U.S. patent application number 09/097194 was filed with the patent office on 2001-07-05 for peptides used as agonists and/or inhibitors of amyloid formation and cytotoxicity and also for use in alzheimer's disease, in type ii diabetes mellitus and in spongiform encephalophathies.
Invention is credited to BERNHAGEN, JURGEN, HERWIG, BRUNNER, KAPURNIOTU, AFRODITI.
Application Number | 20010007015 09/097194 |
Document ID | / |
Family ID | 7832754 |
Filed Date | 2001-07-05 |
United States Patent
Application |
20010007015 |
Kind Code |
A1 |
KAPURNIOTU, AFRODITI ; et
al. |
July 5, 2001 |
PEPTIDES USED AS AGONISTS AND/OR INHIBITORS OF AMYLOID FORMATION
AND CYTOTOXICITY AND ALSO FOR USE IN ALZHEIMER'S DISEASE, IN TYPE
II DIABETES MELLITUS AND IN SPONGIFORM ENCEPHALOPHATHIES
Abstract
Certain peptide molecules can be used as the basic structures
(template molecules) for inhibiting and analysing amyloid formation
and cytotoxicity in amyloid illnesses. These peptides have an
effect on the molecules which are responsible for the amyloid
illnesses (for their part amyloid-forming peptides and proteins).
The peptides are thus either inhibitors themselves or agonists of
amyloid formation and cytotoxicity or can serve as a template for
identifying and producing further inhibitors and agonists and can
be used as molecular tools during analysis. The peptide molecules
have generally 3-15 amino acids, and preferably a maximum of 10
amino acids, and at least an active peptide sequence GA, preferably
GAI, and even more preferably one selected from the group
consisting of GAIL, FGAIL, NFGAIL, NNFGAIL, SNNFGAIL, NFGAILSS and
SNNFGAILSS, or the group consisting of GAII, KGAII, NKGAII,
SNKGAII, GSNKGAII, NKGAIIGL and GSNKGAIIGL, or the group consisting
of AGAVV, AAGAVV, VYYGAVV, HVAAGAVV, AAGAWGG and HVAAGAVVGG. The
peptide sequence generally has at least one hydrogen molecule, and
preferably every second hydrogen molecule, of an amide bond
replaced by a methyl group.
Inventors: |
KAPURNIOTU, AFRODITI;
(TUBINGEN, DE) ; BERNHAGEN, JURGEN; (TUBINGEN,
DE) ; HERWIG, BRUNNER; (STUTTGART, DE) |
Correspondence
Address: |
A JAMES RICHARDSON
1000 CAPITAL CENTER SOUTH
201 NORTH ILLINOIS STREET
INDIANAPOLIS
IN
46204
|
Family ID: |
7832754 |
Appl. No.: |
09/097194 |
Filed: |
June 12, 1998 |
Current U.S.
Class: |
530/300 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 43/00 20180101; C07K 14/575 20130101; A61P 25/00 20180101;
A61P 25/28 20180101; C07K 5/1008 20130101; C07K 14/47 20130101;
C07K 14/4711 20130101 |
Class at
Publication: |
530/300 |
International
Class: |
A61K 038/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 17, 1997 |
DE |
197 25 619.8 |
Claims
In the claims:
1. A peptide acting as at least one of an agonist and inhibitor of
at least one of amyloid formation and cytotoxicity comprising 3-15
amino acids and at least an active peptide sequence GA.
2. The peptide according to claim 1, comprising a maximum of 10
amino acids.
3. The peptide according to claim 1, comprising an active peptide
sequence GAI.
4. The peptide according to claim 3, wherein the peptide is
selected from the group consisting of GAIL, FGAIL, NFGAIL, NNFGAIL,
SNNFGAIL, NFGAILSS and SNNFGAILSS.
5. The peptide according to claim 3, wherein the peptide is
selected from the group consisting of GAII, KGAII, NKGAII, SNKGAII,
GSNKGAII, NKGAIIGL and GSNKGAIIGL.
6. The peptide according to claim 1, wherein the peptide is
selected from the group consisting of AGAVV, AAGAVV, VYYGAVV,
HVAAGAVV, AAGAVVGG and HVAAGAVVGG.
7. The peptide according to claim 1, wherein in the peptide
sequence at least one hydrogen molecule of an amide bond is
replaced by a methyl group.
8. The peptide according to claim 7, wherein every second hydrogen
molecule is replaced by a methyl group.
9. The peptide according to claim 2, comprising an active peptide
sequence GAI.
10. The peptide according to claim 9, wherein the peptide is
selected from the group consisting of GAIL, FGAIL, NFGAIL, NNFGAIL,
SNNFGAIL, NFGAILSS and SNNFGAILSS.
