonstrated.
The typical fibrillar structure as seen by electron microscopy
visualization (FIG. 21a), the very strong green birefringence upon
staining with CR (FIG. 22a), and the typical anti-parallel
.beta.-sheet structure (FIG. 23a), all indicate that the DFNKF
pentapeptide is a very potent amyloid forming agent. Other
pentapeptides capable of self-assembling were shown in hereinabove.
Yet, in terms of the degree of birefringence and electron
microscopy morphology, the hCT fragment seems to be the
pentapeptide with the highest amyloidogenic potential similar to
the potent amyloidogenic fragment of the .beta.-amyloid (A.beta.)
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 IAPP, NFLVH and
FLVHS, can form amyloid fibrils, but their common denominator, the
tetrapeptide FLVH, could not form such fibrils (see Examples
1-5).
Example 22
[0367] Identification of an Amyloidogenic Peptide from
Lactotransferrin
[0368] 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
Lactotransfenin-derived peptide, LFNQTG (SEQ ID NO: 32) were
studied.
[0369] Materials and Experimental Procedures--Described in Examples
7 and 10.
[0370] Results--To characterize the ability of the
Lactotransferrin-derived peptide to form fibrilar supramolecular
ultrastructures, negative staining electron microscopy analysis was
effected. As shown in FIG. 25, under mild conditions, filamentous
structures were observed for the selected peptide, suggesting that
LFNQTG of Lactotransferrin is important for the polypeptide
self-assembly. These results further substantiate the ability of
the present invention to predict amyloidogenic peptide
sequences.
Example 23
Identification of an Amyloidogenic peptide from Serum Amyloid A
Protein
[0371] Fragments of Serum amyloid A proteins (GenBank Accession No.
gi:134167) were found in amyloid-state in cases of Chronic
inflammation amyloidosis (Westermark et al. (1992) Biochem.
Biophys. Res. Commun. 182: 27-33). Based on the proposed role of
aromatic residues in amyloid self-assembly, the amyloidogenic
features of a Serum amyloid A protein-derived peptide, SFFSFL (SEQ
ID NO: 33) were studied.
[0372] Materials and Experimental Procedures--Described in Examples
7 and 10.
[0373] Results--To characterize the ability of the Serum amyloid A
protein-derived peptide to form fibrilar supramolecular
ultrastructures, negative staining electron microscopy analysis was
effected. As shown in FIG. 26, under mild conditions, filamentous
structures were observed for the selected peptide, suggesting that
SFFSFL of serum amyloid A protein is important for the polypeptide
self-assembly. These results further substantiate the ability of
the present invention to predict amyloidogenic peptide
sequences.
Example 24
Identification of an Amyloidogenic Peptide from BriL
[0374] The human BRI gene is located on chromosome 13. The amyloid
fibrils of the BriL gene product (GenBank Accession No.
gi:12643343) are associated with neuronal dysfunction and dementia
(Vidal et al (1999) Nature 399, 776-781). Based on the proposed
role of aromatic residues in amyloid self-assembly, the
amyloidogenic features of a BriL-derived peptide, FENKF (SEQ ID NO:
34) were studied.
[0375] Materials and Experimental Procedures--Described in Examples
7 and 10.
[0376] Results--To characterize the ability of the BriL-derived
peptide to form fibrilar supramolecular ultrastructures, negative
staining electron microscopy analysis was effected. As shown in
FIG. 27, under mild conditions, filamentous structures were
observed for the selected peptide, suggesting that FENKF of BriL is
important for the polypeptide self-assembly. These results further
substantiate the ability of the present invention to predict
amyloidogenic peptide sequences.
Example 25
Identifcation of an Amyloidogenic Peptide from Gelsolin
[0377] Fragments of Gelsolin proteins (GenBank Accession No.
gi:4504165) were found in amyloid-state in cases of Finnish
hereditary amyloidosis [Maury and Nurmiaho-Lassila (1992) Biochem.
Biophys. Res. Commun. 183: 227-31]. Based on the proposed role of
aromatic residues in amyloid self-assembly, the amyloidogenic
features of a Gelsolin-derived peptide, SFNNG (SEQ ID NO: 35) were
studied.
[0378] Materials and Experimental Procedures--Described in Examples
7 and 10.
[0379] Results--To characterize the ability of the Gelsolin-derived
peptide to form fibrilar supramolecular ultrastructures, negative
staining electron microscopy analysis was effected. As shown in
FIG. 28, under mild conditions, filamentous structures were
observed for the selected peptide, suggesting that SFNNG of BriL is
important for the polypeptide self-assembly. These results further
substantiate the ability of the present invention to predict
amyloidogenic peptide sequences.
Example 26
Identification of an Amyloidogenic Peptide from Serum amyloid P
[0380] 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 ID NO: 36) were studied.
[0381] Materials and Experimental Procedures--Described in Examples
7 and 10.
[0382] Results--To characterize the ability of the Serum amyloid
P-derived peptide to form fibrilar supramolecular ultrastructures,
negative staining electron microscopy analysis was effected. As
shown in FIG. 29, under mild conditions, filamentous structures
were observed for the selected peptide, suggesting that LQNFTL of
Serum arnyloid P is important for the polypeptide self-assembly.
These results further substantiate the ability of the present
invention to predict amyloidogenic peptide sequences.
Example 27
Identification of an Amyloidogenic Peptide from Immunoglobulin
Light Chain
[0383] 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, TLIFGG (SEQ ID NO: 37)
were studied.
[0384] Materials and Experimental Procedures--Described in Examples
7 and 10.
[0385] Results--To characterize the ability of the immunoglobulins
light chain-derived peptide to form fibrilar supramolecular
ultrastructures, negative staining electron microscopy analysis was
effected. As shown in FIG. 30, under mild conditions, filamentous
structures were observed for the selected peptide, suggesting that
TLIFGG of the immunoglobulin light chain is important for the
polypeptide self-assembly. These results further substantiate the
ability of the present invention to predict amyloidogenic peptide
sequences.
Example 28
Identification of an Amyloidogenic Peptide from Cystatin C
[0386] Amyloid fibril formation by Cystatin C (GenBank Accession
No. gi:4490944) is associated with hereditary cerebral amyloid
angiopathy [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 Cystatin
C-derived peptide, RALDFA (SEQ ID NO: 38) were studied.
[0387] Materials and Experimental Procedures--Described in Examples
7 and 10.
[0388] Results--To characterize the ability of the Cystatin
C-derived peptide to form fibrilar supramolecular ultrastructures,
negative staining electron microscopy analysis was effected. As
shown in FIG. 31, under mild conditions, filamentous structures
were observed for the selected peptide, suggesting that RALDFA of
the Cystatin C is important for the polypeptide self-assembly.
These results further substantiate the ability of the present
invention to predict amyloidogenic peptide sequences.
Example 29
Identification of an Amyloidogenic Peptide from Transthyretin
[0389] Amyloid fibril formation by Transthyretin (GenBank Accession
No. gi:72095) is associated with familial amyloid polyneuropathy
(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 Transthyretin-derived peptide,
GLVFVS (SEQ ID NO: 39) were studied.
[0390] Materials and Experimental Procedures--Described in Examples
7 and 10.
[0391] Results--To characterize the ability of the
Transthyretin-derived peptide to form fibrilar supramolecular
ultrastructures, negative staining electron microscopy analysis was
effected. As shown in FIG. 32, under mild conditions, filamentous
structures were observed for the selected peptide, suggesting that
GLVFVS of Transthyretin is important for the polypeptide
self-assembly. These results further substantiate the ability of
the present invention to predict amyloidogenic peptide
sequences.
