Peptides for Therapy and Diagnosis of Alzheimer's Disease.

2012·0.18 MB·English

by  Funke, Susanne Aileen

Current Pharmaceutical Design, 2012, 18, 755-767 755
Peptides for Therapy and Diagnosis of Alzheimer’s Disease
Susanne Aileen Funke1 and Dieter Willbold1,2*
1Forschungszentrum Jülich, ICS-6, 52425 Jülich, Germany; 2Institut für Physikalische Biologie, Heinrich-Heine-Universität, 40225
Düsseldorf, Germany
Abstract: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with devastating effects. The greatest risk factor to de-
velop AD is age. Today, only symptomatic therapies are available. Additionally, AD can be diagnosed with certainty only post mortem,
whereas the diagnosis “probable AD” can be established earliest when severe clinical symptoms appear. Specific neuropathological
changes like neurofibrillary tangles and amyloid plaques define AD. Amyloid plaques are mainly composed of the amyloid-(cid:2) peptide
(A(cid:2)). Several lines of evidence suggest that the progressive concentration and subsequent aggregation and accumulation of A(cid:2) play a
fundamental role in the disease progress. Therefore, substances which bind to A(cid:2) and influence aggregation thereof are of great interest.
An enormous number of organic substances for therapeutic purposes are described. This review focuses on peptides developed for diag-
nosis and therapy of AD and discusses the pre- and disadvantages of peptide drugs.
Keywords: Alzheimer’s disease, amyloid-(cid:2), therapy, diagnosis, peptides.
INTRODUCTION addressed for therapy development. As a consequence, the majority
of AD therapeutic research has been focused on the A(cid:2) peptide.
Alzheimer’s disease (AD) is a devastating neurodegenerative
disorder and the most common cause of dementia. The clinical Less effort has been directed towards the development and valida-
tion of tau-targeted therapeutic compounds. A number of tau fibril
characteristics are difficulties with memory, apathy and depression,
formation inhibitors, derived from multiple chemical classes, have
impaired judgment, disorientation, confusion and other. The great-
est risk factor for AD is age. In 2007, AD affected 27 million peo- been identified, as reviewed elsewhere [15]. To date, only one tau
fibrillization inhibitor, the phenothiazine methylene blue [16], has
ple world-wide with steadily increasing tendency. By 2050, the
entered in vivo evaluation. Promising first results have been pre-
prevalence is estimated to quadruple, thereby raising significant
economic problems, not to mention the suffer of each affected indi- sented at the ICAD meeting in 2008, but up to now have not been
published in a peer-reviewed journal. Currently, only one peptide
vidual [1].
compound addressing tau pathology is known. Davunetide (DAP) is
The pathological hallmarks of AD are the presence of neurofi-
an eight amino acid peptide derived from the activity-dependent
brillary tangles and amyloid deposits in the brain of the patient, as
neuroprotective protein ADNP. It decreases tau phosphorylation
already defined by Alois Alzheimer in 1907 [2]. Neurofibrillary
and A(cid:2) levels in tau transgenic mice and 3 x transgenic (tg)-AD
tangles are aggregates of paired helical filament composed of the
mice. The intranasal formulation AL-108 is currently in clinical
abnormally phosphorylated and (cid:2)-folded tau protein. Tau is a hy-
development [17-21].
drophilic microtubule binding protein which is expressed in six
human isoforms of 352 to 441 amino acid residues [3-5]. Therapy approaches targeting A(cid:2) include reduction of A(cid:2) pro-
duction by inhibitors or modulators of the (cid:2)- or (cid:3)-secretases, A(cid:2)
A(cid:2) is the major component of the amyloid plaques. It consists
immunotherapy, and inhibition or modulation of A(cid:2) polymerization
of 39 to 43 amino acid residues. A(cid:2), especially A(cid:2)1-42, is prone to
[22, 23]. Examples for the latter are scyllo-inositol [24, 25], amino-
aggregation and undergoes formation from monomers to oligomers,
propane sulfonic acid (Tramiprosate) [26], PBT-1 [27], polyphenol
larger intermediate forms like protofibrils, and, insoluble fibrils and
(-) epigallocatechin-3-gallate (EGCG) [28, 29], oligomeric acylated
plaques [6]. A(cid:2) is derived from the amyloid precursor protein
aminopyrazoles [30] and several more.
(APP) by sequential activities of the (cid:2)- and (cid:3)-secretases [7-9]. As
originally suggested by the amyloid cascade hypothesis, it appears Peptides, which are specified as (linear) molecules consisting of
two or more (<100) amino acids residues, are today reasonable
likely that A(cid:2) peptides and their aggregated forms initiate cellular
alternatives to chemical pharmaceuticals. They are key regulators of
events leading to the pathologic effects of AD. According to a pre-
vious version of the amyloid cascade hypothesis, fibrillar forms of biological functions and offer high biological activity associated
with high specificity and low toxicity. The peptide market is grow-
A(cid:2), deposited in amyloid plaques, have been thought to be respon-
ing fast due to an increased number of therapeutic targets, improved
sible for neuronal dysfunction [7-11]. More recent studies support
that diffusable A(cid:2) oligomers including protofibrils, prefibrillar delivery methodologies, the establishment of large biological and
synthetic peptide libraries, and high throughput screening or selec-
aggregates and so called A(cid:2)-derived diffusible ligands (ADDLs),
tion. Today, 67 therapeutic peptides are on the market, 150 in clini-
are the major toxic species during disease development and pro-
gression [12-14]. cal phases and more than 400 in the pre-clinics. In spite of this pro-
gress, the development of peptide drugs can be severely hampered
Currently, only palliative therapies for AD are available. Ace-
by their short half-life in vivo. In general, peptides are rapidly de-
tylcholine inhibitors like Donepezil, Galantamine and the NMDA
graded by proteases, and their nature implies problems for admini-
receptor antagonist Memantine have been approved for clinical use
stration and delivery, especially to the brain. These problems can at
as treatment of cognitive symptoms.
least partially be overcome as peptide chemistry permits a variety
Although it is still controversial if A(cid:2) is the causative agent of of methods for peptide modification or the use of D-enantiomeric
AD, inhibition of A(cid:2) production and aggregation are often amino acid residues [31-33].
A variety of small peptides that inhibit aggregation of A(cid:2) and
*Address correspondence to this author at the Forschungszentrum Jülich, reduce its toxic effects were already described and a fraction of
ICS-6, D-52425 Jülich, Germany; Tel: [phone]; them shown to be effective in AD rodent animal models. Addition-
Fax: [phone]; E-mail: [email protected] ally, A(cid:2) binding peptides, developed for a suitable use in in vivo
1873-4286/12 $58.00+.00 © 2012 Bentham Science Publishers
756 Current Pharmaceutical Design, 2012, Vol. 18, No. 6 Funke and Willbold
imaging methods and possibly useful for early diagnosis of AD, strated to bind A(cid:2) with an affinity in the submicromolar range.
were described. Both types of peptides, designed for different ap- Employing surface plasmon resonance, binding to A(cid:2) oligomers
plications, are reviewed in this article. All peptides discussed in this and fibrils, but not to monomers could be demonstrated. D1 stained
article and some more are listed in Tables 1 and 2A to 2F, but al- amyloid plaques in the brain tissue sections derived from AD pa-
though we scanned the literature exhaustively, this list does not tients, whereas other, non-A(cid:2) amyloidogenic deposits, were not
claim to be complete. stained [45, 46]. D1 was also tested for its in vivo binding charac-
teristics in APP/PS1 transgenic mice. Upon direct injection into the
PEPTIDES DEVELOPED FOR AD DIAGNOSIS BY IN VIVO brain, D1 bound very specifically to A(cid:2)1-42, staining all dense
IMAGING METHODS (SEE TABLE 1) deposits in the brain but not diffuse plaques, which contain mainly
Today, the specificity of AD diagnosis can already be improved of A(cid:2)1-40 and are not AD specific [47]. This demonstrated that D1
using glucose metabolism sensing positron emission tomography might be suitable for further development into a molecular probe to
(PET) experiments [34] and perfusion single photon emission com- monitor A(cid:2)1-42 plaque load in the living brain.