11. The peptide according to claim 9, wherein the peptide is
selected from the group consisting of GAII, KGAII, NKGAII, SNKGAII,
GSNKGAII, NKGAIIGL and GSNKGAIIGL.
12. The peptide according to claim 2, wherein the peptide is
selected from the group consisting of AGAVV, AAGAVV, VYYGAVV,
HVAAGAVV, AAGAVVGG and HVAAGAVVGG.
13. A composition acting as at least one of an agonist and
inhibitor of at least one of amyloid formation and cytotoxicity in
Type II diabetes mellitus comprising a peptide comprising 3-15
amino acids and an active peptide sequence GA.
14. The composition according to claim 13, wherein the peptide
comprises a maximum of 10 amino acids.
15. The composition according to claim 13, wherein the peptide
comprises an active peptide sequence GAI.
16. The composition according to claim 13, wherein the peptide is
selected from the group consisting of GAIL, FGAIL, NFGAIL, NNFGAIL,
SNNFGAIL, NFGAILSS and SNNFGAILSS.
17. A composition acting as at least one of an agonist and
inhibitor of at least one of amyloid formation and cytotoxicity in
Alzheimer's disease comprising a peptide comprising 3-15 amino
acids and at least an active peptide sequence GA.
18. The composition according to claim 17, wherein the peptide
comprises an active peptide sequence GAI.
19. The composition according to claim 17, wherein the peptide is
selected from the group consisting of GAII, KGAII, NKGAII, SNKGAII,
GSNKGAII, NKGAIIGL and GSNKGAIIGL.
20. An agonist of at least one of amyloid formation and
cytotoxicity comprising a peptide comprising 3-15 amino acids and
at least an active peptide sequence GA.
21. The agonist according to claim 20, wherein the agonist acts as
agonist of at least one of amyloid formation and cytotoxicity in
spongiform encephalopathies.
22. The agonist according to claim 21, wherein the peptide
comprises a maximum of 10 amino acids.
23. The agonist according to claim 21, wherein the peptide is
selected from the group consisting of AGAVV, AAGAVV, VYYGAVV,
HVAAGAVV, AAGAVVGG and HVAAGAVVGG.
24. An inhibitor of at least one of amyloid formation and
cytotoxicity comprising a peptide comprising 3-15 amino acids and
at least an active peptide sequence GA.
25. The inhibitor according to claim 24, wherein the inhibitor
inhibits at least one of amyloid formation and cytotoxicity in
spongiform encephalopathies.
26. The inhibitor according to claim 25, wherein the peptide
comprises a maximum of 10 amino acids.
27. The inhibitor according to claim 25, wherein the peptide is
selected from the group consisting of AGAVV, AAGAVV, VYYGAVV,
HVAAGAVV, AAGAVVGG and HVAAGAVVGG.
Description
[0001] The invention relates to peptides with 3-15 amino acids
which function as agonists and/or inhibitors in amyloid formation
and/or toxicity and which can be therefore used in various related
clinical pictures.
[0002] New strategies and active agents for the therapy and
diagnosis of the above-mentioned amyloid illnesses are being
researched world-wide. However, there is still no means of treating
these illnesses with medicines/pharmaceuticals. According to
predominant expert opinion, certain amyloid proteins, which are
specific to each illness, are causally responsible for the
occurrence of amyloid illnesses because of their amyloid genesis or
aggregation. The mechanism for amyloid genesis and the associated
cell death (cytotoxicity) in these illnesses is widely unknown and
correspondingly, highly specific inhibitors have hence not been
identified. Pharmaceuticals for treating amyloid illnesses on the
basis of such inhibitors have therefore also not been
developed.
[0003] In exactly the same way, the protein-chemical,
technical-analytical problems, which are caused by amyloid
formation, (formation of insoluble protein aggregates, so-called
amyloid structures) have up to now not permitted the analysis of
amyloid formation. This has contributed to the fact that the
mechanism for amyloid formation is still widely unexplained. For
analysing the formation of amyloids, constructive diagnostic
methods (fast in vitro tests for evaluating the amount, duration
and quality of the amyloid structures) still therefore do not exist
either. For example, a diagnosis for Alzheimer's can only be
performed symptomatically (increasing forgetfulness or similar) or
post mortem. Reliable blood tests, for example, cannot be performed
during the lifetime of the patient.
[0004] On this basis it is therefore the object of the invention to
propose appropriate peptides, which can function as agonists and/or
inhibitors of amyloid formation and/or cytotoxicity.
