C-terminal tetrapeptides inhibit aβ42-induced neurotoxicity primarily through specific interaction at the N-Terminus of Aβ42

Article abstract of DOI:10.1021/jm200982p

Inhibition of amyloid β-protein (Aβ)-induced toxicity is a promising therapeutic strategy for Alzheimer's disease (AD). Previously, we reported that the C-terminal tetrapeptide Aβ(39-42) is a potent inhibitor of neurotoxicity caused by Aβ42, the form of Aβ most closely associated with AD. Here, initial structure-activity relationship studies identified key structural requirements, including chirality, side-chain structure, and a free N-terminus, which control Aβ(39-42) inhibitory activity. To elucidate the binding site(s) of Aβ(39-42) on Aβ42, we used intrinsic tyrosine (Y) fluorescence and solution-state NMR. The data suggest that Aβ(39-42) binds at several sites, of which the predominant one is located in the N-terminus of A 42, in agreement with recent modeling predictions. Thus, despite the small size of Aβ(39-42) and the hydrophobic, aliphatic nature of all four side-chains, the interaction of Aβ(39-42) with Aβ42 is controlled by specific intermolecular contacts requiring a combination of hydrophobic and electrostatic interactions and a particular stereochemistry. (Figure presented)

Full text of DOI:10.1021/jm200982p

Article
pubs.acs.org/jmc
C-Terminal Tetrapeptides Inhibit Aβ42-Induced Neurotoxicity
Primarily through Specific Interaction at the N-Terminus of Aβ42
Huiyuan Li,† Zhenming Du,∥ Dahabada H. J. Lopes,† Erica A. Fradinger,†,⊥ Chunyu Wang,∥
and Gal Bitan*,†,‡,§
†Department of Neurology, David Geffen School of Medicine, ‡Brain Research Institute, and §Molecular Biology Institute, University
of California, Los Angeles, 635 Charles E. Young Drive South, Los Angeles, California 90095-7334, United States
∥Department of Biology, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, New York 12180, United States
S
* Supporting Information
ABSTRACT: Inhibition of amyloid β-protein (Aβ)-induced
toxicity is a promising therapeutic strategy for Alzheimer’s
disease (AD). Previously, we reported that the C-terminal
tetrapeptide Aβ(39−42) is a potent inhibitor of neurotoxicity
caused by Aβ42, the form of Aβ most closely associated with
AD. Here, initial structure−activity relationship studies
identified key structural requirements, including chirality,
side-chain structure, and a free N-terminus, which control
Aβ(39−42) inhibitory activity. To elucidate the binding site(s)
of Aβ(39−42) on Aβ42, we used intrinsic tyrosine (Y)
fluorescence and solution-state NMR. The data suggest that
Aβ(39−42) binds at several sites, of which the predominant one is located in the N-terminus of Aβ42, in agreement with recent
modeling predictions. Thus, despite the small size of Aβ(39−42) and the hydrophobic, aliphatic nature of all four side-chains, the
interaction of Aβ(39−42) with Aβ42 is controlled by specific intermolecular contacts requiring a combination of hydrophobic
and electrostatic interactions and a particular stereochemistry.