puted tomography (SPECT) [35]. The appearance of amyloid In 2008, Larbanoix et al. selected A(cid:2)1-42 binding peptides
plaques probably occurs many years before cognitive symptoms using a random disulfide constrained heptapeptide phage display
appear [36, 37]. Therefore, in vivo detection and quantification of library. Two clones (see sequences in Table 1) were enriched. The
amyloid species in the brains of patients during the course of the K -values for the phage clones, on which several peptide copies are
d
disease, for early diagnosis and the evaluation of the effects of AD- displayed, were in the picomolar range. After peptide synthesis
therapies, is an emerging field in AD research. The best characteri- including biotinylation, the binding affinities dropped to the mi-
zation of amyloid plaque load in the brain can be expected from cromolar range. Nevertheless, preliminary in vivo studies in trans-
imaging approaches using amyloid ligands as contrast agents. To genic mice, in which the functionalized peptides were used as con-
date, many A(cid:2) binding contrast substances failed due to intolerable trast agents, showed high contrast effects in APP /PS1
V717I A246E
unspecific binding or poor distribution in the brains of animals. transgenic mice, but not in wildtype controls. For the experiments,
Only a few PET ligands have been applied to clinical studies (for a however, the blood brain barrier (bbb) had to be permeabilized
review, see ref. [38]). The most prominent and best studied is Pitts- artificially with 25 % mannitol [48]. Very recently, Larbanoix et al.
burgh compound B (PIB) [39], a benzothiazole derivative binding designed another linear hexapeptidic phage display library based on
fibrillar A(cid:2). Novel A(cid:2) binding substances, suitable for in vivo im- the A(cid:2)1-42 amino acid sequence and selected against aggregated
aging, are urgently needed. Small A(cid:2) binding peptides with favor- A(cid:2)1-42 as a target. Two of 26 selected clones, presenting highest
able drug properties could easily be coupled to radionucleides or binding affinities to A(cid:2)1-42, were translated to synthetic peptides
other markers for imaging of amyloid plaques in living AD patients. with biotine label (Pep1: LIAIMA and Pep2: IFALMG, correspond-
Phage display technologies allow the identification of peptide ing A(cid:2) fragment IIGLMV31-36) and presented lower Kdvalues (still
ligands for a given target molecule out of a huge library of different in the micromolar range) as the peptides described in the first arti-
peptides expressed on the surface of bacteriophages. Presentation of cle. Pep1 and Pep2 were highly hydrophobic and are expected to
the peptide library on the surface of bacteriophages ("phage dis- pass the bbb very well. The peptides did not show any sign of toxic-
play") as a fusion of peptide and a phage coat protein allows the ity in cell culture. The specific interaction of both peptides with
physical link between the presented peptide and the DNA sequence amyloid plaques in human brain tissue was demonstrated by immu-
coding for its amino acid sequence. Diversity of the pep- nohistochemistry [49].
tides/proteins can be introduced by combinatorial mutagenesis of
THERAPEUTIC PEPTIDES (SEE TABLES 2A-D)
the fusion gene. Extremely large numbers of different peptides can
be constructed, replicated, selected and amplified in a process A(cid:2)-sequence Derived Peptides (see Table 2A)
called "biopanning". In 2003, Kang et al. have employed phage In 1996, Tjernberg and coworkers searched for an A(cid:2) ligand to
display selection to identify two 20-amino acid peptides specifically interfere with A(cid:2)-self interaction and polymerization. The strategy
binding to the amyloid form of A(cid:2)1-40, but not to monomeric A(cid:2).
was to identify binding sequences within A(cid:2) and, based on their
One of the peptides (amino acid sequence primary structures, to synthesize A(cid:2) derived peptide ligands. The
DWGKGGRWRLWPGASGKTEA) could be produced recombi- short A(cid:2) fragment KLVFF (A(cid:2)16-20) was identified to bind full
nantly in E. coli as a fusion protein with thioredoxin, as well as the
length A(cid:2) and to prevent the fibrillization thereof. Alanine substitu-
chemically synthesized version. The recombinant thiopeptide bound tion experiments revealed that amino acids Lys16, Leu17 and
A(cid:2)1-40 amyloid with a Kdof 60 nM, determined by ELISA. Both Phe20 were critical for A(cid:2) interference [50]. A molecular modeling
versions specifically stained amyloid plaques in brain tissue slices
study suggested that the association of full length A(cid:2) and the
of AD patients. The authors discussed the molecules as potential KLVFF peptide lead to the formation of atypical antiparallel (cid:2)-
probes for in vivo imaging as well as potential carrier molecules to sheet structures stabilized by Lys16, Leu17 and Phe20 [51].
deliver other therapeutic molecules like antioxidants, chelators, and
The effects of KLVFF containing or derived synthetic peptides
plaque degrading compounds to the desired location of action [40].
was further approved in a variety of studies, e.g. by the one of Ma-
To select for an A(cid:2)-binding D-enantiomeric peptide, which
tsunaga et al. [52], see Table 2A. Additionally, it was proposed that
specifically binds to fibrillar A(cid:2) species and plaques, aggregated D- conjugates, bearing several copies of the KLVFF sequence or the
enantiomeric A(cid:2) was used as a target in a mirror phage. Mirror
retro-inverso version thereof, linked to dendrimers or to branched
phage display allows the use of phage display to identify peptides
poly(ethylene glycol) moieties, possess superior affinity and effi-
that consist solely of D-amino acids. D-enatiomeric peptides are ciency [53, 54]. In 2008, Austen et al. designed KLVFF derived
highly resistant to proteases, which can dramatically increase serum
compounds to address very early aggregation intermediates of A(cid:2),
and saliva half-life. Additionally, D-peptides can be absorbed sys-
i.e. A(cid:2) oligomers. The idea was to add water soluble amino acids
temically after oral administration. D-peptide immunogenicity is residues to KLVFF, thereby generating the peptides OR1
reported to be reduced in comparison to L-peptides [41-43]. In the
(RGKLVFFGR) and OR2 (RGKLVFFGR-amid). Both peptides
selection process, the A(cid:2)1-42 D-enantiomer was used as a target for
inhibited A(cid:2) fibrillogenesis, whereas OR2 additionally inhibited
selection of peptides displayed on the surface of M13 bacterio- oligomer formation and A(cid:2) toxicity on SY5Y cells, supporting the
phages for those that bind best to D-A(cid:2)1-42. For reasons of symme-
idea that particularly oligomers are responsible for the cytotoxic
try, the D-enantiomeric form of the selected 12-mer peptide will
effects of A(cid:2) [55]. Unlike OR2, the retro-inverso D-enantiomeric
also bind to the native L-form of A(cid:2)1-42 [44]. The most representa- version (RI-OR2) of the peptide was highly resistant to proteolysis
tive peptide in the selection procedure, denoted D1, was demon-
and stable in human plasma and brain extracts [56]. Additionally, it
Alzheimer’s Therapy Current Pharmaceutical Design, 2012, Vol. 18, No. 6 757
Table 1. Peptides Selected for In vivo Imaging
Name Sequence Description D/L Results Reference
none DWGKGGRWRLWPGASGKTEA Selected by phage L Binds amyloid form of A(cid:2)40, labels amy- Kang et al., 2003
and display loid plaques in AD brains slices, discussed [40]
PGRSPFTGKKLFNQEFSQDQ as carrier protein for plaque treatment and
in vivo imaging
D1/ ACI-80: QSHYRHISPAQV Selected by mirror D Aggregate specific, stains human A(cid:2) Wiesehan et al.,
image phage dis- plaques selectively, stains plaques in mice 2003 [46]
ACI-
play ex vivo
80 Van Groen et al.,
2009 [47]
none C-IPLPFYN-C / Selected by phage L K for A(cid:2)42 in micromolar range, specific Larbanoix et al.,
d
display interaction with plaques on brain sections 2008 [48]
C-FRHMTEQ-C
(immunochemistry), encouraging prelimi-
nary MRI in vivo study in mice after open-
ing of bbb by mannitol
Pep1 LIAIMA Selected by phage L K for A(cid:2)42 in micromolar range, specific Larbanoix et al.,
d
display, library interaction with plaques in human brain 2011 [49]
Pep2 IFALMG
based on A(cid:2) se- sections (immunochemistry),
quence
inhibit A(cid:2) aggregation
bbb: blood brain barrier. D/L: describes peptide conformation.