[0005] The object is resolved by the characterising features of
claim 1. The sub-claims indicate advantageous further developments.
The application of the peptides is given in claims 7 to 9.
[0006] According to the invention, peptides with 3-15 amino acids
are proposed, which contain at least the active sequence GA. It has
been shown, that these peptide molecules function as inhibitors
and/or agonists of those amyloid peptides/proteins, which cause the
amyloid illnesses Alzheimer's disease, Type II diabetes mellitus
and spongiform encephalopathies (Creutzfeld Jacob disease, scrapie,
BSE). The peptide molecules according to the invention are in a
position to inhibit the amyloid genesis or aggregation of the
amyloid peptides/proteins of amyloid peptide or _-AP (in
Alzheimer's disease), amyline/IAPP (in Type II diabetes mellitus)
and prion protein (in spongiform encephalopathies). When inhibition
of the amyloid genesis of illness-inducing peptides/proteins is
achieved, the cyto-toxic effect, which is caused by aggregation of
_-AP, amyline/IAPP or prion protein, on tissue cells is
inhibited.
[0007] The invention offers the following advantages relative to
the state of the art:
[0008] Simple chemosynthesis to a high degree of purity, and use,
according to current methods, of fixed phase peptide synthesis (the
peptides described here are shorter, as a rule, than the potential
inhibitors described up till now and consist of only one type of
chemical component [=amino acids]).
[0009] high biological stability, which can be increased even more
by the simple chemical inclusion of unnatural amino acids.
[0010] broad biological and therefore potentially therapeutic
applicability (the high homology level between the corresponding
sequences of amyloid-forming peptides/proteins makes possible the
overlapping application of inhibitors in all three illnesses).
[0011] few side-effects and little antigenicity when used as a
therapeutic (the peptides, on the basis of their small size, have a
small tendency to induce immune reactions in the patient); other
inhibitor candidates which are described in the literature (see
above antibodies or higher molecular serum components) are approx.
200-300 times larger than the peptides which are described
here.
[0012] high biological activity in vitro and thus high predictable
biological activity in vivo.
[0013] thus high predictability of therapeutic application.
[0014] It has been shown that, for the three areas of application
described at the beginning, various peptides respectively are
particularly appropriate, said peptides all having homology amongst
one another (FIG. 1). The individual peptides for the three groups
are described subsequently in greater detail.
[0015] The appropriate peptide sequence, which is adequate for
amyloid formation and cytotoxicity of IAPP, is the sequence FGAIL
(one-letter code) which comprises the amino acid residues 23-27 of
IAPP. Lengthening this sequence in the direction of the N terminus
of IAPP results equally in small (<10 amino acid residues)
peptide fragments which can form fibrils (an arranged aggregate
structure, which is typical of amyloid illnesses) and has
cytotoxicity. For the spontaneous aggregation, i.e. production of
an oversaturated peptide solution of these peptides, a
concentration is required which is 100-100 times greater compared
with IAPP. These sequences were successfully used as small
molecular inhibitors of amyloid formation of IAPP, since they
contain the shortest peptide sequence and that which is necessary
for the aggregation of IAPP. Underlying this effect is the
aggregation mechanism of IAPP and, in equal measure, other amyloid
forming peptides (such as _-AP and the prion protein). The
aggregation and amyloid formation results thus from an
intermolecular _- folding leaf formation, for which intermolecular
(between the molecular chains) hydrogen bridges and hydrophobic
reciprocal effects are necessary between the side chains of certain
amino acid residues. This _-folding leaf structure leads to a
non-covalent bond firstly between two and subsequently between
several IAPP molecules which consequently form insoluble
aggregates/amyloid structures. The effect mechanism (inhibition) of
the peptides according to the invention lies in bonding the
aggregation-promoting, intermolecular reciprocal effects between
two IAPP molecules. By means of this, it has been achieved recently
that the peptides themselves are involved in these reciprocal
effects with IAPP. Thus competition (agonism) occurs between IAPP
and the peptides for the free "bonding points" of IAPP. Their
potential use as illness-diagnostics is also based on the agonistic
effect of these peptides. Because of the fact that the soluble form
of the peptides or lower concentrations (micromolar) neither
aggregate nor have cytotoxicity, their application as inhibitors
and Type II diabetes diagnostics is made possible.