Aβ(32−37) with varying extent of N-methylation were found
to retard β-sheet and fibril formation and reduce Aβ
neurotoxicity.21
INTRODUCTION
■
Neurotoxic oligomers of amyloid β-protein (Aβ) are believed
to be the main cause of Alzheimer’s disease (AD).1−4 Two
predominant forms of Aβ, comprising 40 (Aβ40) or 42 (Aβ42)
amino acid residues, are produced in vivo. Aβ42 has been
shown to be more neurotoxic than Aβ405 and to follow a
different pathway of oligomerization.6,7 Aβ42 forms higher-
order metastable oligomers than Aβ40, and this tendency
correlates with structural stabilization of the C-terminus of
Aβ42 mediated by the presence of I41 and A42.6,8−10
Inhibition of Aβ aggregation by short peptides derived from
the sequence of Aβ itself has been used by a number of groups,
primarily along the idea of “β-sheet breaker” peptides that
interfere with formation of the characteristic β-sheet-rich
amyloid fibrils. The most utilized sequence for this line of
investigation has been the central hydrophobic cluster of Aβ
(CHC, residues 17−21),11−16 which is a key region in Aβ
fibrillogenesis.17 Utilizing a similar strategy, recently, rationally
designed aminopyrazole-based β-sheet breakers were found to
inhibit Aβ assembly and toxicity, with the most effective
inhibitor being a conjugate of aminopyrazole and the CHC-
derived sequence LPFFD.18 Using a different Aβ region for
inhibitor design, modified Aβ42 C-terminal fragments, GVVIA-
NH2 and RVVIA-NH2, were designed as β-sheet breakers and
partially protected SH-SY5Y neuroblastoma cells from Aβ42
neurotoxicity in cell viability19 but not electrophysiological
assays.20 In a different study, hexapeptides derived from
As evidence emerged ascribing pathogenic primacy to Aβ
oligomers rather than fibrils,22 inhibitor-design efforts have
shifted toward inhibition of Aβ oligomerization. Guided by the
principle of self-recognition and considering the critical role of
the C-terminal region of Aβ42 in self-assembly,6,8 we prepared
C-terminal fragments (CTFs) of the general formula Aβ(x−
42), x = 28−39, and evaluated their capability to disrupt the
assembly and neurotoxicity of Aβ42.23 Of the 12 CTFs tested,
the shortest one, Aβ(39−42), had surprisingly high activity.
Aβ(39−42) was found to inhibit Aβ42-induced neurotoxicity in
differentiated rat pheochromocytoma (PC-12) cells with half-
maximal (IC50) values of 16 ± 5 and 47 ± 14 μM using the (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) reduction and lactate dehydrogenase (LDH) release
assays, respectively.23 In addition, Aβ(39−42) significantly
rescued mouse primary hippocampal neurons from Aβ42-
induced inhibition of miniature excitatory postsynaptic current
frequency.23 The data suggested that Aβ(39−42) inhibited
Aβ42-induced toxicity both in the early stage of synaptic
activity and in later stages of metabolism deficits and cell death.
Received: July 22, 2011
Published: November 16, 2011
© 2011 American Chemical Society
8451
dx.doi.org/10.1021/jm200982p | J. Med. Chem. 2011, 54, 8451−8460

Journal of Medicinal Chemistry
Article
In follow-up dynamic light scattering studies, we found that
Aβ(39−42) stabilized oligomers with a hydrodynamic radius of
6 ± 3 nm and 30 ± 10 nm, which we interpreted as resulting
from formation of heterooligomers comprising both Aβ42 and
Aβ(39−42).23,24 Computer modeling of Aβ(39−42) coas-
sembled with Aβ42 supported the formation of hetero-
oligomers and suggested that Aβ(39−42) binds near the N-
terminal region, Aβ(2−4), and sequesters this region from the
aqueous milieu.23,25
The amphipathic nature and small size of Aβ(39−42) make
its pharmacokinetic characteristics close to recommended
values of drug-like criteria, including Lipinski’s Rule of 526
and topological polar surface area (tPSA)27 (Table 1),
Table 2. Sequences, Masses, and IC50 Values of Aβ(39−42)
and Derivatives
calculated
mass
observed
mass
IC50 (μM)
(MTT)
IC50 (μM)
(LDH)
sequence
[M + H]
VVIA
AVIA
VAIA
VVAA
401.5
373.5
373.5
359.4
401.5
443.6
400.5
401.2
373.0
373.2
359.1
401.2
443.4
400.1
21 ± 6
53 ± 10
15 ± 3
nd
16 ± 3
22 ± 5
14 ± 2
nd
a
vvia
nd
nd
Ac-VVIA
nd
nd
VVIA-
NH2
28 ± 7
30 ± 5
AIVV
401.5
443.5
400.5
401.3
443.1
400.2
14 ± 1
nd
22 ± 3
nd
Ac-AIVV
Table 1. Physicochemical Characteristics of Aβ(39−42)
AIVV-
NH2
20 ± 4
16 ± 3
recommended value
a
Lipinski’s rule of 526
Aβ(39−42)
Lower-case letters represent D-configuration. nd: not determined.
criterion
MW
<500
<5
400.5
−0.3
5
ClogP
H-bond donors
H-bond acceptors
tPSA (Å2)
≤5
≤10
9
≤14027
150.62
supporting its development as a drug lead. Toward this end,
here we performed structure−activity relationship (SAR)
studies to delineate structural features important for inhibitory
activity and characterized the binding of Aβ(39−42) to Aβ42
using intrinsic fluorescence and two-dimensional (2D)
solution-state NMR. The data suggest that Aβ(39−42) protects
cells against Aβ42-induced toxicity predominantly via specific
interaction at the N-terminus of Aβ42.