Example 30
Identifwation of an Amyloidogenic Peptide from Lysozyme
[0392] Amyloid fibril formation by Lysozyme (GenBank Accession No.
gi:299033) is associated with familial non-neuropathic 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 Lysozyme-derived peptide, GTFQIN
(SEQ ID NO: 40) were studied.
[0393] Materials and Experimental Procedures--Described in Examples
7 and 10.
[0394] Results--To characterize the ability of the Lysozyme-derived
peptide to form fibrilar supramolecular ultrastructures, negative
staining electron microscopy analysis was effected. As shown in
FIG. 33, under mild conditions, filamentous structures were
observed for the selected peptide, suggesting that GTFQIN of
Lysozyme is important for the polypeptide self-assembly. These
results further substantiate the ability of the present invention
to predict amyloidogenic peptide sequences.
Example 31
Identification of an Amyloidogenic Peptide from Fibrinogen
[0395] Amyloid fibril formation by Fibrinogen (GenBank Accession
No. gi: 1 1761629) is associated with hereditary renal 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 Fibrinogen-derived peptide, SGIFTN
(SEQ ID NO: 41) were studied.
[0396] Materials and Experimental Procedures--Described in Examples
7 and 10.
[0397] Results--To characterize the ability of the Fibrinogen
-derived peptide to form fibrilar supramolecular ultrastructures,
negative staining electron microscopy analysis was effected. As
shown in FIG. 34, under mild conditions, filamentous structures
were observed for the selected peptide, suggesting that SGIFIN of
Fibrinogen is important for the polypeptide self-assembly. These
results further substantiate the ability of the present invention
to predict amyloidogenic peptide sequences.
Example 32
Identification of an Amyloidogenic Peptide from Insulin
[0398] Amyloid fibril formation by Insulin (GenBank Accession No.
gi:229122) is associated with injection-localized 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 amytoidogenic features of an insulin-derived peptide, ERGFF
(SEQ ID NO: 42) were studied.
[0399] Materials and Experimental Procedures--Described in Examples
7 and 10.
[0400] Results--To characterize the ability of the Insulin-derived
peptide to form fibrilar supramolecular ultrastructures, negative
staining electron microscopy analysis was effected. As shown in
FIG. 35, under mild conditions, filamentous structures were
observed for the selected peptide, suggesting that ERGFF of insulin
is important for the polypeptide self-assembly. These results
further substantiate the ability of the present invention to
predict amyloidogenic peptide sequences.
Example 33
[0401] Identification of an Amyloidogenic Peptide from
Prolactin
[0402] Amyloid fibrils formation by prolactin (GenBank Accession
No. gi:4506105) is associated with pituitary-gland 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 prolactin-derived peptide, RDFLDR
(SEQ ID NO: 43) were studied.
[0403] Materials and Experimental Procedures--Described in Examples
7 and 10.
[0404] Results--To characterize the ability of the
prolactin-derived peptide to form fibrilar supramolecular
ultrastructures, negative staining electron microscopy analysis was
effected. As shown in FIG. 36, under mild conditions, filamentous
structures were observed for the selected peptide, suggesting that
RDFLDR of prolactin is important for the polypeptide self-assembly.
These results further substantiate the ability of the present
invention to predict amyloidogenic peptide sequences.
Example 34
Identfication of an Amyloidogenic Peptide from
Beta-2-microglobulin
[0405] 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-derived peptide, SNFLN (SEQ ID NO: 44) were
studied.
[0406] Materials and Experimental Procedures--Described in Examples
7 and 10.
[0407] Results--To characterize the ability of the
beta-2-microtublin-derived peptide to form fibrilar supramolecular
ultrastructures, negative staining electron microscopy analysis was
effected. As shown in FIG. 37, under mild conditions, filamentous
structures were observed for the selected peptide, suggesting that
SNFLN of beta-2-microtublin is important for the polypeptide
self-assernbly. These results further substantiate the ability of
the present invention to predict amyloidogenic peptide
sequences.
Example 35
Inhibition of Amyloid Formation an Amyloidogenic Peptide Identified
According to the Teachings of the Present Invention
[0408] The ability of amyloidogenic peptides of IAPP, 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).
[0409] The degree of amyloid fibrils formation with and without the
inhibitor was assessed using thioflavin T (ThT) as molecular
indicator. The degree of fluorescence of the ThT dye is directly
correlated with the amount of amyloid fibrils in the solution
[LeVine H 3rd. (1993) Protein Sci. 2:404-410. IAPP solutions (4
.mu.M hIAPPin 10 mM Tris buffer pH 7.2), were incubated in the
presence or absence of 40 .mu.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
.mu.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, Mass.).
As a control 10 mM Tris buffer pH 7.2 were diluted into the ThT
solution and fluorescence was determined as described.
[0410] Result--As shown in FIG. 38, while the IAPP alone showed
high levels of ThT fluorescence as expected for amyloidogenic
protein, there was a significant increase in fluorescence in the
presence of the inhibitory peptide. Thus, these results validate
the NFLVH sequence as the amyloidogenic determinant in the IAPP
polypeptide.
Example 36
Significance of Hydrophobic Residues in Amyloid Assembly
[0411] The significance of an aromatic residue in the basic
amyloidogenic unit of IAPP has been demonstrated in Examples 1-5.
As described, substitution of a phenylalanine to an alanine
abolished the ability of an amyloidogenic fragment (NAGAIL, SEQ ID
NO: 9) to form amyloid fibrils in vitro. Based on this observation,
the remarkable occurrence of aromatic residues in other short
amyloid related sequences (Examples 12-35), and the well-known role
of .pi.-stacking in processes of self-assembly in chemistry and
biochemistry, it was suggested that stacking of aromatic residues
may play a role in the process of amyloid fibrils formation [Gazit
(2002) FASEB J. 16:77-83].
[0412] The study was further extended to indicate whether the
phenylalanine residue is critical due to aromaticity thereof, or
rather due to its hydrophobic nature. The effect of phenylalanine
substitution with hydrophobic residues on the self assembly of the
basic amyloidogenic unit of AIPP (i.e., NFGAIL peptide) was
addressed.
[0413] The list of peptides used in the study and designation
thereof is presented in Table 6, below. TABLE-US-00006 TABLE 6
Amino acid substitution and coordinates on hIAPP Peptide sequence
SEQ ID NO: WT 22-29 NH.sub.2-NFGAILSS --COOH 46 F > I 22-29
NH.sub.2-NIGAILSS --COOH 47 F > L 22-29 NH.sub.2-NLGAILSS --COOH
48 F > V 22-29 NH.sub.2-NVGAILSS --COOH 49 F > A 22-29
NH.sub.2-NAGAILSS --COOH 89
[0414] It will be appreciated that while these 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 .beta.-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 .beta.-sheet rich
amyloid fibrils.
Example 37
Characterization of the Aggregation Kinetics of Hydrophobically
Modified hIAPP Peptide Fragments as Monitored by Turbidity
Measurements
[0415] Experimental Procedures--Effected as described in Example
8.
[0416] Results
[0417] To get insight into the aggregation potential of the
hydrophobically-modified IAPP-derived peptide analogues, turbidity
assay was performed. Freshly made stock solutions of the wild-type
peptide and the various peptide mutants were made in DMSO. The
peptides were then diluted to a buffer solution and the turbidity
was monitored by following the absorbance at 405 nm as a function
of time. As shown in FIG. 39, significant increase in turbidity was
observed for the wild-type NFGAILSS octapeptide within minutes
following dilution thereof into the aqueous solution. The shape of
the aggregation curve resembled that of a saturation curve, with a
rapid increase in turbidity in the first hour, followed by a much
slower increase in turbidity over the entire incubation time
monitored. This probably reflects a rapid aggregation process, with
the number of free building blocks as the rate limiting factor. In
contrast, none of the analogue peptides revealed any significant
aggregative behavior and the turbidity of all the hydrophobic
analogues as well as the alanine-substituted analogues remained
very low for at least 24 hours (FIG. 39).