Table 2A. Peptides Influencing A(cid:2) Aggregation and Toxicity. A(cid:2)-sequence Derived Peptides
Name Sequence Description D/L Results Reference
A(cid:2)(16-20) KLVFF Based on A(cid:2) sequence L Prevents A(cid:2) fibrilization, identification Tjernberg et al., 1996, 1997
of key amyloidogenic region [50, 51]
A(cid:2)- (15-22) Include Based on A(cid:2) sequence L Inhibit A(cid:2) aggregation in vitro Matsunaga et al., 2004 [52]
A(cid:2)- (16-23) KLVFF
A(cid:2)- (17-24)
K4 KLVFF- A(cid:2) sequence conjugate L Inhibitory effect of compound is potenti- Chafekar et al., 2007 [53]
dendrimer ated in comparison to monomer
none KLFVV retro- A(cid:2) sequence conjugate D Inhibitory effect on A(cid:2) aggregation is Zhang et al., 2003 [54]
inverso version potentiated
linked to
branched hex-
americ PEG
OR1, OR2 RGKLVFFGR Based on A(cid:2)-(16-20) L/D Inhibit A(cid:2) oligomerization, aggregation Austen et al., 2008; Matha-
or region and toxicity. Retro-inverso-peptide resis- ru et al., 2010 [55, 57];
tant to proteolysis and more active Taylor et al., 2010 [56]
RGKLVFFGR-
NH
2
A(cid:2)- (31-35) RIIGL Based on A(cid:2) sequence L Inhibits A(cid:2) aggregation in vitro Fülöp et al., 2004 [61]
IIGL
none A(cid:2)(x-42) C-terminal A(cid:2) sequence L Stabilize A(cid:2) in non-toxic oligomers, Fradinger et al., 2008 [62]
X=28-39 derived peptides inhibit A(cid:2) neurotoxicity
A(cid:2)12-28P A(cid:2)(12-28) Peptides block A(cid:2)-Apo D Inhibition of aggregation in vitro, reduc- Sadowski et al., 2004 [66]
E4 interaction by compet- tion of plaque formation in tg mice
V18P mutation,
end-protected ing for the binding site
D/L: describes peptide conformation. PEG: poly (ethylene glycol). A(cid:2)-derived peptide sequences are written in bold. Tg: transgenic
758 Current Pharmaceutical Design, 2012, Vol. 18, No. 6 Funke and Willbold
Table 2B. Peptides Influencing A(cid:2) Aggregation and Toxicity. (cid:2)-sheet Breaking Peptides Based on the KLVFF Sequence
Name Sequence Description D/L Results Reference
nn KLVFF based, (cid:4)-sheet breaker L Enhance fibrillization of A(cid:4) oligomers Ghanta et al., 1996; Pallitto
with chain of and therefore reduce A(cid:4) toxicity. Effec- et al., 1999; Lowe et al.,
charged amino tiveness of inhibitor is dependent on its 2001; Kim et al., 2004;
acids as disrup- surface tension modifying properties Moss et al., 2003; Kim et
tion motif al., 2003; Gibson et al.,
2005 [69-75]
A(cid:4)16-20e KLVFF with No hydrogen bond can be L Inhibit A(cid:4) aggregation, disassembles Gordon et al., 2003 [77, 78]
ester sub- formed fibrils.
stitution
Expected to hydrolyze rapidly in vivo
AMY-1 KLVFF based (cid:4)-sheet breaker (cid:2),(cid:2)- L Inhibition of fibrillization, globular Etienne et al., 2006 [79]
AMY-2 disubstituted amino acids aggregates are formed
P1, P2 KLVF-(cid:3)A-I- Disruption of aggregation L Inhibit A(cid:4) aggregation Rangachari et al., 2009 [81]
(cid:3)A and by different local confir-
mation
KF- (cid:3)A-(cid:3)A-
(cid:3)A-F
D/L: describes peptide conformation. (cid:3)Ala:(cid:2),(cid:4)-dehydroalanine.A(cid:4)-derived peptide sequences are written in bold.
Table 2C. Peptides influencing A(cid:2) aggregation and toxicity. (cid:2)-sheet breaking peptides based on proline
Name Sequence Description D/L Results Reference
iA(cid:4)5 Ac-LPFFD- Proline (cid:4)-sheet breaker L/D A(cid:4) fibril inhibition and de-fibrillization Soto et al., 1996; Soto et
amid in vitro. Reduction of plaque load and al., 1998; Poduslo et al.,
A(cid:4) induced pathological processes in rat 1998; Sigurdsson et al.,
model and in AD tg mice. Improvement 2000; Permanne et al.,
of rat spatial memory impairments 2002; Chacon et al., 2004
[68, 83-86, 135]
iA(cid:4)5 LPFFD- Proline (cid:4)-sheet breaker L Methylation of amide nitrogen increased Adessi et al., 2003 [131]
derivatives in vitro and in vivo stability while main-
taining iA(cid:4)5 activity in vitro
iA(cid:4)-PEG LPFFD-PEG Proline (cid:4)-sheet breaker L Biological activity of iA(cid:4) (in vivo) is not Rocha et al., 2009 [136]
5 5
affected by PEG. Aimed to improve
pharmacological properties, especially in
vivo degradation
none LPYFD Proline (cid:4)-sheet breaker L Decreased neurite degeneration, tau Datki et al., 2004 [87]
aggregation and cell viability reduction
induced by A(cid:4)
LPYFDa LPYFDamid Proline (cid:4)-sheet breaker, L Protects neurons in vitro and in vivo after Szegedi et al., 2005; Juhász
amidated intraperitonial administration to rat mod- et al., 2009 [88, 89]
els
D/L: describes peptide conformation. Tg: transgenic. PEG: poly (ethylene glycol).
Alzheimer’s Therapy Current Pharmaceutical Design, 2012, Vol. 18, No. 6 759
Table 2D. Peptides Influencing A(cid:2) Aggregation and Toxicity. (cid:2)-sheet Breaking Peptides Based on Methyl-Amino Acids
Name Sequence Description D/L Results Reference
none N-methylated First methylated (cid:2)-sheet L Prevent A(cid:2) aggregation in Hughes et al., 2000 [93, 94]
GSNKGAIIGLM breakers vitro, inhibit cell toxicity
A(cid:2)16-22m KLVFF based e.g. Methylated (cid:2)-sheet breaker, L Prevent A(cid:2) fibril forming, Gordon et al., 2001, 2002
A(cid:2)16-20m KNmeLVNMEFFNMeAE N-methyl groups in alternat- disassemble fibrils in vitro [78, 95]
ing positions
inL (all L) LKLVFF based, Methylated (cid:2)-sheet breaker, L/D Reduces A(cid:2) toxicity in cell Cruz et al., 2004; Grillo-
culture Bosch et al., 2009 [96, 97]
in D (all D) N-methyl-20F single N-methyl-amino acids
inrD (retro-inverso)
PPI-1019 Methyl-LVFFL Methylated (cid:2)-sheet breaker D Completed phase I and II Jhee et al., 2003 [98, 99]
“Apan” human clinical trials
e.g. SEN304 e.g. “meptide”, D Highly active inhibitors of Kokkoni et al., 2006 [100]
A(cid:2) aggregation and toxicity
D-chGly-D-Tyr-D- methylated (cid:2)-sheet breaker
chGly-D-chGly-D-
mLeu
D/L: describes peptide conformation. chGly: cyclohexylglycine; mLeu: N-methylleucine; NMe: N-methylated amino acids. A(cid:2)-derived peptide sequences are written in bold.
Table 2E. Peptides Influencing A(cid:2) Aggregation and Toxicity. Other (cid:2)-sheet Breaking Peptides
Name Sequence Description D/L Results Reference
e.g. e.g. Cholyl- (cid:2)-sheet breaker L/D A(cid:2) specific, inhibit A(cid:2) aggregation Findeis et al., 1999, 2001,
PPI-368 LVFFA A(cid:2)-binding sequences potently, reduces A(cid:2) cell toxicity 2002 [98, 101, 102]
with cholyl-bulky group
PPI-457
A(cid:2)-(38-42) GVVIA, Based on A(cid:2) sequence, L Inhibit A(cid:2) aggregation and toxicity Hetényi et al., 2002 [132]
RVVIA amidated at C-termius
Trp-Aib (cid:2)-sheet breaker D Inhibition of A(cid:2) oligomer formation in Frydman-Marom et al.,
vitro, effective in AD tg mice after oral 2009 [103]
application
none EIVY-rest (cid:2)-sheet breaker L Modulate A(cid:2) aggregation, depending of Sun et al., 2009 [76]
see text in subchapter solvent disruptive amino acid sequence.