RESULTS
■
Structure−Activity Relationship Study of Aβ(39−42).
To guide future rational development of Aβ(39−42) as a drug
lead, we asked what structural characteristics were important for
the inhibitory activity and what specific interactions controlled
the binding of Aβ(39−42) to Aβ42. In search of the answers
for these questions, we synthesized a series of Aβ(39−42)
derivatives, including A substitution of the first three residues
(AVIA, VAIA, VVAA), an inverso-peptide (vvia, lower-case
letters represent D-configuration), the N-terminally and C-
terminally protected analogues Ac-VVIA, VVIA-NH2, a retro-
peptide (AIVV), and N-terminally and C-terminally protected
versions of the retro-peptide (Ac-AIVV, AIVV-NH2) (Table 2).
We began evaluating the new derivatives by testing if these
peptides themselves were toxic to differentiated PC-12 cells
using the MTT assay. The results showed that all the
derivatives were not toxic (Figure 1, white bars). Notably,
Aβ(39−42) and the analogues, AVIA, VAIA, VVIA-NH2, AIVV,
and AIVV-NH2 caused a significant increase of 10−35% in cell
viability relative to control cells.
Next, we screened the Aβ(39−42) derivatives for inhibition
of Aβ42-induced neurotoxicity in single-dose experiments.
Differentiated PC-12 cells were incubated with Aβ42 for 24 h in
the absence or presence of 10-fold excess of each derivative,
and cell viability was assessed using the MTT assay. Among the
nine derivatives tested, the same five analogues that increased
cell viability on their own showed statistically significant
attenuation of Aβ42-induced toxicity (Figure 1, black bars),
similar to the parent peptide. The A-substituted sequences,
AVIA and VAIA, showed similar inhibitory activity to that of
Figure 1. Evaluation of inhibitory activity of Aβ(39−42) analogues.
Aβ42 (10 μM, gray bar), mixtures of Aβ42:Aβ(39−42) analogues at
1:10 concentration ratio (black bars), Aβ(39−42) analogues alone at
100 μM (white bars), or control medium containing NaOH at the
same concentration as in the peptide solutions (dotted bar) were
incubated with differentiated PC-12 cells for 24 h and cell viability was
measured using the MTT assay. The data are shown as mean ± SEM
of at least three independent experiments with six replicates per data
point (n ≥ 18). Statistical significance was calculated and compared
with Aβ42 alone by using ANOVA followed by Dennett’s multiple-
comparison tests (**p < 0.01, ***p < 0.001), NS = non-significant.
Aβ(39−42), whereas VVAA lost inhibitory activity suggesting
that the side-chains of V39 (full-length Aβ numbering) and V40
were relatively insensitive to structural changes, but the side-
chain of I41 was important for inhibition. However, the
observation that inhibitory activity was maintained in the retro
sequence, AIVV, suggested that a bulky hydrophobic side-chain
in position 41, such as I or V, might be sufficient for the
inhibitory activity. The loss of activity in the inverso-peptide
(vvia) indicated that the chirality of Aβ(39−42) was required
for inhibition of toxicity. The analogues in which the N-
terminus was acetylated, Ac-VVIA and Ac-AIVV, showed no
inhibitory activity, whereas the analogues with amidated C-
terminus, VVIA-NH2 and AIVV-NH2, were as active as Aβ(39−
8452
dx.doi.org/10.1021/jm200982p | J. Med. Chem. 2011, 54, 8451−8460

Journal of Medicinal Chemistry
Article
Figure 2. Aβ(39−42) selectively inhibits Aβ42-induced toxicity. Aβ42 (10 μM) or staurosporine (ST, 0.2 μM) in the absence or presence of
different Aβ(39−42) concentrations were (A) incubated with differentiated PC-12 cells for 24 h and cell viability was determined using MTT assay,
and (B) incubated with differentiated PC-12 cells for 48 h and cell death was measured using LDH assay. (C) α-Synuclein (15 μM), alamethicin (4
μM), or Aβ42 (10 μM) in the absence or presence of different Aβ(39−42) concentrations were incubated with differentiated PC-12 cells for 24 h,
and viability was determined using MTT assay. The data represent mean ± SEM from at least three independent experiments with five replicates per
data point (n ≥ 15).