[0418] To determine whether the non-aggregative behavior of the
hydrophobic analogues is a result of extremely slow kinetics,
peptide analogue solutions were incubated for 1 week in the same
experimental conditions and endpoint turbidity values were
determined. As shown in FIG. 30, some low degree of turbidity was
observed with the NIGAILSS, and lower extent for the NLGAILSS,
NAGAILSS, and NVGAILSS peptides in decreasing order of turbidity.
However, even for the NIGAILSS, the degree of turbidity was
significantly lower as compared to the wild-type NFGAILSS protein
(FIG. 40). Moreover, there was no correlation between aggregation
potential and hydrophobicity or .beta.-sheet forming tendency,
since the lower degree of aggregation was observed with the
substitution to the highly hydrophobic and .beta.-sheet former,
valine. The slight decrease in the endpoint turbidity value of the
NFGAILSS wild-type peptide, as compared to the values obtained
after 24 hours incubation, could reflect the formation of very
large aggregates that adhere to the cuvette surface.
Example 38
Utrastructural Analysis of Hydrophobically Modified hIAPP Peptide
Fragments
[0419] Electron microscopy analysis was effected as described in
Example 10.
[0420] 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 (FIGS. 41a-b). Some amorphous aggregates
could be also seen with the modified fragments (FIGS. 41c-f).
However, those structures were significantly less abundant on the
microscope grid. Larger aggregative structures were observed with
the more hydrophobic substitutions as compared to the alanine
analogues. Yet, unlike the ordered fibrillar structures that were
seen with the NFGAILSS peptide, as mentioned above, these
aggregates were quite rare and did not have ordered structures
(FIGS. 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.
Example 39
Determination of the Specific Function of Phenylalanine in the IAPP
Self Assembly
[0421] To determine the specific role of the phenylalanine residue
in IAPP-self assembly, a membrane-based binding assay was preformed
in order to systematically explore the molecular determinants that
facilitate the ability of the full-length hIAPP to recognize the
"basic amyloidogenic unit". To this end, the ability of MBP-IAPP
(see Example 6) to interact with an array of peptides in which the
phenylalanine position was systematically altered (SEQ ID NOs.
91-110), was addressed.
[0422] Materials and Experimental Procedures--see Examples 6-7.
[0423] Results
[0424] A peptide array corresponding to the SNNXGAILSS motif (SEQ
ID NO: 90), where X is any natural amino-acid but cysteine was
constructed. As shown in FIG. 42a, binding of MBP-IAPP was clearly
observed to peptides which contained the aromatic tryptophan and
phenylalanine residues at the X position (FIG. 42a). Interestingly,
binding was also observed upon substitution of phenylalanine with
basic amino acids such as arginine and lysine. In contrast, no
binding was observed with any of the hydrophobic substitutions of
the position, even after long exposure of the membrane (FIG.
42b).
[0425] The short exposure binding was assessed using densitometry
(FIG. 42c). It will be appreciated though, that the measured
binding should be interpreted as semiquantitative since the
coupling efficiency during synthesis and therefore the amount of
peptide per spot may vary. In this case, however, the marked
difference in binding between the various peptide variants was very
clear.
[0426] Taken together, all these observations substantiate the role
of aromatic residues in the acceleration of amyloid formation
processes.
Example 40
Design and Configuration of .alpha.-aminoisobutyric Acid (Aib)
Substituted Amyloid Forming Peptides
[0427] The minimal amyloid forming region of IAPP polypeptide
(i.e., IAPP 14-20, see Table 3 above) was selected as the target
sequence for designing inhibitors which are able to bind thereto,
block it and prevent aggregation thereof.
[0428] Experimental approach
[0429] To abolish the aggregation ability of amyloid forming
peptides, .beta.-sheet breakers are incorporated into the target
sequence, such that the peptides cannot display a .beta.-sheet
conformation by which the monomers are stacked together to form
fibrils. .alpha.-aminoisobutyric acid (Aib) is an unnatural amino
acid which contains two methyl residues attached to C.sub..alpha.
of the carboxylic group. Unlike natural amino acids, this molecule
does not have a hydrogen atom attached to the C.sub..alpha.. This
affects widely the sterical properties of the amino acid especially
with respect to the .phi. and .psi. angels of the amide bond. While
alanine has a wide range of allowed .phi. and .psi. conformations,
Aib, which is a .alpha.-methylated alanine has limited .phi. and
.psi. conformations. FIG. 43a shows the conformational map of Aib
derived from the superposition of the Ramachandran plots of
L-alanine and D-alanine. As is evident for FIG. 43a, the allowed
angels are limited to small regions and the overall structure is
much more suitable for an .alpha.-helix conformation rather than a
.beta.-strand conformation.
[0430] Hence, Aib can be used to prevent .beta.-sheet conformation
which is central to amyloid aggregation. Notably, a comparison
between the Ramachandran plots of Aib and proline shows that Aib is
a more potent .beta.-sheet breaker than proline (FIG. 43a).
[0431] Two peptides including IAPP amyloid forming regions (i.e.,
ANFLVH and ANFLV, SEQ ID NOs: 124 and 126, respectively) were
synthesized to include Aib substituting the alanine and the leucine
residues. The newly synthesized peptides including the following
amino acid sequences Aib-NF-Aib-VH (SEQ ID NO: 125) and
Aib-NF-Aib-V (SEQ ID NO: 127), are illustrated in FIGS. 43b-c,
respectively.
[0432] Peptides Synthesis--Peptides were synthesized by Peptron,
Inc. (Taejeon, Korea) using solid-phase techniques. The correct
identity of the peptides was confirmed by ion spray
mass-spectrometry using a HP 1100 series LC/MSD. The purity of the
peptides was confirmed by reverse phase high-pressure liquid
chromatography (RP-HPLC) on a C.sub.18 column, using a 30 minutes
linear gradient of 0 to 100% acetonitrile in water and 0.1%
trifluoroacetic acid (TFA) at flow rate of 1 ml/min.
[0433] Peptide solutions--freshly prepared stock solutions were
prepared by dissolving the lyophilized form of the peptides in
dimethyl sulfoxide (DMSO) at a concentration of 100 mM. To avoid
any pre-aggregation, fresh stock solutions were prepared for each
experiment. Peptide stock solutions were diluted into microtubes as
follows: 5 .mu.L of peptides stock solutions added to 95 .mu.L of
10 mM Tris, pH 7.2 (hence the final concentration of the peptide
was 5 mM in the presence of 5% DMSO).
Example 41
Ultrastructural Analysis of Wild-type and Aib Modified hIAPP
Peptides
[0434] The fibrillogenic potential of the peptides described in
Example 40 above, was assessed by electron microscopy analysis.
[0435] Materials and Experimental procedures
[0436] Transmission Electron Microscopy--a 10 .mu.L sample of 5 mM
peptide solution in 10 mM Tris buffer, pH 7.2 aged for 4 days (for
wt peptides) and for 10 days (for modified peptides) was placed on
400-mesh copper grids (SPI supplies, West Chester Pa.) covered by
carbon-stabilized Fornvar film. After 1 minute, excess fluid was
removed, and the grid was then negatively stained with 2% uranyl
acetate in water for another 2 minutes. Samples were viewed in a
JEOL 1200EX electron microscope operating at 80 kV.