[0016] The following peptide sequences are synthesised and used as
inhibitors of amyloid formation: GAIL, FGAIL, NFGAIL, NNFGAIL,
SNNFGAIL, NGGAILSS, SNNFGAILSS, the individual letters for the
amino acids exist according to the one letter code. Furthermore,
H-molecules of the amide bond of the above peptide sequences are
replaced by a methyl group, to bond the aggregation of peptides
which is induced by_folding leaf formation. The methyl groups were
introduced at every second amide bond, however varying the number
of methyl groupings. The peptide analogues produced contain a
number of N-methyl substituted amide bonds, which extend between
one and half of the present amide bonds per molecule. Underlying
the design of this class of aggregation inhibitors lies the idea
that, by introducing the methyl group at every second amide bond,
the dimerisation or the non-covalent bond of the small molecular
peptide sequences of IAPP are not impaired; the otherwise
consequent non-covalent expansion of the_folding leaf structures,
which leads to aggregation, is however comprehensively switched
off. In the following, representative examples of this substance
class which were used successfully as aggregation inhibitors of
IAPP, are named:
[0017] (N-Me)-GA-(N-Me) IL, F(N-Me)-GA-(NME) IL,
NF(N-Me)-GA-(N-Me)IL,
[0018] NNF(N-Me)-GA-(N-Me)IL, SNNF (N-Me)-GA-(N-Me)IL,
[0019] NF(N-Me)-GA-(N-Me)ILSS, SNNF
(N-Me)-GA-(N-Me)-GA-(N-Me)IL,
[0020] G(N-Me)-AI(N-Me)-L, FG(N-Me)-AL(N-Me)-L,
NFG(N-Me)-AI(N-Me)-L,
[0021] NNFG (N-Me) -AI(N-Me) -L, SNNFG(N-Me) -AI(N-Me)-L,
[0022] NFG(N-Me)AI(N-Me)-LSS, SNNFG(N-Me)-AI(N-Me)-LSS,
FGA(N-Me)-IL,
[0023] NFGA(N-Me)-IL, NNFGA(N-Me) -IL, SNNFGA(N-Me) -IL,
NFGA(N-Me)-ILSS,
[0024] SN(N-Me)-NFGAILSS, N-Me)-SN(N-Me)-NFGAILSS, (
[0025] N-Me)-SN(N-Me)-NF(N-Me)-GAILSS etc.
[0026] In the appropriate peptide, which is used for amyloid
formation and cytotoxicity of _-AP, sequence regions of _-AP, which
are found between the amino acids 25 and 34, are of concern. In
analogy to the sequence 23-27 of IAPP, the sequence of 28-32
(KGAII) in _-AP was found to be the appropriate substance for
forming amyloids and for neurotoxicity of the entire molecule _-AP.
On the basis of the same considerations as in the case of the IAPP
amyloid forming inhibitors, the following were synthesised to IAPP
homologue _-AP sequences and used as inhibitors of _-AP amyloid
formation and _-AP neuro-toxicity: GAII, KGAII, NKGAII, SNKGAII,
GSNKGAII, NKGIIGL, GSNKGAIIGL. Furthermore, as is described above,
analogues were also produced with substituted amide bonds. The
structures of these analogues were designed according to the same
principle as the N-methylated inhibitors of IAPP amyloid formation
(see above). Representative examples of this substance class are
listed in the following:
[0027] (N-Me)GA-(N-Me)II, K(N-Me)-GA-(N-Me)II,
NK(N-Me)-GA-(N-Me)II,
[0028] SNK(N-Me)-GA- (N-Me) II, GSNK(N-Me) -GA- (N-Me)II, NK(N-Me)
-GA- (N-Me)IIGL,
[0029] GSNK(N-Me)-GA-(N-Me)II, G(NMe)AI(N-Me)-I,
KG(N-Me)-AI(N-Me)-I,
[0030] NKG(N-Me)-AI(NMe)-I, SNKG(N-Me)-AI(N-Me)-I,
GSNKG(N-Me)-AI(N-Me)-I,
[0031] NKG(N-Me)-AI(N-Me)-IGL, GSNKG(N-Me) -AI (N-Me)-IGL,
KGA(N-Me) -II,
[0032] NKGA(N-Me)-II, SNKGA(N-Me)-II, GSNKG- A(N-Me)-IIGL,
[0033] GS(N-Me)-NKGAIIGL, (N-Me)-GS(N-Me) - NKGAIIGL,
[0034] (N-ME)-GS(N-Me)-GAIIGL etc.