“KLVFF derived (cid:2)-sheet Discussed in this article with KLVFF-
breakers” based (cid:2)-sheet breakers
D/L: describes peptide conformation. AIB: (cid:3)-aminoisobutyric acid; tg: transgenic. A(cid:2)-derived peptide sequences are written in bold.
was reported that retro-inversions of OR1 and OR2 increase the most effective inhibitors of A(cid:2) induced cell toxicity. The peptides
inhibitory effects of the peptides [57]. additionally prevented the disturbance of synaptic activity by A(cid:2)
oligomers. To investigate the in vitro mechanism of action, dy-
In 2004, Fülop et al. developed an A(cid:2) aggregation inhibitor
based on the A(cid:2)31-34 sequence IIGL, which also plays a funda- namic light scattering, photo-induced cross-linking and discrete
molecular dynamics were applied and gave clear hints that the pep-
mental role in A(cid:2) aggregation and cytotoxicity [58-60]. As in the
tides inhibit A(cid:2) induced toxicity by stabilizing A(cid:2) in non-toxic
study described above, the strategy was to link a solubilizing amino
acid residue to the original sequence RIIGL. In contrast to oligomers [62].
propionyl-IIGL, another derivative of the same sequence, PIIGL The inheritance of the apolipoprotein (apo) E4 allele has been
did not self-aggregate and was not toxic to cells in culture. RIIGL identified as a major genetic risk factor for sporadic AD [63]. All
inhibited the formation of A(cid:2) fibrils and reduced A(cid:2) cytotoxicity Apo isoforms are discovered to act as pathological chaperones and
[61]. propagate A(cid:2) fibril formation, with apo E4 being the most efficient
In 2008, Fradinger et al. prepared a series of A(cid:2) C-terminal isoform [64, 65]. Sadowski et al. investigated weather blocking of
the interaction between apo E4 and A(cid:2) can have therapeutic effects
fragments (A(cid:2)x-42; x = 28-39). The authors of the article tested the
hypothesis that C-terminal peptides of A(cid:2) should possess high af- [66]. In earlier studies, Ma et al. demonstrated that the synthetic
finity to full length A(cid:2) and might disrupt oligomer formation, as the peptide A(cid:2)12-28 can be used as inhibitor of apo E4-A(cid:2) interaction,
inhibiting A(cid:2) fibril formation in vitro [67]. Sadowski et al. modi-
C-terminus is supposed to be a key region controlling A(cid:2) aggrega-
tion. Cell viability assays identified A(cid:2)31-42 and 39-42 to be the fied the A(cid:2)12-28 sequence: substitution of valine at position 18 to
proline rendered the peptide non-amyloigogenic and untoxic
760 Current Pharmaceutical Design, 2012, Vol. 18, No. 6 Funke and Willbold
Table 2F. Peptides Influencing A(cid:2) Aggregation and Toxicity. Peptides Selected Using Combinatorial Libraries
Name Sequence Description D/L Results Reference
DP1 – DP8 6 residues from: “Decoy” peptides of D Eliminate calcium effect of A(cid:2)1- Blanchard et al., 97,
combinatorial library 42 2000 [106, 107]
Ala, Ile, Val, Ser, Thr, Gly
None Several with 4 groups of consensus Out of combinatorial L Inhibit A(cid:2) aggregation Schwarzman et al.,
sequences: library 2005 [108]
++H+(H/+)
or
++XX+
or
(D/E)LVH
or
+LVLF
None 12 mers Phage Display with two L Ligands bind targets in different Orner et al., 2006
libraries. aggregation states, partly affect [109]
Parent molecule: aggregation
KLVFFKKKKKK
Target: Monomeric and
fibrillar A(cid:2).
ANA1 6 or 15 mers Novel selection involving L Inhibit A(cid:2) toxicity Taddei et al., 2008
ANA2 e.g. TNPNRRNRTPQMLKR phage display and coun- [110]
(ANA1) terselection against rat A(cid:2)
ANA3
1A, 1B, 2 MSNKGASIGLMAGDVDIADSHA Combinatorial library and L Inhibitory effect on A(cid:2) aggrega- Bain et al., 2009 [111]
or selection based on en- tion, de-fibrillization
zyme EGFP
MSNKGASNALMAGDGDIADSHS
or
MQKLDVVAEDAGSNK
D3 RPRTRLHTHRNR Mirror image phage dis- D Modulates A(cid:2) oligomerization, Van Groen et al.,
play effective in tg mouse model after 2008; Funke et al.,
oral application 2010 [112, 113]
JM169 Trimer-TEG-D3 Hybrid compound D3 and D In vitro more effective than trimer Müller-Schiffmann et
nonpeptidic (cid:2)-sheet or D3 al., 2010 [115]
breaker
1-8 e.g. RAPMGGR or Panning of heptapeptide L Suppress A(cid:2)1-42 37/48 kDa oli- Kawasaki et al., 2010
RRPVVGR library against soluble gomer formation, keep monomer [128]
A(cid:2)1-42 stable
GN- GNLLTLD Peptide homologous to L Modulates aggregation, prevents Paula-Lima et al.,
peptide sequence from apo A-I, hippocampal neuronal cultures 2009 [119]
selected by phage display from A(cid:2) induced degeneration
D-4F Ac-DWFKAFYDKVAEKFKEAF- Apo A-I mimetic peptide D Inhibits A(cid:2) deposition and im- [120]
NH prover cognitive performance of
2
AD tg mice after oral application
D/L: describes peptide conformation. TEG: triethyleneglycol. +: positively charged amino acids. H: hydrophobic amino acid. X: hydrophobic or polar amino acid
whereas the affinity of the peptide to apo E4 was not affected. The
use of D-amino acids and end-protection increased the serum half- (cid:2)-sheet Breaking Peptides (see Tables 2B-2E)
life of the peptide substantially. A(cid:2)12-28P blocked A(cid:2)-apoE4 in- The first (cid:2)-sheet breaker peptides were reported by Soto et al.
teraction and reduced A(cid:2) fibrillogenesis and toxicity in vitro. The in 1996 [68]. In general, the term (cid:2)-sheet breaker describes com-
peptide was bbb-permeable and inhibited A(cid:2) deposition in AD pounds, containing an A(cid:2) recognition or binding motif which pro-
transgenic mice [66]. vides specificity, combined with an A(cid:2) oligomer or fibril disrupting
Alzheimer’s Therapy Current Pharmaceutical Design, 2012, Vol. 18, No. 6 761
motif which can consist of charged amino acids, prolines, methy- proteases [68]. In later studies, iA(cid:4)5 was shown to inhibit A(cid:4) cyto-
lated amino acids, cholyl-groups and others. By far the most A(cid:4) toxicity in cell culture. It also reduced A(cid:4) fibrillogenesis in a rat
aggregation inhibiting peptides are (cid:4)-sheet breaker, demonstrating brain model of amyloidogenesis. Male Fischer-334 rats were in-
the effectiveness of these compounds. In most cases, the A(cid:4) recog- jected with freshly solubilized A(cid:4)1-42 directly into the amygdala
nition domain is based on the A(cid:4) sequence. and the animals were sacrificed 8 days later. Co-injection of iA(cid:4)5
in 20 % molar excess lead to significantly reduced plaque deposi-
(cid:2)-sheet Breaking Peptides Based on the KLVFF Sequence (see tion [83]. In addition, it was shown that iA(cid:4)5 induced disassembly
Table 2B) of fibrils, reduced A(cid:4) induced histopathological changes like neu-
Starting in 1996, Ghanta et al. designed A(cid:4) binding hybrid ronal shrinkage and the extent of interleukin-1(cid:4) positive microglia
peptides based on the KLVFF-binding sequence in addition to a cells surrounding A(cid:4) deposits [84]. The chronic intraperitoneal
disruption domain consisting of a chain of charged amino acids like administration of the peptide to the rat model described above lead
KKKKKK or RRRRRR. Interestingly, some of the hybrid peptides to a significant improvement of spatial learning acquisition in Mor-
accelerated A(cid:4) aggregation, but reduced A(cid:4) toxicity. Presumably, ris water maze tests and working memory tests. To perform these
the peptides speeded up the association of potentially toxic A(cid:4) oli- studies, iA(cid:4)5 was end protected by N-terminal acetylation and C-
gomers into less toxic A(cid:4) fibrils as demonstrated by a range of terminal amidylation [85], as the non-protected peptide was unsta-
biochemical and biophysical methods. The ability of the com- ble in blood and proteolytically degraded very rapidly [86]. In
pounds to increase solvent tension was a very strong predictor on 2002, two different AD transgenic mouse models (double transgen-
the effect on A(cid:4) aggregation. Chains of charged amino acids with- ics overexpressing human APP with London mutation V717I and
out A(cid:4) recognition motif, used in the assays as control, did not human PS1 with A246E mutation, and single transgenics only
exert comparable strong effects [69-75]. In a similar study, Sun et overexpressing human APP V717I) were used to demonstrate that
al. investigated hybrid peptides composed of the critical A(cid:4) binding intraperitoneal injected, end-protected iA(cid:4)5 reduced amyloid
domain EIVY and solvent disruptive sequences poly E, K or R. The plaque formation, neuronal cell death and brain inflammatory proc-
hybrid peptides EIVY-EEEE and EIVY-KKKK enhanced A(cid:4) fibril- esses. Pharmacokinetic studies in mice and rats demonstrated good
lization, whereas EIVY-RRRR inhibited A(cid:4) aggregation and al- stability of the end-protected peptide as well as high capability of
tered the morphology of A(cid:4) fibrils to amorphous aggregates. The the peptide to cross the bbb. The mechanism of peptide action re-
ability of the hybrid peptides to interfere with A(cid:4) aggregation was mained unclear, but administration of large doses of the peptide did
also discussed in the context of their abilities to change the surface- not lead to antibody production in the treatment and evaluation
tension in solutions [76], in Table 2E. period [86].