[0437] Results
[0438] The ability of Aib containing peptides to form amyloid
fibrils was examined in comparison to native IAPP peptides. Aged
solutions of the Aib-modified and wild-type peptides were examined
under electronic microscope (EM) using negative staining. As is
shown in FIGS. 44a-b, both native peptides, ANFLVH (FIG. 44a) and
ANFLV (FIG. 44a), formed fibrillar structures with high resemblense
to the fibrils formed by the full-length IAPP protein. On the other
hand, no fibrillar structures were evident for the Aib containing
peptides, Aib-NF-Aib-VH (FIG. 44c) and Aib-NF-Aib-V (FIG. 44d),
even after longer periods of incubation, while amorphous aggregates
were still evident, suggesting that the Aib substituted peptides of
the present invention are incapable of fibril formation.
Example 42
Examination of the Amyloidogenic Properties of Wild-type and Aib
Substituted IAPP Peptides by Congo Red Binding Assay
[0439] Materials and Experimental Procedures
[0440] Congo Red Staining and Birefringence--A 10 .mu.L suspension
of 5 mM peptide solution in 10 mM Tris buffer, pH 7.2 aged for 10
days was allowed to dry overnight on a glass microscope slide.
Staining was performed by the addition of a 10 .mu.L suspension of
saturated Congo Red (CR) and NaCl in 80% ethanol (v/v) solution.
The solution was filtered via 0.2 .mu.m filter. The slide was then
dried for a few hours. Birefringence was determined with a SZX-12
Stereoscope (Olympus, Hamburg, Germany) equipped with cross
polarizers. 100.times. magnification is shown.
[0441] Results
[0442] Congo red staining was used to assess the aamyloidogenic
properties of Aib modified peptides. Slides were examined under the
microscope using cross-polarizers. FIGS. 45a-b show a typical
yellow-green birefringence for both ANFLVH and ANFLV peptides (FIG.
45a and b, respectively). However, in accordance with the EM
studies, the Aib-modified peptides ,Aib-NF-Aib-VH and Aib-NF-Aib-V,
exhibited no birefringence suggesting that Aib modified peptides
can not form amyloid fibrils (FIGS. 45c-d).
Example 43
[0443] Secondary Structure Analysis of Aib-modifted IAPP Peptide
Fragments
[0444] Fourier transform infrared spectroscopy (FT-IR) was effected
to determine the secondary structure of the Aib-modified hIAPP.
[0445] Materials and Experimentalprocedures
[0446] Fourier Transform Infrared Spectroscopy--Infrared spectra
were recorded using a Nicolet Nexus 470 FT-IR spectrometer with a
DTGS detector. Samples of two week aged peptide solutions, were
suspended on a CaF.sub.2 plate and dried by vacuum. The peptide
deposits were resuspended with D.sub.2O and subsequently dried to
form thin films. The resuspension procedure was repeated twice to
ensure maximal hydrogen to deuterium exchange. The measurements
were taken using a 4 cm.sup.-1 resolution and 2000 scans averaging.
The transmittance minima values were determined by the OMNIC
analysis program (Nicolet).
[0447] Results
[0448] FT-IR spectroscopy was used to elucidate the internal
conformation of the observed structures (see Examples 42 and 43,
above). As shown in FIGS. 46a-b upon incorporation of Aib, IAPP
peptides displayed a sharp change in the IR spectra. Whereas the
ANFLVH and ANFLV peptides spectra were typical of .beta.-sheet
spectra, with minima at 1630 cm.sup.-1 and 1632 cm.sup.-1
respectively, the Aib-NF-Aib-VH and Aib-NF-Aib-V peptides displayed
minima at 1670 cm.sup.-1 and 1666 cm.sup.31 1 , respectively, which
are characteristic to a random coil conformation.
[0449] Taken together, these results suggest a fundamental
difference between the native IAPP peptide and the Aib containing
peptides. Whereas the native peptides are highly amiloidogenic, the
modified Aib containing peptides are not able to form amyloid
fibrils (Examples 41-43).
Example 44
Aib Modified Peptides Inhibit Amyloid Formation by IAPP
Polypeptide
[0450] The degree of amyloid fibril formation with and without the
Aib inhibitor was assessed using thioflavin T (ThT) as molecular
indicator. The degree of fluorescence of the ThT dye is directly
correlated with the amount of amyloid fibrils in the solution
[LeVine H 3rd. (1993) Protein Sci. 2:404-410].
[0451] Materials and Experimental procedures
[0452] Fourier Transform Infrared Spectroscopy--IAPP solutions (4
.mu.M peptide in 10 mM Tris buffer pH 7.2), were incubated with or
without 40 .mu.M of the various peptide solutions at room
temperature. Fibril formation was assessed by a ten fold diluation
of the solutions into a solution which contained 3 .mu.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 Perkin
Elmar LS50B spectroflurimeter. As a control a 10 mM Tris buffer pH
7.2 was diluted into the ThT solution and fluorescence was
determined as described.
[0453] Results
[0454] As shown in FIG. 47, all Aib-modified peptides were able to
inhibit the assembly of full-length IAPP polypeptide, suggesting
that Aib modification of amyloid forming peptides can serve as
strong inhibitory tool in various therapeutic applications.
Example 45
Di- and Tri-aromatic Peptides can Inhibit Aggregation of IAPP
Polypeptide
[0455] In order to study the ability of short aromatic amino acid
sequences to inhibit the assembly of amyloid polypeptides and to
address the ability of .beta.-sheet breaker amino acids to
facilitate such inhibition, an array of tetra-, tri- and dipeptides
was synthesized and assayed.
[0456] Experimental Procedures
[0457] Peptide synthesis--The list of peptides used in the present
and Examples 46-47, which follow and designation thereof is
presented in Table 7, below. Peptide synthesis is described in
Examples 46-47, which follow. TABLE-US-00007 TABLE 7 Amino acid
Identification sequence SEQ ID NO: EG01 D-Phe-D-Phe-D-Pro 128 EG02
Aib-D-Phe-D-Asn-Aib 129 EG03 D-Phe-D-Asn-D-Pro 130 EG04
Aib-Asn-Phe-Aib 131 EG05 Gln-Lys-Leu-Val-Phe-Phe 132 EG06 Tyr-Tyr
133 EG07 D-Phe-D-Phe-D-Pro 112 EG08 Aib-D-Phe-D-Asn-Aib 113 EG09
Aib-Asn-Phe-Aib 114 EG10 Tyr-Tyr 115 EG11 Tyr-Tyr-NH2 116 EG12
Aib-Phe-Phe 117 EG13 Asn-Tyr-Aib 118 EG14 Asn-Tyr-Pro 119 EG15
D-Pro-D-Tyr-D-Asn 120 .beta.-amino- isobutyric acid (Aib) EG16
D-Tyr-Aib 121 EG17 D-Pro-D-Tyr 122 EG18 D-Tyr-D-Pro 123 EG19
Asn-Tyr-Tyr-Pro 134 EG20 Tyr-Tyr-Aib 135 EG21 Aib-Tyr-Tyr 136 EG22
Aib-Tyr-Tyr-Aib 137 EG23 D-Asn-Tyr-Tyr-D-Pro 138 EG24 Pro-Tyr-Tyr
139 EG25 Tyr-Tyr-Pro 140 EG26 Pro-Tyr-Tyr-Pro 141 EG27 D-Tyr-D-Tyr
142 EG28 D-Pro-Aib 143 EG29 D-Phe-D-Pro 144 EG30 D-Trp-Aib 145 EG31
D-Trp-D-Pro 146 d-F-P D-Phe-Pro 147 P-d-F Pro-D-Phe 148
[0458] ThT fluorescence assay--see Example 44 above.