[0035] The most suitable sequence, which is adequate for amyloid
formation and cytotoxicity of PrP is AGAVV. This is to do with a
partial sequence from the sequence 110-119 PrP. Further sequences
for use as aggregation and toxicity inhibitors of PrP are GAIL,
AAGAVV, VYYGAVV, HVAAGAVV, AAGAVVGG, HVAAGAVVGG. The corresponding
N-methylated peptide sequences--analagous to the IAPP derivatives
(see above)--are also suitable as aggregation and toxicity
inhibitors. Some representative examples of N-methylated analogues
are presented in the following:
[0036] (N-Me)-GA-(N-Me)VV, A(N-Me)-GA-(N-Me)VV,
AA(N-Me)-GA-(N-Me)VV,
[0037] AAGA(N-Me)-VVGG, HV(N-Me)-AAGAVVGG,
(N-Me)-HV(N-Me)-AAGAVVGG,
[0038] (N-Me)HV(N-Me)-AA(N-Me)-GAVVGG etc.
[0039] This invention is therefore concerned with peptide molecules
which can be used as the basic structures (template molecules) for
inhibiting and analysing amyloid formation and cytotoxicity in
amyloid illnesses. In this respect, these peptides have an effect
on the molecules which are responsible for the amyloid illnesses
(for their part amyloid-forming peptides and proteins). The
peptides are thus either inhibitors themselves or agonists of
amyloid formation and cytotoxicity or can serve as a template for
identifying and producing further inhibitors and agonists and can
be used as molecular tools during analysis.
[0040] These peptides can be used as pharmaceutical inhibitors of
amyloid formation and cytotoxicity or as molecular tools for
analysing amyloid formation and cytotoxicity in amyloid illnesses.
Thus, this is to do with potential pharmaceuticals and analyses for
the treatment and diagnosis of the following illnesses which also
occur in humans:
[0041] Alzheimer's disease
[0042] Type II diabetes mellitus
[0043] Spongiform encephalopathies
[0044] (e.g. Creutzfeld-Jacob disease, scrapie, BSE)
[0045] The diagnostic use comprises two aspects:
[0046] Use as a molecular tool for further researching the
mechanism for amyloid formation in these illnesses (use in research
laboratories and R and D laboratories)
[0047] Potential use as a reagent for diagnosing amyloid illnesses
or the prediction of such illnesses (diagnostic market).
[0048] The invention is explained subsequently in greater detail
with the aid of an embodiment example.
[0049] NFGAIL: Production, characterisation, aggregation, testing
for fibril formation, testing for cytotoxicity, testing for the
inhibitor effect on the aggregation of IAPP.
[0050] Production and Characterisation
[0051] NFGAIL was produced using current methods of fixed phase
peptide synthesis. The Wang anchor was used and the Fmoc/tBu
synthesis strategy was adopted. For one 1 mMol application, 4 mMol
protected amino acids, 4 mMol of TBTU and 6 mMol of DIEA in DMF
were used per coupling step. The splitting of the temporary
protected groups (Fmoc) was achieved by means of 25% piperidine in
DMF and, in order to split the peptide from the anchor while
simultaneously splitting the permanent protected groups of the side
chains, 95% TFA was used (reaction time 2 hours). The pipe product
was cleaned using preparative RP-HPLC and characterised by FAB-MS
and amino acid analysis.
[0052] Aggregation and Testing for Fibril Formation
[0053] The aggregation properties were tested in 10 mM Of phosphate
butter pH 7.4. Firstly, a highly concentrated (125-250 mM) parent
solution of the peptide in DMSO was produced. The peptide is
pipetted from the solution directly into the aggregation solution
while being gently stirred. A 5 mM peptide solution aggregated
spontaneously and thus did not have an "Aggregation-Lag-Time". On
the other hand, aggregate formed only after 9.5 hrs. from a
solution of 3.75 mM NFGAIL (Aggregation-Lag-Time: 9.5 hrs) (FIG.
2). Furthermore, aggregation was accomplished immediately, when
pre-prepared NFGAIL fibrils (nucleation centres) were added to the
3.75 mM solution (fibril concentration 0.375 mM). It was shown
thus, that the aggregation of these peptides operates according to
the so-called "nucleation dependent polymerisation mechanism".
Recently it has been considered of great importance, since the
opinion prevails in the literature, that amyloid formation in vitro
should operate using this mechanism. After complete aggregation,
the deposit was isolated by centrifuging and examined by electron-
and polarisation- microscopy after congo red staining. In this way,
the fibrillar and amyloid structure of the aggregate was confirmed
(FIG. 3).
[0054] Testing for Cytotoxicity
[0055] Suspensions of the peptide aggregate (produced as described
as above) were tested by means of a Rat Insulinoma (RIN5 mf) and a
human astroglioma-cell series (HTB-14) for toxicity. The newly
dissolved peptides were also tested for cytotoxicity. It thus be
shown, that the aggregated form of NFGAIL is cyto-toxic, the
soluble form of the peptide having no toxicity (FIG. 4).
* * * * *