Figure 3. Intrinsic fluorescence of Aβ42 analogues in the presence of Aβ(39−42) or its derivatives. (A) Primary structure of Aβ42 and its Y-
substituted analogues. Hyphens indicate residues identical to WT Aβ42. In WT Aβ42, Y is at position 10. The substitutions, in which a single Y
residue substituted the original residues at positions (n) 1, 20, 30, or 42, and the native Y10 was substituted by F, are simply named [Yn]Aβ42. (B)
Representative electron micrograph of freshly prepared 5 μM Aβ42. The scale bar represents 100 nm. (C) Intrinsic fluorescence intensity of 5 μM
Aβ42 and each Y-substituted analogue. (D) Intrinsic fluorescence of Aβ42 analogues in the presence of Aβ(39−42), Ac-VVIA, or VVIA-NH2
normalized to the percentage of each Aβ42 analogue alone. The data are shown as mean ± SEM of four independent experiments with at least 10
measurements per data point. Statistical significance was analyzed using ANOVA followed by Bonferroni’s and Dennett’s multiple-comparison tests
(*p < 0.05, ***p < 0.001).
42), indicating that a free N-terminal amino group was essential
for the activity, whereas the C-terminus could be modified to
provide protection from carboxyexopeptidases.
Further characterization showed that all the active derivatives
inhibited Aβ42-induced toxicity dose-dependently (Table 2 and
Supporting Information Figure S1). The differences among the
IC50 values of the Aβ(39−42) derivatives in the MTT assay
were relatively small and not statistically significant, except for
the IC50 of AVIA, 53 ± 10 μM, which was significantly higher
(p = 0.0081, Student’s t test) than that of Aβ(39−42), 21 ± 6
μM. The differences among the IC50 values found in the LDH
assay for all the Aβ(39−42) derivatives were statistically
insignificant.
8453
dx.doi.org/10.1021/jm200982p | J. Med. Chem. 2011, 54, 8451−8460

Journal of Medicinal Chemistry
Article
Aβ(39−42) Specifically Inhibits Aβ42-Induced Toxic-
ity. Because Aβ(39−42) and some of its analogues caused
increased cell viability relative to cells treated with cell culture
medium alone, we asked whether the observed inhibition of
Aβ42-induced toxicity was mediated, at least partially, by a
mechanism that did not involve interaction with Aβ42. To
address this question, we compared the effect of Aβ(39−42) on
neurotoxicity induced by Aβ42 and several other toxins. For
initial examination we used staurosporine, a nonselective
protein-kinase inhibitor that induces apoptosis in multiple cell
types.28 Differentiated PC-12 cells treated with 0.2 μM
staurosporine or 10 μM Aβ42 showed similar decrease in cell
viability in both the MTT (Figure 2A) and the LDH (Figure
2B) assays. As expected, Aβ(39−42) showed dose-dependent
inhibition of Aβ42-induced toxicity. In contrast, Aβ(39−42)
had no effect on staurosporine-induced cell death.
One mechanism by which Aβ42 is thought to cause toxicity
is disruption of the cell membrane leading to leakage of ions
and/or other metabolites, either due to formation of non-
specific channels29 or via perturbation of the phospholopid
bilayer conductance without channel formation.30 To examine
whether Aβ(39−42) protected the cells against membrane
perturbation, we examined next its ability to protect against
alamethicin, a fungal peptide antibiotic, which potently induces
voltage-dependent ion channel formation in phospholipid
membranes.31 In addition, we used another amyloidogenic
protein, α-synuclein, for which similar mechanisms of toxicity
to Aβ42 have been proposed.32 Differentiated PC-12 cells
treated with 15 μM α-synuclein, 4 μM alamethicin, or 10 μM
Aβ42 showed similar decrease in cell viability in MTT assay
(Figure 2C). Addition of increasing concentrations of Aβ(39−
42) resulted in dose-dependent inhibition of the toxicity
induced by Aβ42, as observed in previous experiments (Figure
2A and Supporting Information Figure S1). In contrast, only
weak protection from α-synuclein- or alamethicin-induced
toxicity was observed, suggesting that nonspecific protection
was a minor component of the inhibitory effect of Aβ(39−42),
whereas the major mechanism was mediated through direct and
specific interaction with Aβ42.