[0459] Results
[0460] The inhibitory peptides were assayed using standard ThT
fluorescence assay (as described). FIG. 48 shows endpoint values
after IAPP aggregation reaches a plateau, following 142 hours of
incubation. The aggregation assay was preformed in the presence of
IAPP polypeptide (4 .mu.M) and the inhibitory peptides (40
.mu.M).
[0461] As is clearly shown in FIG. 48, short aromatic amino acid
sequences mediate recognition to IAPP polypeptide while inhibiting
aggregation thereof. The best inhibitor was the Aib-Phe-Phe peptide
(EG12) in which the Aib residue is conjugated to the shortest
recognition element which can form amyloid-related structures
[Reches and Gazit, Science (2003) 300(2619):625-7]. D-Tyr-D-Pro
(EG18) and D-Tyr-D-Aib (EG16) displayed a significant inhibitory
effect on IAPP aggregation substantiating the ability of a single
aromatic residue to mediate molecular-recognition. Interestingly,
Aib displayed a higher inhibitory activity than Proline.
Furthermore, the significant difference in activity between peptide
EG17 and EG18 which differ in the position of the .beta.-sheet
breaker amino acid relatively to the aromatic amino acid, suggests
a role for the order and not only the composition of the
peptides.
Example 46
Selection of Best Performing IAPP Fibrilization Inhibitors and
Criteria for Selecting Same
[0462] Peptides Synthesis--Peptide synthesis (excluding EG5, EG6,
and EG7, D-Phe-Pro, and D-Pro-Phe), was effected by using
solid-phase synthesis (Peptron, Inc., Taejeon, Korea). Peptide
identity was confirmed by ion spray mass-spectrometry. Peptide
purity was confirmed by reverse phase high-pressure liquid
chromatography (RP-HPLC). EG5, EG6, and EG7, D-Phe-Pro, and
D-Pro-Phe were purchased from Bachem (Bubendorf, Switzerland).
Islet amyloid polypeptide (IAPP) was purchase from CalBiochem (La
Jolla Calif., USA).
[0463] Thioflavin T fluorescence assay--hIAPP fibrillization was
monitored by Thioflavin T dye binding assay. hIAPPI.sub.1-37 stock
solution was diluted to a final concentration of 4 .mu.M in 10
.mu.M sodium acetate buffer (pH 6.5) with or without inhibitors (40
.mu.M), and a fmal concentration of HFIP of 1% (vol). Immediately
after dilution, sample was centrifuged for 20 minutes in
20,000.times.g at 4.degree. C. and the supernatant fraction was
used for fluorescence measurements. In every measurement Tht was
added to a 1 ml sample at a final concentration of 3 .mu.M and
measurements were effected by using Perkin-Elmer (excitation 450
nm, 2.5 nm slit; emission 480 nm 10 nm slit). Background was
subtracted from all samples.
[0464] Results
[0465] Identification of potent inhibitors and rules for inhibitor
design--In order to identify small peptide inhibitors of IAPP
fibrillization and to optimize a minimal length of the inhibitors
and maximal stability thereof, iterative cycles of peptide
selection were effected using D-amino acids analogues.
[0466] The first round of selection shown in FIG. 49a demonstrated
that peptides as short as tripeptides can efficiently inhibit the
formation of amyloid by IAPP. Comparison of EG01 and EG03 suggests
that presence of Asn residue within the short peptide does not
contribute to the inhibition of amyloid formation and further
supports the use of aromatic moieties along with beta-breakers for
optimal inhibition. The effective inhibition of IAPP fibrillization
by EG05, a known inhibitor of amyloid formation [Tjemberg et al.
(1996) J. Biol. Chem. 271: 8545-854], suggests a generic inhibition
of amyloid formation by aromatic residues. Indeed, the similar
activity of the EG05 and the much shorter EG08 (Aib conjugated to
the Phe-Phe) element clearly suggest that the generic inhibition of
EG05 stems from its aromatic nature.
[0467] The second round of selection shown in FIG. 49b demonstrated
that effective inhibition could be achieved not only by tripeptide
but also by dipeptides (EG16, EG17, EG18). In all cases the
conjugation of a D-isomer to an aromatic moiety was used. The
difference between EG16 and EG17 was further studied in the next
round to establish rules for the design of inhibitors. FIG. 49c
shows the results of a third round of selection. The comparison in
the third round (FIG. 49c) of EG20 vs. EG 21 (d-F-P vs. P-d-F)
along with the results obtained by comparing EG16 and EG17 in the
second round, and the results obtained by comparing EG24 and EG35
(in the forth round--FIG. 1d), suggest a general formula for the
design of dipeptide inhibitors as set forth in: (Aromatic D or
L)-(beta-breaker). These selection steps further provided a general
formula for tripeptide inhibitors as set forth in: (Aromatic D or
L)-(Aromatic D or L)-(beta-breaker).
[0468] The forth round also demonstrated the inhibition potential
of four putuavely metabolically stable dipeptides EG28, EG29, EG30,
EG31. Again, stressing the value of beta-breakers and aromatic
amino acids for the inhibitory sequences.
Example 47
Inhibition of .beta.-amyloid Polypeptide Fibril Formation by
D-Trp-Aib Peptides Synthesis--See Example 46 above. Recombinant
.beta.-amyloid (A.beta. 1-40, >98% Pure) was purchase from
rPeptide (Athens Ga., USA).
[0469] Thioflavin T fluorescence assay--Fibrillization of
A.beta.1-40 was monitored by Thioflavin T dye binding assay.
A.beta. 1-40 stock solution was diluted to a final concentration of
5 .mu.M in 100 mM NaCl, 10 mM sodium phosphate buffer (pH 7.4) with
or without inhibitors. In every measurement Tht was added to 0.1 ml
sample to a final concentration of 0.3 .mu.M ThT and 0.4 .mu.M
polypeptide. Measurements were effected using Jobin Yvon FluroMax-3
(excitation 450 nm, 2.5 mn slit; emission 480 nm 5 nm slit,
integration time 1 second). Background was subtracted from all
samples.
[0470] Transmission Electron Microscopy--10 .mu.L samples were
placed on 400-mesh copper grids (SPI supplies, West Chester Pa.)
covered with carbon-stabilized Formvar film. Following 1 minute,
excess fluid was removed, and the grids were negatively stained
with 2% uranyl acetate in water for another two minutes. Samples
were viewed by a JEOL 1200EX electron microscope operating at 80
kV.
[0471] Results
[0472] Fluorescence measurements of inhibition--To test whether the
D-Trp-Aib can inhibit amyloid formation by molecules other than
IAPP, Alzheimer's .beta.-amyloid 1-40 (A.beta.) was served as a
model system. As shown in FIG. 50, in the absence of inhibitor,
A.beta. displayed a lag phase in fibrilization of about 100 hours
which was followed by a fast enhancement in fluorescent levels. In
the presence of 10 .mu.M of EG30, the lag time was significantly
increased and the overall fluorescence upon reaching a plateau was
significantly lower than that abserved without the inhibitor.
[0473] This further suggests that aromatic inhibitors may serve as
generic amyloid inhibitors and a common mechanism of assembly
[Gazit (2002) FASEB J. 16, 77-83].
[0474] Ultrastructural analysis--To get information on the
mechanism of inhibition, samples taken from the fluorsence assay
upon its termination assay were viewed by electron microscopy (FIG.
51a-c). Distinct and well-defined amyloid fibers were present in
samples of A.beta. not including the inhibitor (FIG. 51a). In
contrast, in the presence of EG30, A.beta. was detected mostly as
amorphous aggregates (FIG. 51b) or as fragmented fibrils (FIG.