In 2003, Gordon et al. replaced the amide bonds of A(cid:4)16-20 In 2004, Datki and Coworkers investigated the effects of a
with ester bonds in an alternating fashion. The ester peptide A(cid:4)16- LPFFD based peptide, amino acid sequence LPYFD, on the A(cid:4)
20e was monomeric under solvation conditions, inhibited A(cid:4) ag- induced changes on neuroblastoma cells. Neurite degeneration and
gregation and disassembled existing fibrils. A(cid:4)16-20e, could, how- tau aggregation were significantly decreased. Additionally, A(cid:4)
ever, build dimers. These data demonstrated that interference with induced cell toxicity was reduced [87]. The pentapeptide LPYFD-
backbone hydrogen bonding is therapeutically attractive [77, 78]. amid protected neurons against A(cid:4) toxicity in vitro and in vivo.
Intraperitoneally administered LPYFD-amid crossed the blood
Other (cid:4)-sheet breakers based on the KLVFF sequence are
brain barrier in rats at least to a certain extent, and protected against
AMY-1 and AMY-2. Both contain alpha, alpha-disubstituted amino
synaptotoxic effects of A(cid:4) up to 3.5 h after i.p. injection [88, 89].
acids at alternating positions and arrest A(cid:4) fibril growth. Instead,
large globular aggregates are formed [79]. Rangachari et al. inves-
Methylated (cid:2)-sheet Breaking Peptides (see Table 2D)
tigated the (cid:3),(cid:4)-dehydroalanine ((cid:3)Ala) containing peptides P1
(KLVF-(cid:3)A-I(cid:3)A) and P2 (KF-(cid:3)A-(cid:3)A-(cid:3)A-F). The design of P2 was Several teams have studied the effects of N-methyl amino acid
based on the experience that peptides containing the FxxxxF motif incorporation into peptides. For the resulting compounds, the term
“meptides” was established. The first methylated (cid:4)-sheet breaker
bind to the groove composed of the GxxxG motif (amino acids
G33-G37) in A(cid:4) fibrils [80] (cid:3),(cid:4)-dehydro-amino acid residues are was described by Hughes et al. After it has been suggested that
known to be strong inducers of specific peptide conformations. A(cid:4)25-25, amino acid sequence GSNKGAIIGLM, resembles a bio-
logically active region of A(cid:4) that forms large (cid:4)-sheet fibrils and is
Additionally, they increased resistance to proteolysis in vivo. Both
peptides under investigation inhibited A(cid:4) aggregation [81]. highly cytotoxic [90-92], the authors used it as a full length model
for A(cid:4)1-42 and synthesized six N-methylated derivatives to prove
Proline Based (cid:2)-sheet Breaker (see Table 2C) that those could prevent A(cid:4) wildtype aggregation and cytotoxicity.
As the derivatives were homologous to A(cid:4), they were expected to
One peptide compound which was extensively investigated in
bind the wildtype form and to prevent further addition of A(cid:4)
vitro, as well as in animal models, was introduced 1996 by Soto et
monomers. It was assumed that N-methylation could block hydro-
al [68]. The authors developed a peptide partially homologous to
gen bonding at the outer edge of the assembling amyloid, disrupting
the central hydrophobic region of A(cid:4) (amino acids 17-21: LVFFA),
peptide-peptide interactions that promote A(cid:4) fibrillization. As ex-
containing the amino acid proline to prevent the formation of (cid:4)-
pected, N-methylated peptide variants had significant influence on
sheet structure and to inhibit A(cid:4) amyloid formation. Proline has
A(cid:4) aggregation, as investigated using a variety of biophysical
special characteristics and is a well-known (cid:4)-sheet blocker [82]. To
methods. Notably N-methyl-Gly-33 was shown to be amenable to
increase the solubility of the peptide compound, charged amino
inhibit A(cid:4) aggregation and cytotoxicity. The localization of the N-
acids were added to the ends. The so called “inhibitor of fibrillo-
methyl group was very critical as some of the other peptides did not
genesis” (iA(cid:4)1: RDLPFFPVPID) did not aggregate itself, inhibited
prevent A(cid:4) aggregation, but altered fibril morphology [93, 94].
amyloid formation and disassembled existing fibrils in vitro. The
relative dissociation constant of iA(cid:4)1 was determined by fluores- In 2001, Gordon et al. described the synthesis and biochemical
cence spectroscopy to be approximately 80 nM. In order to enhance characterization of rationally designed “meptides” based on the
bbb permeability and to reduce generation of immune responses in KLVFF sequence, containing N-methyl amino acids in alternating
vivo, the peptide was shortened, and the derivatives iA(cid:4)3 (seven positions of the sequence. One of the compounds, termed A(cid:4)16-
amino acids) and iA(cid:4)5 (five amino acids) were proven to be similar 22m (NH2-K(Me-L)V(Me-F)F(Me-A)E-CONH2) was shown to be
good or even better A(cid:4) aggregation inhibitors in vitro than the basic highly soluble in aqueous media and monomeric in buffer solution.
compound, compared e.g. in ThT assays. The D-enantiomeric ver- It inhibited A(cid:4) fibrillization and disassembled preformed A(cid:4) fibrils
sion of iA(cid:4)1 was as effective as the L-version, and more resistant to in vitro. Inhibition was sequence specific and dependent of N-
762 Current Pharmaceutical Design, 2012, Vol. 18, No. 6 Funke and Willbold
methylization. Protease resistance of the methylated peptide was Peptides Selected in Combinatorial Libraries
increased in comparison to the unmethylated A(cid:2)16-22 peptides Combinatorial peptide libraries offer a powerful technique to
[78]. The A(cid:2)16-20m peptide, a truncated version of the peptide select highly specific peptide ligands for different pharmacological
described above, was synthesized in order to eliminate the charged
interesting targets [44, 105].
Glu residue, providing the inhibitor with a net positive charge.
Already in 1997, Blanchard and colleaques selected so-called
A(cid:2)16-20m was effective to inhibit A(cid:2) polymerization and to dis-
“decoy peptides” using a combinatorial library of approximately
sassemble preformed fibrils. In addition, it was highly water soluble
43000 individual sequences composed of the D-amino acids Ala,
despite its composition of hydrophobic amino acids. The peptide
Ile, Val, Ser, Thr and Gly. The 6 residue peptides were chosen by
passed spontaneously model phospholipid bilayers and cell mem-
their ability to complex with tagged A(cid:2)25-35 peptide and had (cid:2)-
branes, suggesting promising pharmacological properties [95].
sheet forming potential, associating with A(cid:2) and blocking aggrega-
In 2004, Cruz et al. developed the peptide “inL”, based on the
tion. Some of the selected peptides abolished the calcium influx,
KLVFF A(cid:2) recognition element. The authors added an additional
caused by aggregated A(cid:2)25-35 or A(cid:2)1-42 in cell culture [106, 107].