Binding Site(s) of Aβ(39−42) on Aβ42. Originally, the
hypothesis that led us to examine Aβ42 CTFs as inhibitors of
Aβ42 assembly and toxicity was based on the principle of self-
recognition and we predicted that the CTFs would bind to the
C-terminus of Aβ42.23,33 However, our previous investigation
of the mode of interaction between the CTFs and Aβ42
suggested that different CTFs might inhibit Aβ42-induced
toxicity by distinct mechanisms23,24 and might bind Aβ42 at
sites other than the C-terminus.25 Therefore, here we used two
complementary methods to elucidate the binding site(s) of
Aβ(39−42), the shortest CTF in original series, on Aβ42.
Characterization of the Interaction between Aβ(39−42)
and Aβ42 by Intrinsic Y Fluorescence. Elucidation of binding
sites for inhibitors of aberrant protein self-assembly is a difficult
task because the self-assembly typically occurs among
disordered monomers and produces metastable oligomers in
which the degree of order still is low. To explore potential
binding site(s) of Aβ(39−42) on Aβ42, we took advantage of
the intrinsic fluorescence of Y residues, which enables rapid
signal detection at low concentrations under which minimal or
no aggregation occurs during the time of the experiment (∼30
min), thus measuring binding to monomers and low-order
oligomers.
In addition to wild-type (WT) Aβ42, in which a single Y
residue is at position 10, we used analogues in which Y
substituted the original residues at positions 1, 20, 30, or 42,
and the native Y10 was substituted by the fluorometrically
silent F. The sequences of WT Aβ42 and its Y-substituted
analogues are shown in Figure 3A. These analogues were used
previously to study Aβ42 folding and assembly.34,35 Morpho-
logical studies by electron microscopy (EM) showed that all the
Y-substituted Aβ42 analogues formed fibrils, and the fibril
morphologies observed were similar to those formed by WT
Aβ42.34 Secondary structure dynamics examination by circular
dichroism spectroscopy showed that qualitatively all the Y-
substituted analogues were predominately disordered initially
and then displayed characteristic statistical coil to α-helix to β-
sheet transitions during oligomerization and fibril formation.34
Exposure to the aqueous milieu is known to decrease Y
fluorescence intensity without altering the wavelength of
maximum emission (λmax). In addition, the fluorescence of
the phenol group in the Y side-chain can be quenched by
exposure to hydrated carbonyl groups or through hydrogen-
bond formation with peptide carbonyls or with carboxylate
groups in aspartate or glutamate side-chains.34,36 In our
experimental system, an increase in Y fluorescence upon
addition of Aβ(39−42) would suggest a decrease in solvent
exposure, possibly indicating binding of the tetrapeptide to, or
in the vicinity of, the Y residue. Alternatively, an increase in
fluorescence could be interpreted as arising from increase in
intra- or intermolecular interactions within or between Aβ42
monomers, respectively. However, we reasoned that changes in
fluorescence resulting from global folding and/or assembly of
Aβ42 likely would affect Y residues in multiple positions,
whereas specific, local binding of Aβ(39−42) would lead to
increased Y fluorescence only in a specific position.
To minimize Aβ aggregation during the assay, all the samples
were pretreated with 1,1,1,3,3,3-hexafluoroisopropanol
(HFIP),37,38 and measurement of fluorescence was initiated
immediately following rehydration. In addition, aliquots were
monitored by EM. During the time of fluorescence measure-
ments (∼30 min), all the peptides formed quasiglobular
structures with diameters ranging from ∼7 to 15 nm and no
fibrils were observed. A representative electron micrograph of
Aβ42 prepared under these conditions is shown in Figure 3B.
The fluorescence intensity of Aβ42 and its Y-substituted
analogues is shown in Figure 3C. Consistent with a previous
report,34 the observed trend suggested that the degree of
exposure to the aqueous solvent decreased gradually from the
N- to the C-terminus.