51c). This suggests that even those fibrils generated in the
presence of the inhibitor were short and probably
dysfunctional.
[0475] Thus, the D-Trp-Aib compound offers a unique combination of
pharmaceutical properties in an extremely small molecule:
[0476] 1. Hetero-aromatic interactions: The interaction between the
tryptophan indole and the aromatic recognition interfaces of the
growing amyloid fibrils (Gazit, 2002) allows specific and oriented
binding that directly and precisely blocks further homo-molecular
self-assembly of the growing chain.
[0477] 2. The Aib conformational restriction: The conjugation of
the .beta.-aminoisobutyric acid (Aib), an amino acid with
exceptional geometrical constrain, induce a very strong
.beta.-sheet breakage effect, a key measure to halt the growing of
amyloid fibrils. This .beta.-breakage strategy shows clear a
advantage as compared to prior art (proline introduction) in terms
of size and complexity of the molecule.
[0478] 3. A stable D-isomer conformation: The inhibitor is built of
a D-amino acid and a non-chiral Aib moiety. This results in the
formation of a non-cleavable peptide bond and thus with a
presumably high physiological stability.
[0479] 4. Peptide bond stacking: In spite of its small size, a
typical peptide bond (although isomerically stable one) is retained
within the molecule. The unique planar characteristic of such a
peptide bond allows a specific and geometrically-constrained
stacking of the molecule on the growing amyloid chain, due to their
partially planar resonating structures with the exact gemetry that
is consistant with .beta.-strand interaction.
[0480] 5. Electrostatic repulsion: The existence of charged terimi
results in electrostatic repulsion of further binding monomers.
Such repulsion is achieved by introduction of a charge aspartic
acid in other peptide inhibitors [Soto et al. (2003) J. Biol. Chem
278:13905] as there is a need to block the termini of these
peptides to decrease proteolyic degradation. The non-native stable
isomeric configuration of the D-Trp-Aib allows the retention of
charged termini within the minimal framework resulting in a
significantly small molecule.
[0481] 6. Bulky hydrophobic moiety: The trypotophan indole group
offers a unique bulkiness and hydrophobic nature in a very small
molecular system. It is well established that tryptophan moieties
have among the highest membrane partition coefficients. It is
speculated that this property should have a key advantage for oral
bioavailability and blood brain barrier (BBB) transfer.
[0482] 7. Antioxidant activity: The indole group is also known to
act as antioxidant by the scavenging of free-radicals. Indeed some
of the drug candidates for treating AD are based on this property
(e.g. indole-3-propionic acid of MindSet). However, while such
molecules are effective in the protection of neural cells from AD
related oxidation stress, they lack the unique fibrillization
inhibitory properties of the D-Trp-Aib molecular frame.
[0483] 8. Small size: D-Trp-Aib is a remarkably small active
molecule. All the unique properties that were described in the
previous paragraphs (1-7) are maintained within a molecule of less
than 300 Da. The small size of this non-cleavable molecule suggests
an oral bioavailability, long half-life, and transfer of the BBB,
while maintaining low immunogenic potential.
[0484] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0485] 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
150 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) Any aromatic
amino acid misc_feature (2)..(2) Any amino acid, but 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
9 PRT Artificial sequence Synthetic peptide 14 Asn Phe Leu Val His
Ser Ser Asn Asn 1 5 15 7 PRT Artificial sequence Synthetic peptide
15 Asn Phe Leu Val His Ser Ser 1 5 16 6 PRT Artificial sequence
Synthetic peptide 16 Phe Leu Val His Ser Ser 1 5 17 5 PRT
Artificial sequence Synthetic peptide 17 Asn Phe Leu Val His 1 5 18
5 PRT Artificial sequence Synthetic peptide 18 Phe Leu Val His Ser
1 5 19 4 PRT Artificial sequence Synthetic peptide 19 Phe Leu Val
His 1 20 8 PRT Artificial sequence Synthetic peptide 20 Asn Phe Gly
Ser Val Gln Val Phe 1 5 21 6 PRT Artificial sequence Synthetic
peptide 21 Asn Phe Gly Ser Val Gln 1 5 22 5 PRT Artificial sequence
Synthetic peptide 22 Asn Phe Gly Ser Val 1 5 23 5 PRT Artificial
sequence Synthetic peptide 23 Phe Gly Ser Val Gln 1 5 24 4 PRT
Artificial sequence Synthetic peptide 24 Gly Ser Val Gln 1 25 4 PRT
Artificial sequence Synthetic peptide 25 Phe Gly Ser Val 1 26 6 PRT
Artificial sequence Synthetic peptide 26 Asn Ala Gly Ser Val Gln 1
5 27 5 PRT Artificial sequence Synthetic peptide 27 Asp Phe Asn Lys
Phe 1 5 28 4 PRT Artificial sequence Synthetic peptide 28 Phe Asn
Lys Phe 1 29 4 PRT Artificial sequence Synthetic peptide 29 Asp Phe
Asn Lys 1 30 3 PRT Artificial sequence Synthetic peptide 30 Asp Phe
Asn 1 31 5 PRT Artificial sequence Synthetic peptide 31 Asp Ala Asn
Lys Phe 1 5 32 6 PRT Artificial sequence Synthetic peptide 32 Leu
Phe Asn Gln Thr Gly 1 5 33 6 PRT Artificial sequence Synthetic
peptide 33 Ser Phe Phe Ser Phe Leu 1 5 34 5 PRT Artificial sequence
Synthetic peptide 34 Phe Glu Asn Lys Phe 1 5 35 5 PRT Artificial
sequence Synthetic peptide 35 Ser Phe Asn Asn Gly 1 5 36 6 PRT
Artificial sequence Synthetic peptide 36 Leu Gln Asn Phe Thr Leu 1
5 37 6 PRT Artificial sequence Synthetic peptide 37 Thr Leu Ile Phe
Gly Gly 1 5 38 6 PRT Artificial sequence Synthetic peptide 38 Arg
Ala Leu Asp Phe Ala 1 5 39 6 PRT Artificial sequence Synthetic
peptide 39 Gly Leu Val Phe Val Ser 1 5 40 6 PRT Artificial sequence
Synthetic peptide 40 Gly Thr Phe Gln Ile Asn 1 5 41 6 PRT