Lys to the N-terminus, in order to increase solubility, and an N-
Schwarzman and al. selected inhibitors of amyloid formation by
methyl-20F in order to block A(cid:2) aggregation [96]. The peptide
screening of a FliTrx random peptide library. 12 residue peptides
inhibited A(cid:2) toxicity in cell culture very efficiently. In 2009, the
were displayed on the surface of E. coli by fusion to a flaggelar
corresponding D-peptide “inD”, as well as the retro-inverso peptide
protein. Synthetic A(cid:2)1-42 was used as a target molecule in five
“inrD”, was investigated. Both D-enantiomeric peptides were more
rounds of biopanning. Four groups of A(cid:2) binding peptides were
resistant against protease degradation, as expected. The retro-
isolated (see Table 2F), two of which were enriched by positively
inverso peptide was shown to be a more effective inhibitor of A(cid:2)
charged amino acids. Most clones were shown to bind monomeric
aggregation than the other two peptide versions [97].
A(cid:2), but exhibited very low binding to fibrillar A(cid:2) 1-42 [108].
The peptidic inhibitor PPI-1019, also known as Apan, is de-
Orner et al. identified peptides that bind to A(cid:2) in either mono-
rived from the D-enantiomeric Cholyl-LVFFA-NH2 (see chapter
meric or fibrillar state using phage display approaches with mono-
other (cid:2)-sheet breaking peptides). The cholyl-group was replaced by
meric or fibrillar A(cid:2) as targets, respectively. Two libraries were
a methyl-group and the C-terminal residue was changed from D-
designed, guided by the group’s previous studies on the KLVFFK
alanine to D-leucine. PPI-1019 completed phase I and II clinical 6
peptide (see above). The first library displayed sequences with the
trials and was found to be safe, well tolerated and amenable to cross
PoPoPoKLVFFPoPoPo motif, where Po indicates a residue with
bbb. After peptide administration, levels of A(cid:2)1-40 in the CSF
polar side chain. The second library contained sequences with
increased, which might be discussed as a sign for A(cid:2) clearance out
XXXKLpLpArArPoPoPoPo motif, where X is any amino acid, and
of the brain [98, 99].
Lp and Ar indicate residues with lipophilic and aromatic side
In 2006, Kokkoni et al. optimized “meptides” based on the
chains. Peptides with selectivity for monomeric versus fibrillar A(cid:2)
KLVFF sequence in a large approach based on five peptide librar- could be identified and most of the selected peptides bound to
ies. Peptide length, methylation sites, end-blocking, side chain iden-
A(cid:2)10-35 with higher affinity than the parent peptide Ac-
tity and chirality were varied. The most interesting compound,
KLVFFKKKKK-OH. Peptides selected for A(cid:2) monomer binding
judged by A(cid:2) fibrillization and cell cytotoxicity inhibition activity, did not affect aggregation, whereas peptides selected to bind fibril-
was D-[(chGly)-(Tyr)-(chGly)-(chGly)-(mLeu)]-NH and rules
2 lar A(cid:2) increased the aggregation of A(cid:2) dramatically, altering the
could be stated to predict peptide performance. Ideal inhibitors
morphology of the resulting aggregate. This effect was clearly cor-
should be D-peptides, possess a free N- but an amidated C-terminus related with affinity of the peptides to the N-terminal part of A(cid:2)
and residues one to four should be large, branched hydrophobic side
[109].
chains. Only one methylated amino acid was essential [100].
Taddei et al. reported on an approach with the aim to inhibit the
Other (cid:2)-sheet Breaking Peptides (see Table 2E) catalytic production of H2O2 by A(cid:2), which is dependent on A(cid:2)’s
superoxide dismutase (SOD)-like activity. A phage display proce-
In 1999, Findeis et al. started the development of new (cid:2)-sheet
dure with 6- and 15mer peptide libraries was applied to select for
breaking peptides derived from 15-residue A(cid:2) peptides. The anti-
peptides that target the active site of human A(cid:2)’s SOD-like activity,
A(cid:2) fibrillization activity of the peptides was enhanced by modifica-
in order to prevent its interaction with redox-active metal ions. As
tion of their amino terminus, where different organic reagents were
the SOD-like activity site is not present in rat A(cid:2), a counterselec-
attached. In subsequent libraries, the size of the inhibitors as well as
tion step with rat A(cid:2) was employed in the phage display procedure.
the amino acid sequences were optimized, and finally, the lead
A(cid:2)1-40 or A(cid:2)1-42 were used as targets. 25 peptides which bound to
compound cholyl-LVFFA-OH, designated PPI-368, was identified.
A(cid:2) were identified, and two of the three most enriched peptides,
PPI-368, the acyl-D-amino acid analogue PPI-433 and the amide
named amyloid neutralizing agents (ANA) 1 to 3, were shown to
analogue PPI-457 inhibited A(cid:2) polymerization potently. The latter
significantly reduce A(cid:2)’s SOD-like activity in cell culture. A 15-
two were stable in monkey CSF for at least 24 h. Unfortunately,
mer peptide additionally reduced A(cid:2) toxicity in cell culture and
hepatic first –pass elimination was reported for all compounds after
seemed to be comparably potent as the known A(cid:2) metal-mediated
biodistribution studies were performed [101, 102].
redox activity inhibitor Clioquinol [110].
In 2009, Fryman-Marom et al. introduced the D-enantiomeric
A very sophisticated system was used by Baine et al. to select
(cid:2)-sheet breaker NH -D-Trp-Aib-OH, which combined an indole
2 for peptides that inhibit A(cid:2) aggregation in two combinatorially
and (cid:3)-aminobutyric acid (Aib) [103]. Aib has been shown to induce
diverse peptide libraries. The goal was to select peptides which bind
helical conformations and to disrupt (cid:2)-sheet structures, the
the two hydrophobic patches of A(cid:2) and block aggregation by highly
Ramachandran plot indicating that Aib even has the potential to be
charged and polar aspartatic acid residues. A(cid:2)1-42 was genetically
an better (cid:2)-sheet breaker than proline [104]. NH -D-Trp-Aib-OH
2 fused to EGFP. When expressed in E. coli, aggregation of A(cid:2) inhib-
specifically interacted with (untoxic) low molecular weight A(cid:2)
its the correct folding of EGFP and therefore its fluorescent proper-
oligomers and inhibited their growth to larger, celltoxic A(cid:2) forms
ties. In the selection process, randomized peptides were co-
in vitro. The compound was stable, safe, orally bioavailable, and
expressed with A(cid:2)-EGFP. Peptides which were resistant to degra-
crossed the bbb in the range of 4 to 8%, depending on the route of
dation by cellular proteases and inhibited A(cid:2) aggregation permitted
administration. It reduced the amount of plaques in the brains of
EGFP to be folded properly. Colonies with the brightest fluores-
AD transgenic mice and improved their cognitive performance
cence were chosen for further characterization. Three candidate
[103].
peptides were selected and characterized, being capable to inhibit
Alzheimer’s Therapy Current Pharmaceutical Design, 2012, Vol. 18, No. 6 763
A(cid:2) aggregation. One of them even disaggregated preformed A(cid:2) CONCLUSION
fibrils [111]. During the past years, several peptide inhibitors of A(cid:2) aggrega-
Recently a highly specific D-enantiomeric ligand for A(cid:2) has tion have been investigated for their applicability as new therapeu-
been identified using a mirror image phage display approach with a tic lead compounds. In conclusion, it must be stated that only very
huge randomized 12-mer peptide library (> 1 billion different pep- few, iA(cid:2)5 [84-86], A(cid:2)12-28P [66], LPYFDa [88, 89], trp-Aib
tides). The dominant peptide sequence RPRTRLHTHRNR was [103], D-4F [120] and D3 [47, 112, 113] were proven to be effec-
obtained, referred to as D3. D3 modulated A(cid:2) aggregation and in- tive in rodent mouse models. Only one compound, PPI-1019, is
hibited A(cid:2) toxicity in cell culture. In vitro data clearly demon- tested in clinical trials. Despite the high diversity of peptides, com-
strated that D3 is able to precipitate toxic A(cid:2) oligomers into large, bined with their simplicity, high specificity, low toxicity and high
high-molecular-weight, nontoxic, ThT negative, nonamyloidogenic biological activity [31, 33], peptides are susceptible to proteolytic
amorphous aggregates that fail to act as seeds in A(cid:2) fibril formation degradation and in general do not circulate for more than a few
assays. D3 did not increase the concentration of monomeric A(cid:2). minutes in blood [129]. Additionally, peptides and do not cross
Computational simulations of an A(cid:2) nonamer in the presence and membranes very well [130].