The fluorescence intensity of Aβ42 analogues (5 μM) mixed
with Aβ(39−42) (50 μM) is shown in the second bar of each
group in Figure 3D. To facilitate the comparison among the
five Aβ42 analogues, the fluorescence intensity in the presence
of Aβ(39−42) in each case was normalized to the fluorescence
of the analogue in the absence of Aβ(39−42) (first bar of each
group in Figure 3D). We found that upon addition of Aβ(39−
42), the fluorescence of [Y1]Aβ42 increased by 117 ± 3%. In
contrast, the fluorescence of WT Aβ42, [Y20]Aβ42,
[Y30]Aβ42, and [Y42]Aβ42 did not change significantly
upon addition of Aβ(39−42). These results suggested that
Aβ(39−42) bound mainly at the N-terminus of Aβ42.
Because the N-terminus of Aβ contains several charged
residues, we hypothesized that the charged amino and carboxyl
groups in Aβ(39−42) might be important for its binding to the
N-terminus of Aβ42. To test this hypothesis, we examined the
8454
dx.doi.org/10.1021/jm200982p | J. Med. Chem. 2011, 54, 8451−8460

Journal of Medicinal Chemistry
Article
Figure 4. Aβ(39−42) binding site on Aβ42 determined by solution-state 2D NMR. Aβ42 resonances were measured in the absence (red) or
presence (cyan) of 8-fold excess Aβ(39−42). (A) 2D 13C−1H HSQC spectrum of Aβ42 in the absence or presence of Aβ(39−42) at 4 °C.
Hβ(Cβ)CO experiments detected upfield movement and reduced intensity of chemical shifts for the side-chains of D1, D7, and D23 upon addition of
Aβ(39−42). (B) 2D 15N−1H HSQC spectra of Aβ42 in the absence or presence of Aβ(39−42) detected upfield movement and reduced intensity of
chemical shifts for the Nε-Hε crosspeak of R5.
effect of Aβ(39−42) analogues in which the N- or C-termini
were blocked by acetylation (Ac-VVIA) or amidation (VVIA-
NH2), respectively, on Y fluorescence of Aβ42 and its Y-
substituted analogues. Representative spectra are shown in
Supporting Information Figure S2. The results are shown in the
third and fourth bars of each group in Figure 3D, respectively.
Upon addition of Ac-VVIA or VVIA-NH2, the fluorescence of
[Y1]Aβ42 increased by 48 ± 3% and 62 ± 6%, respectively.
These values were 2.4- and 1.8-times lower than with
unmodified Aβ(39−42), suggesting that both the carboxyl
and amino groups of Aβ(39−42) contributed to the interaction
with Aβ42. The larger loss of affinity caused by blocking the N-
terminus relative to blocking the C-terminus of Aβ(39−42) is
consistent with the loss of inhibitory activity observed for the
N-terminally acetylated analogue (Figure 1). Small effects on
fluorescence were observed in two other positions. The
fluorescence of Aβ42 decreased by 19 ± 3% upon addition of
Ac-VVIA, and the fluorescence of [Y30]Aβ42 decreased by 24
± 3% upon addition of VVIA-NH2. These data suggested that
removing either one of the charges in Aβ(39−42) decreased
the affinity of the peptide for the putative Aβ42 N-terminal
binding site and increased affinity for alternative binding sites.
8455
dx.doi.org/10.1021/jm200982p | J. Med. Chem. 2011, 54, 8451−8460

Journal of Medicinal Chemistry
Article
Analysis of the interaction of other Aβ(39−42) analogues
with [Y1]Aβ42 using internal Y fluorescence showed that the
fluorescence increase induced by the N-terminally acetylated
and C-terminally amidated retro sequences (Ac-AIVV and
AIVV-NH2), and by the inverso-peptide (vvia) also were
significantly lower than those of Aβ(39−42) (Supporting
Information Figure S3), in agreement with the low inhibitory
activity of these analogues. Not all the fluorescence results
correlated directly with the inhibition data presented in Figure
1, presumably because small sequence perturbation might affect
the binding mode of the tetrapeptide derivatives. Exploring the
binding sites of all the derivatives was beyond the scope of this
study, which was limited to delineation of the major binding
sites of the lead compound and the effect of the structural
changes that were found to have the greatest effect on the
inhibitory activity, namely the charge and chirality in Aβ(39−
42) analogues, which also had the greatest effect on binding to
the N-terminal region of Aβ42.