Artificial sequence Synthetic peptide 41 Ser Gly Ile Phe Thr Asn 1
5 42 5 PRT Artificial sequence Synthetic peptide 42 Glu Arg Gly Phe
Phe 1 5 43 6 PRT Artificial sequence Synthetic peptide 43 Arg Asp
Phe Leu Asp Arg 1 5 44 5 PRT Artificial sequence Synthetic peptide
44 Ser Asn Phe Leu Asn 1 5 45 7 PRT Artificial sequence Synthetic
peptide 45 Asn Phe Leu Val His Pro Pro 1 5 46 8 PRT Artificial
sequence Synthetic peptide 46 Asn Phe Gly Ala Ile Leu Ser Ser 1 5
47 8 PRT Artificial sequence Synthetic peptide 47 Asn Ile Gly Ala
Ile Leu Ser Ser 1 5 48 8 PRT Artificial sequence Synthetic peptide
48 Asn Leu Gly Ala Ile Leu Ser Ser 1 5 49 8 PRT Artificial sequence
Synthetic peptide 49 Asn Val Gly Ala Ile Leu Ser Ser 1 5 50 24 DNA
Artificial sequence Single strand DNA oligonucleotide 50 aaatgcaaca
ccgcgacctg cgcg 24 51 30 DNA Artificial sequence Single strand DNA
oligonucleotide 51 acccagcgcc tggcgaactt tctggtgcat 30 52 30 DNA
Artificial sequence Single strand DNA oligonucleotide 52 agcagcaaca
actttggcgc gattctgagc 30 53 33 DNA Artificial sequence Single
strand DNA oligonucleotide 53 agcaccaacg tgggcagcaa cacctattaa tga
33 54 18 DNA Artificial sequence Single strand DNA oligonucleotide
54 tcgttgtgca taattact 18 55 30 DNA Artificial sequence Single
strand DNA oligonucleotide 55 ccgcgctaag actcgtcgtg cttgcacccg 30
56 33 DNA Artificial sequence Single strand DNA oligonucleotide 56
cgcttgaaag accacgtatc gtcgttgttg aaa 33 57 36 DNA Artificial
sequence Single strand DNA oligonucleotide 57 tttacgttgt ggcgctggac
gcgctgggtc gcggac 36 58 114 DNA Artificial sequence Modified IAPP
cDNA for expression in bacteria 58 atgaaatgca acaccgcgac ctgcgcgacc
cagcgcctgg cgaactttct ggtgcatagc 60 agcaacaact ttggcgcgat
tctgagcagc accaacgtgg gcagcaacac ctat 114 59 56 DNA Artificial
sequence Single strand DNA oligonucleotide 59 gggtttccat gggccatcac
catcaccatc acgaaaaatg caacaccgcg acctgc 56 60 35 DNA Artificial
sequence Single strand DNA oligonucleotide 60 gggtttgcgg ccgctcatta
ataggtgttg ctgcc 35 61 10 PRT Artificial sequence Synthetic peptide
61 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln 1 5 10 62 10 PRT
Artificial sequence Synthetic peptide 62 Cys Asn Thr Ala Thr Cys
Ala Thr Gln Arg 1 5 10 63 10 PRT Artificial sequence Synthetic
peptide 63 Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu 1 5 10 64 10 PRT
Artificial sequence Synthetic peptide 64 Thr Ala Thr Cys Ala Thr
Gln Arg Leu Ala 1 5 10 65 10 PRT Artificial sequence Synthetic
peptide 65 Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn 1 5 10 66 10 PRT
Artificial sequence Synthetic peptide 66 Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe 1 5 10 67 10 PRT Artificial sequence Synthetic
peptide 67 Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu 1 5 10 68 10 PRT
Artificial sequence Synthetic peptide 68 Ala Thr Gln Arg Leu Ala
Asn Phe Leu Val 1 5 10 69 10 PRT Artificial sequence Synthetic
peptide 69 Thr Gln Arg Leu Ala Asn Phe Leu Val His 1 5 10 70 10 PRT
Artificial sequence Synthetic peptide 70 Gln Arg Leu Ala Asn Phe
Leu Val His Ser 1 5 10 71 10 PRT Artificial sequence Synthetic
peptide 71 Arg Leu Ala Asn Phe Leu Val His Ser Ser 1 5 10 72 10 PRT
Artificial sequence Synthetic peptide 72 Leu Ala Asn Phe Leu Val
His Ser Ser Asn 1 5 10 73 10 PRT Artificial sequence Synthetic
peptide 73 Ala Asn Phe Leu Val His Ser Ser Asn Asn 1 5 10 74 10 PRT
Artificial sequence Synthetic peptide 74 Asn Phe Leu Val His Ser
Ser Asn Asn Phe 1 5 10 75 10 PRT Artificial sequence Synthetic
peptide 75 Phe Leu Val His Ser Ser Asn Asn Phe Gly 1 5 10 76 10 PRT
Artificial sequence Synthetic peptide 76 Leu Val His Ser Ser Asn
Asn Phe Gly Ala 1 5 10 77 10 PRT Artificial sequence Synthetic
peptide 77 Val His Ser Ser Asn Asn Phe Gly Ala Ile 1 5 10 78 10 PRT
Artificial sequence Synthetic peptide 78 His Ser Ser Asn Asn Phe
Gly Ala Ile Leu 1 5 10 79 10 PRT Artificial sequence Synthetic
peptide 79 Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser 1 5 10 80 10 PRT
Artificial sequence Synthetic peptide 80 Ser Asn Asn Phe Gly Ala
Ile Leu Ser Ser 1 5 10 81 10 PRT Artificial sequence Synthetic
peptide 81 Asn Asn Phe Gly Ala Ile Leu Ser Ser Thr 1 5 10 82 10 PRT
Artificial sequence Synthetic peptide 82 Asn Phe Gly Ala Ile Leu
Ser Ser Thr Asn 1 5 10 83 10 PRT Artificial sequence Synthetic
peptide 83 Phe Gly Ala Ile Leu Ser Ser Thr Asn Val 1 5 10 84 10 PRT
Artificial sequence Synthetic peptide 84 Gly Ala Ile Leu Ser Ser
Thr Asn Val Gly 1 5 10 85 10 PRT Artificial sequence Synthetic
peptide 85 Ala Ile Leu Ser Ser Thr Asn Val Gly Ser 1 5 10 86 10 PRT
Artificial sequence Synthetic peptide 86 Ile Leu Ser Ser Thr Asn
Val Gly Ser Asn 1 5 10 87 10 PRT Artificial sequence Synthetic
peptide 87 Leu Ser Ser Thr Asn Val Gly Ser Asn Thr 1 5 10 88 10 PRT
Artificial sequence Synthetic peptide 88 Ser Ser Thr Asn Val Gly
Ser Asn Thr Tyr 1 5 10 89 8 PRT Artificial sequence Synthetic
peptide 89 Asn Ala Gly Ala Ile Leu Ser Ser 1 5 90 10 PRT Artificial
sequence Peptide array consensus sequence misc_feature (4)..(4) Any
amino acid, but cysteine 90 Ser Asn Asn Xaa Gly Ala Ile Leu Ser Ser
1 5 10 91 8 PRT Artificial sequence Synthetic peptide 91 Asn Ala
Gly Ala Ile Leu Ser Ser 1 5 92 8 PRT Artificial sequence Synthetic
peptide 92 Asn Ala Gly Ala Ile Leu Ser Ser 1 5 93 8 PRT Artificial
sequence Synthetic peptide 93 Asn Asp Gly Ala Ile Leu Ser Ser 1 5
94 8 PRT Artificial sequence Synthetic peptide 94 Asn Glu Gly Ala
Ile Leu Ser Ser 1 5 95 8 PRT Artificial sequence Synthetic peptide
95 Asn Phe Gly Ala Ile Leu Ser Ser 1 5 96 8 PRT Artificial sequence
Synthetic peptide 96 Asn Gly Gly Ala Ile Leu Ser Ser 1 5 97 8 PRT
Artificial sequence Synthetic peptide 97 Asn His Gly Ala Ile Leu
Ser Ser 1 5 98 8 PRT Artificial sequence Synthetic peptide 98 Asn