absence of D3 proved strong interactions between the arginine-rich
Several strategies were already applied to overcome high prote-
D3 and negatively charged groups of A(cid:2), which were expected to
ase susceptibility of peptides and to improve bbb permeability.
compensate the charge on the A(cid:2) surface and reduce solubility and Different chemical modifications, including incorporation of con-
promote the aggregation of A(cid:2). Moreover, D3 binding also showed
formationally constrained amino acids, or modifications of the pep-
effects on the topology of the A(cid:2) oligomers, which induced a large
tide backbone, have been performed. For example, Adessi et al.
twist and facilitated the formation of nonfibrillar aggregates [112, introduced a methyl-group at the nitrogen atom of one amid bond in
113]. Van Groen et al. demonstrated the usage of FITC-labelled D3
iA(cid:2)5, resulting in a 10-fold higher in vivo life-time in comparison
for both in vitro and in vivo staining of A(cid:2)-1-42 in the brains of
to the unmodified iA(cid:2)5 [131]. Moreover, end-protection is com-
transgenic AD-model mice [47]. Additionally, D3 was proven to monly used to shield peptides against proteolytic degradation as
have notable bbb permeability in an in vitro bbb cell culture model
well as to increase bbb permeability [66, 89, 132]. Another promis-
which further demonstrated the therapeutic potential of D3 [114].
ing strategy to improve the peptide stability is the use of D-
Most recently, oral treatments of A(cid:2) transgenic mice with D3 enantiomeric amino acids which are considered to be rather prote-
yielded significant cognitive improvement, reduction of plaque load
ase resistant in vivo and in addition often less immunogenic than the
and plaque-related inflammation [112]. In 2010, Müller-Schiffmann
respective L-peptides [42, 66, 133]. Additionally, D-peptides can be
et al. reported on the D3 hybrid compound JM169, which combined taken up systematically after oral administration [134].
the D-enantiomeric peptide with a (cid:2)-sheet breaking compound via a
It was assumed for a long time that A(cid:2) deposited in extracellu-
linker substance. The authors demonstrated that the hybrid com-
lar amyloid fibrils and plaques were the major pathogenic species in
pound was more efficient in vitro than the sum of its components
AD. However, over the past decade, accumulating evidence sug-
and had novel properties [115].
gests that A(cid:2) oligomers are the toxic moiety responsible for synap-
In 2009, Paula-Lima et al. used a phage display approach to tic dysfunction and neuronal cell loss [12]. At the moment it is un-
select peptides binding to the aggregated form of A(cid:2). One of the
clear whether peptide inhibitors should be targeted to monomeric
identified heptapeptides with the amino acid sequence GNLLTLD A(cid:2), oligomeric A(cid:2) or A(cid:2) fibrils deposited in plaques. It is also un-
(designated GN peptide) was detected to be homologous to the N- clear if A(cid:2)1-40 or A(cid:2)1-42 should be addressed and weather com-
terminal domain of mammalian apolipoprotein A-I. Apo A-I, the
pounds need to cross the bbb in order to be effective. More work is
major protein component of high-density lipoprotein (HDL), has a necessary to elucidate the nature of the most synaptotoxic A(cid:2) spe-
central role in reverse cholesterol transport [116, 117] and anti- cies during AD development and progression. The ongoing research
oxidant as well as anti-inflammatory properties [118]. It was shown
on peptides which target distinct A(cid:2) species, as well as the investi-
that purified human apoA-I and A(cid:2) formed complexes. The interac- gation of their influence on A(cid:2) aggregation and toxicity, will pro-
tion of apo A-I also rendered the morphology of amyloid aggre- vide further understanding of the molecular mechanisms involved
gates [119]. Likewise in 2009, Handattu et al. evaluated the apo A-I
in AD.
mimetic peptide D-4F, synthesized from D-amino acids and co-
administered with pravastatin, as a treatment for AD transgenic ACKNOWLEDGEMENTS
mice [120]. D-4F was developed based on the presence of lipid-
D.W. is supported by the Technology-Transfer Fonds of For-
associating amphipathic (cid:3)-helices in apo A-I and possessed the
schungszentrum Jülich. D.W. and S.A.F. are supported by Deutsche
abilityto avidly bind lipids [121, 122]. Most studies of D-4F were
Forschungsgemeinschaft (Research Training Group 1033).
focused on its potential role in atherosclerosis management [123,
124]. Several studies suggested that AD may have an inflammatory REFERENCES
component similar to atherosclerosis that is associated with very
[1] Brookmeyer R, Johnson E, Ziegler-Graham K, Arrighi HM.
small vessels such as arterioles [125-127]. In the study, groups of
Forecasting the global burden of Alzheimer's disease. Alzheimers &
male mice were treated with D-4F and pravastatin via the drinking Dementia. 2007; 3(3): 186-91.
water. In comparison to the controls, the treated group showed sig- [2] Alzheimer A. Über eine eigenartige Erkrankung der Hirnrinde. All
nificantly increased cognitive behavior, reduced plaque deposition Z psychait. 1907; 64: 146-8.
and reduced inflammatory responses [120]. [3] Goedert M, Spillantini MG, Jakes R, Rutherford D, Crowther RA.
Multiple isoforms of human microtubule-associated protein tau:
Very recently, Kawasaki et al. constructed a random library to
sequences and localization in neurofibrillary tangles of Alzheimer's
obtain peptide inhibitors specific to inhibit formation of soluble
disease. Neuron. 1989; 3(4): 519-26.
37/48 kDa A(cid:2)1-42 oligomers. The random library was based on the
[4] Leroy K, Bretteville A, Schindowski K, Gilissen E, Authelet M, De
LPFFD sequence: XX-P-XXX, where X means any amino acid. Decker R, et al. Early axonopathy preceding neurofibrillary tangles
Novel peptides containing arginine residues were enriched while in mutant tau transgenic mice. Am J Pathol. 2007; 171(3): 976-92.
panning for soluble A(cid:2)1-42. Selected ligands with the strongest [5] Vieira MN, Forny-Germano L, Saraiva LM, Sebollela A, Martinez
affinity to A(cid:2) contained three arginine residues and suppressed AM, Houzel JC, et al. Soluble oligomers from a non-disease related
formation of 37/48 kDa oligomers and kept the monomeric form of protein mimic Abeta-induced tau hyperphosphorylation and
A(cid:2) even after 24 hours of incubation [128]. neurodegeneration. J Neurochem. 2007; 103(2): 736-48.
[6] Finder VH, Glockshuber R. Amyloid-beta aggregation.
Neurodegener Dis. 2007; 4(1): 13-27.
764 Current Pharmaceutical Design, 2012, Vol. 18, No. 6 Funke and Willbold
[7] Haass C, Selkoe DJ. Cellular processing of beta-amyloid precursor and amyloid-{beta} fibrils and reduces cellular toxicity. Proc Natl
protein and the genesis of amyloid beta-peptide. Cell. 1993; 75(6): Acad Sci U S A. 2010.
1039-42. [29] Ehrnhoefer DE, Bieschke J, Boeddrich A, Herbst M, Masino L,
[8] Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, Lurz R, et al. EGCG redirects amyloidogenic polypeptides into
Grzeschik KH, et al. The precursor of Alzheimer's disease amyloid unstructured, off-pathway oligomers. Nat Struct Mol Biol. 2008;
A4 protein resembles a cell-surface receptor. Nature. 1987; 15(6): 558-66.
325(6106): 733-6. [30] Nagel-Steger L, Demeler B, Meyer-Zaika W, Hochdorffer K,
[9] Weidemann A, Konig G, Bunke D, Fischer P, Salbaum JM, Masters Schrader T, Willbold D. Modulation of aggregate size- and shape-
CL, et al. Identification, biogenesis, and localization of precursors distributions of the amyloid-beta peptide by a designed beta-sheet
of Alzheimer's disease A4 amyloid protein. Cell. 1989; 57(1): 115- breaker. Eur Biophys J. 2010; 39(3): 415-22.
26. [31] Danho W, Swistok J, Khan W, Chu XJ, Cheung A, Fry D, et al.
[10] Hardy JA, Higgins GA. Alzheimer's disease: the amyloid cascade Opportunities and challenges of developing peptide drugs in the
hypothesis. Science. 1992; 256(5054): 184-5. pharmaceutical industry. Adv Exp Med Biol. 2009; 611: 467-9.
[11] Selkoe DJ. The molecular pathology of Alzheimer's disease. [32] Estrada LD, Soto C. Disrupting beta-amyloid aggregation for
Neuron. 1991; 6(4): 487-98. Alzheimer disease treatment. Curr Top Med Chem. 2007; 7(1):
[12] Haass C, Selkoe DJ. Soluble protein oligomers in 115-26.
neurodegeneration: lessons from the Alzheimer's amyloid beta- [33] Lien S, Lowman HB. Therapeutic peptides. Trends Biotechnol.
peptide. Nat Rev Mol Cell Biol. 2007; 8(2): 101-12. 2003; 21(12): 556-62.
[13] Shankar GM, Bloodgood BL, Townsend M, Walsh DM, Selkoe DJ, [34] Silverman DH. Brain 18F-FDG PET in the diagnosis of
Sabatini BL. Natural oligomers of the Alzheimer amyloid-beta neurodegenerative dementias: comparison with perfusion SPECT
protein induce reversible synapse loss by modulating an NMDA- and with clinical evaluations lacking nuclear imaging. J Nucl Med.
type glutamate receptor-dependent signaling pathway. J Neurosci. 2004; 45(4): 594-607.
2007; 27(11): 2866-75. [35] Dougall NJ, Bruggink S, Ebmeier KP. Systematic review of the
[14] Shankar GM, Li S, Mehta TH, Garcia-Munoz A, Shepardson NE, diagnostic accuracy of 99mTc-HMPAO-SPECT in dementia. Am J
Smith I, et al. Amyloid-beta protein dimers isolated directly from Geriatr Psychiatry. 2004; 12(6): 554-70.
Alzheimer's brains impair synaptic plasticity and memory. Nat [36] Braak H, Braak E. Frequency of stages of Alzheimer-related lesions
Med. 2008; 14(8): 837-42. in different age categories. Neurobiol Aging. 1997; 18(4): 351-7.
[15] Ballatore C, Brunden KR, Trojanowski JQ, Lee VM, Smith AB, [37] Thal DR, Rub U, Orantes M, Braak H. Phases of A beta-deposition
3rd, Huryn D. Modulation of protein-protein interactions as a in the human brain and its relevance for the development of AD.
therapeutic strategy for the treatment of neurodegenerative Neurology. 2002; 58(12): 1791-800.
tauopathies. Curr Top Med Chem 2011; 11(3): 317-30. [38] Nordberg A. Amyloid imaging in early detection of Alzheimer's
[16] Wischik CM, Edwards PC, Lai RY, Roth M, Harrington CR. disease. Neurodegener Dis. 2010; 7(1-3): 136-8.
Selective inhibition of Alzheimer disease-like tau aggregation by [39] Klunk WE, Jacob RF, Mason RP. Quantifying amyloid beta-peptide
phenothiazines. Proc Natl Acad Sci U S A. 1996; 93(20): 11213-8. (Abeta) aggregation using the Congo red-Abeta (CR-abeta)
[17] Bassan M, Zamostiano R, Davidson A, Pinhasov A, Giladi E, Perl spectrophotometric assay. Anal Biochem. 1999; 266(1): 66-76.
O, et al. Complete sequence of a novel protein containing a [40] Kang CK, Jayasinha V, Martin PT. Identification of peptides that
femtomolar-activity-dependent neuroprotective peptide. J specifically bind Abeta1-40 amyloid in vitro and amyloid plaques
Neurochem. 1999; 72(3): 1283-93. in Alzheimer's disease brain using phage display. Neurobiol Dis.
[18] Gozes I. Tau pathology and future therapeutics. Curr Alzheimer 2003; 14(1): 146-56.
Res. 2010; 7(8): 685-96. [41] Funke SA, Willbold D. Mirror image phage display--a method to
[19] Matsuoka Y, Jouroukhin Y, Gray AJ, Ma L, Hirata-Fukae C, Li HF, generate D-peptide ligands for use in diagnostic or therapeutical
et al. A neuronal microtubule-interacting agent, NAPVSIPQ, applications. Mol Biosyst. 2009; 5(8): 783-6.
reduces tau pathology and enhances cognitive function in a mouse [42] Schumacher TN, Mayr LM, Minor DL, Jr., Milhollen MA, Burgess
model of Alzheimer's disease. J Pharmacol Exp Ther. 2008; 325(1): MW, Kim PS. Identification of D-peptide ligands through mirror-
146-53. image phage display. Science. 1996; 271(5257): 1854-7.
[20] Shiryaev N, Jouroukhin Y, Giladi E, Polyzoidou E, Grigoriadis NC, [43] Wiesehan K, Willbold D. Mirror-image phage display: aiming at
Rosenmann H, et al. NAP protects memory, increases soluble tau the mirror. Chembiochem : a European journal of chemical biology.
and reduces tau hyperphosphorylation in a tauopathy model. [Review]. 2003; 4(9): 811-5.
Neurobiol Dis. 2009; 34(2): 381-8. [44] Gallop MA, Barrett RW, Dower WJ, Fodor SP, Gordon EM.
[21] Stewart AJ, Fox A, Morimoto BH, Gozes I. Looking for novel ways Applications of combinatorial technologies to drug discovery. 1.
to treat the hallmarks of Alzheimer's disease. Expert Opin Investig Background and peptide combinatorial libraries. J Med Chem.
Drugs. 2007; 16(8): 1183-96. 1994; 37(9): 1233-51.
[22] Jakob-Roetne R, Jacobsen H. Alzheimer's disease: from pathology [45] Bartnik D, Funke SA, Andrei-Selmer LC, Bacher M, Dodel R,
to therapeutic approaches. Angew Chem Int Ed Engl. 2009; 48(17): Willbold D. Differently selected D-enantiomeric peptides act on
3030-59. different Abeta species. Rejuvenation research. 2010; 13(2-3): 202-
[23] Nerelius C, Johansson J, Sandegren A. Amyloid beta-peptide 5.
aggregation. What does it result in and how can it be prevented? [46] Wiesehan K, Buder K, Linke RP, Patt S, Stoldt M, Unger E, et al.
Front Biosci. 2009; 14: 1716-29. Selection of D-amino-acid peptides that bind to Alzheimer's disease
[24] McLaurin J, Golomb R, Jurewicz A, Antel JP, Fraser PE. Inositol amyloid peptide abeta1-42 by mirror image phage display.
stereoisomers stabilize an oligomeric aggregate of Alzheimer Chembiochem. 2003; 4(8): 748-53.
amyloid beta peptide and inhibit abeta -induced toxicity. J Biol [47] van Groen T, Kadish I, Wiesehan K, Funke SA, Willbold D. In
Chem. 2000 Jun 16; 275(24): 18495-502. vitro and in vivo staining characteristics of small, fluorescent,
[25] Townsend M, Cleary JP, Mehta T, Hofmeister J, Lesne S, O'Hare Abeta42-binding D-enantiomeric peptides in transgenic AD mouse
E, et al. Orally available compound prevents deficits in memory models. ChemMedChem. 2009; 4(2): 276-82.
caused by the Alzheimer amyloid-beta oligomers. Ann Neurol. [48] Larbanoix L, Burtea C, Laurent S, Van Leuven F, Toubeau G,
2006; 60(6): 668-76. Vander Elst L, et al. Potential amyloid plaque-specific peptides for
[26] Gervais F, Paquette J, Morissette C, Krzywkowski P, Yu M, Azzi the diagnosis of Alzheimer's disease. Neurobiol Aging. 2010;
M,et al. Targeting soluble Abeta peptide with Tramiprosate for the 31(10): 1679-89.
treatment of brain amyloidosis. Neurobiol Aging. 2007; 28(4): 537- [49] Larbanoix L, Burtea C, Ansciaux E, Laurent S, Mahieu I, Vander
47. Elst L, et al. Design and evaluation of a 6-mer amyloid-beta protein
[27] Price KA, Crouch PJ, White AR. Therapeutic treatment of derived phage display library for molecular targeting of amyloid
Alzheimer's disease using metal complexing agents. Recent Pat plaques in Alzheimer's disease: Comparison with two cyclic
CNS Drug Discov. 2007; 2(3): 180-7. heptapeptides derived from a randomized phage display library.
[28] Bieschke J, Russ J, Friedrich RP, Ehrnhoefer DE, Wobst H, Peptides. 2011; 32: 1232-43.
Neugebauer K, et al. EGCG remodels mature {alpha}-synuclein