Ile Gly Ala Ile Leu Ser Ser 1 5 99 8 PRT Artificial sequence
Synthetic peptide 99 Asn Lys Gly Ala Ile Leu Ser Ser 1 5 100 8 PRT
Artificial sequence Synthetic peptide 100 Asn Leu Gly Ala Ile Leu
Ser Ser 1 5 101 8 PRT Artificial sequence Synthetic peptide 101 Asn
Met Gly Ala Ile Leu Ser Ser 1 5 102 8 PRT Artificial sequence
Synthetic peptide 102 Asn Asn Gly Ala Ile Leu Ser Ser 1 5 103 8 PRT
Artificial sequence Synthetic peptide 103 Asn Pro Gly Ala Ile Leu
Ser Ser 1 5 104 8 PRT Artificial sequence Synthetic peptide 104 Asn
Gln Gly Ala Ile Leu Ser Ser 1 5 105 8 PRT Artificial sequence
Synthetic peptide 105 Asn Arg Gly Ala Ile Leu Ser Ser 1 5 106 8 PRT
Artificial sequence Synthetic peptide 106 Asn Ser Gly Ala Ile Leu
Ser Ser 1 5 107 8 PRT Artificial sequence Synthetic peptide 107 Asn
Thr Gly Ala Ile Leu Ser Ser 1 5 108 8 PRT Artificial sequence
Synthetic peptide 108 Asn Val Gly Ala Ile Leu Ser Ser 1 5 109 8 PRT
Artificial sequence Synthetic peptide 109 Asn Trp Gly Ala Ile Leu
Ser Ser 1 5 110 8 PRT Artificial sequence Synthetic peptide 110 Asn
Tyr Gly Ala Ile Leu Ser Ser 1 5 111 6 PRT Artificial sequence
Synthetic peptide 111 Asn Phe Gly Ala Ile Leu 1 5 112 3 PRT
Artificial sequence Synthetic peptide misc_feature (1)..(3)
Stereoisomer D 112 Phe Phe Pro 1 113 4 PRT Artificial sequence
Synthtic peptide misc_feature (1)..(1) D and L methyl alanine
misc_feature (2)..(3) Stereoisomer D misc_feature (4)..(4) D and L
methyl alanine 113 Xaa Phe Asn Xaa 1 114 4 PRT Artificial sequence
Synthetic peptide misc_feature (1)..(1) D and L methyl alanine
misc_feature (4)..(4) D and L methyl alanine 114 Xaa Asn Phe Xaa 1
115 2 PRT Artificial sequence Synthetic peptide 115 Tyr Tyr 1 116 2
PRT Artificial sequence Synthetic peptide misc_feature (2)..(2)
amidated amino acid 116 Tyr Tyr 1 117 3 PRT Artificial sequence
Synthetic peptide misc_feature (1)..(1) D and L methyl alanine 117
Xaa Phe Phe 1 118 3 PRT Artificial sequence Synthetic peptide
misc_feature (3)..(3) D and L methyl alanine 118 Asn Tyr Xaa 1 119
3 PRT Artificial sequence Synthetic peptide 119 Asn Tyr Pro 1 120 3
PRT Artificial sequence Synthetic peptide misc_feature (1)..(3)
Stereoisomer D 120 Asn Tyr Pro 1 121 2 PRT Artificial sequence
Synthetic peptide misc_feature (1)..(1) Stereoisomer D misc_feature
(2)..(2) D and L methyl alanine 121 Tyr Xaa 1 122 2 PRT Artificial
sequence Synthetic peptide misc_feature (1)..(2) Stereoisomer D 122
Pro Tyr 1 123 2 PRT Artificial sequence Synthetic peptide
misc_feature (1)..(2) Stereoisomer D 123 Tyr Pro 1 124 6 PRT
Artificial sequence Synthetic peptide 124 Ala Asn Phe Leu Val His 1
5 125 6 PRT Artificial sequence Synthetic peptide misc_feature
(1)..(1) D and L methyl alanine misc_feature (4)..(4) D and L
methyl alanine 125 Xaa Asn Phe Xaa Val His 1 5 126 5 PRT Artificial
sequence Synthetic peptide 126 Ala Asn Phe Leu Val 1 5 127 5 PRT
Artificial sequence Synthetic peptide misc_feature (1)..(1) D and L
methyl alanine misc_feature (4)..(4) D and L methyl alanine 127 Xaa
Asn Phe Xaa Val 1 5 128 3 PRT Artificial sequence Synthetic peptide
misc_feature (1)..(3) Stereoisomer D 128 Phe Phe Pro 1 129 4 PRT
Artificial sequence Synthetic peptide misc_feature (1)..(1)
Beta-aminoisobutyric acid (Aib) misc_feature (2)..(3) Stereoisomer
D misc_feature (4)..(4) Beta-aminoisobutyric acid (Aib) 129 Xaa Phe
Asn Xaa 1 130 3 PRT Artificial sequence Synthetic peptide
misc_feature (1)..(3) Stereoisomer D 130 Phe Asn Pro 1 131 4 PRT
Artificial sequence Synthetic peptide misc_feature (1)..(1)
Beta-aminoisobutyric acid (Aib) misc_feature (4)..(4)
Beta-aminoisobutyric acid (Aib) 131 Xaa Asn Phe Xaa 1 132 6 PRT
Artificial sequence Synthetic peptide misc_feature (1)..(1)
Beta-aminoisobutyric acid (Aib) misc_feature (4)..(4)
Beta-aminoisobutyric acid (Aib) 132 Gln Lys Leu Val Phe Phe 1 5 133
2 PRT Artificial sequence Synthetic peptide 133 Tyr Tyr 1 134 4 PRT
Artificial sequence Synthetic peptide 134 Asn Tyr Tyr Pro 1 135 3
PRT Artificial sequence Synthetic peptide misc_feature (3)..(3)
Beta-aminoisobutyric acid (Aib) 135 Tyr Tyr Xaa 1 136 3 PRT
Artificial sequence Synthetic peptide misc_feature (1)..(1)
Beta-aminoisobutyric acid (Aib) 136 Xaa Tyr Tyr 1 137 4 PRT
Artificial sequence Synthetic peptide misc_feature (1)..(1)
Beta-aminoisobutyric acid (Aib) misc_feature (4)..(4)
Beta-aminoisobutyric acid (Aib) 137 Xaa Tyr Tyr Xaa 1 138 4 PRT
Artificial sequence Synthetic peptide misc_feature (1)..(1)
Stereoisomer D misc_feature (4)..(4) Stereoisomer D 138 Asn Tyr Tyr
Pro 1 139 3 PRT Artificial sequence Synthetic peptide 139 Pro Tyr
Tyr 1 140 3 PRT Artificial sequence Synthetic peptide 140 Tyr Tyr
Pro 1 141 4 PRT Artificial sequence Synthetic peptide 141 Pro Tyr
Tyr Pro 1 142 2 PRT Artificial sequence Synthetic peptide
misc_feature (1)..(2) Stereoisomer D 142 Tyr Tyr 1 143 2 PRT
Artificial sequence Synthetic peptide misc_feature (2)..(2)
Beta-aminoisobutyric acid (Aib) 143 Pro Xaa 1 144 2 PRT Artificial
sequence Synthetic peptide misc_feature (1)..(2) Stereoisomer D 144
Phe Pro 1 145 2 PRT Artificial sequence Synthetic peptide
misc_feature (2)..(2) Beta-aminoisobutyric acid (Aib) 145 Trp Xaa 1
146 2 PRT Artificial sequence Synthetic peptide misc_feature
(1)..(2) Stereoisomer D 146 Trp Pro 1 147 2 PRT Artificial sequence
Synthetic peptide misc_feature (1)..(1) Stereoisomer D 147 Phe Pro
1 148 2 PRT Artificial sequence Synthetic peptide misc_feature
(2)..(2) Stereoisomer D 148 Pro Phe 1 149 3 PRT Artificial sequence
Synthetic peptide misc_feature (1)..(2) Stereoisomer D misc_feature
(3)..(3) Beta-aminoisobutyric acid (Aib) 149 Cys Trp Xaa 1 150 3
PRT Artificial sequence Synthetic peptide misc_feature (2)..(2)
Stereoisomer D misc_feature (3)..(3) Beta-aminoisobutyric acid
(Aib) 150 Cys Trp Xaa 1
* * * * *