Solution-State NMR Characterization of the Interaction
between Aβ(39−42) and Aβ42. To complement the intrinsic
fluorescence experiments, we used solution-state, 15N−1H and
13C−1H heteronulear single quantum coherence (HSQC)
NMR experiments, which enable detection of residue-specific
signal perturbation upon binding of unlabeled Aβ(39−42) to
15N- or 13C/15N-labeled Aβ42. In preliminary experiments, the
NMR signal was observed to decrease gradually over 24 h due
to self-assembly of Aβ42, as reported previously.39 Nonetheless,
during the relatively short time needed for acquiring HSQC
spectra (∼37 min), perturbation of specific resonances upon
addition of Aβ(39−42) could be observed.
DISCUSSION AND CONCLUSIONS
■
Aβ(39−42) is a promising inhibitor of Aβ42-induced toxicity,
which unlike most peptide-based drug leads, has favorable
physicochemical characteristics. For future development of this
peptide lead toward metabolically stable peptidomimetic
derivatives, a detailed understanding of its mechanism of action
is needed. Here, using a combination of cell cultural and
biophysical methods, we found that the major mechanism by
which Aβ(39−42) inhibits Aβ42-induced toxicity is through
specific interaction with Aβ42, in agreement with computer
modeling predictions,25 DLS,23,24 and ion-mobility-spectrosco-
py−mass-spectrometry findings.40 In addition, Aβ(39−42)
showed a weak, nonspecific protective effect. Interestingly,
contrary to our initial hypothesis, the binding of Aβ(39−42)
appears to occur predominantly at the N-terminus of Aβ42.
Determination of the binding site of compounds that inhibit
Aβ assembly and toxicity is challenging because of the
difficulties associated with high-resolution structural study of
Aβ itself. Co-crystals of Aβ with inhibitors are difficult to
obtain, and the metastable character of Aβ oligomers does not
lend itself easily to high-resolution structure determination. The
combination of our SAR (Figure 1), fluorescence (Figure 3),
and NMR (Figure 4) data, and the weak effect of Aβ(39−42)
on alamethicine- or α-synuclein-induced toxicity (Figure 2) all
suggest that Aβ(39−42) binds to Aβ42 specifically, predom-
inantly at the N-terminus.
Multiple findings support an important role for the N-
terminus of Aβ in mediating assembly and toxicity. Two familial
AD-linked mutations resulting in the English (H6R)41 and
Tottori (D7N)42 variants were found to stabilize ordered
secondary structural elements in Aβ monomers, facilitate Aβ
oligomerization, and produce oligomeric assemblies that are
larger and are more toxic than those of WT Aβ.43 In addition, a
double substitution of the first two N-terminal residues of Aβ,
D1E/A2V, increases protofibril formation substantially.44 Thus,
the N-terminal region plays an important role in Aβ assembly
and toxicity, suggesting that small molecule binding in this
region may inhibit Aβ toxicity. In addition, N-terminally
truncated Aβ analogues, particularly those containing an N-
terminal pyroglutamate (pE), e.g., [pE3]Aβ or [pE11]Aβ, were
found in senile plaques and have been reported to form β-sheet
faster and with higher propensity45,46 and to be more toxic than
WT Aβ.47,48 One mechanism by which Aβ(39−42) may reduce
Aβ42 toxicity is by masking putative enzymatic cleavage sites
and thereby preventing the truncation of the N-terminus.
The observation that the N-terminally acetylated Aβ(39−42)
analogues, Ac-VVIA and Ac-AIVV, did not inhibit Aβ42-
induced toxicity suggests that electrostatic interactions between
the unprotected, positively charged N-terminal amino group of
Aβ(39−42) and negatively charged side-chain groups in Aβ42
might be important for inhibitory activity. The observation of
small chemical shift changes in the resonances of D1 and D7,
but not E3 (Figure 4A), supports the specificity of the binding.
An alternative explanation for the lack of inhibition by the
acetylated peptides may be creation of specific degradation
signals (degrons),49 leading to rapid proteolysis of Ac-VVIA
and Ac-AIVV. However, the large difference between the
perturbation of the intrinsic fluorescence of [Y1]Aβ42 by free
and acetylated analogues (Figure 3D and Supporting
Information Figure S3) supports an important role for
Coulombic interaction involving the amino group of Aβ(39−
42) and negatively charged side chains in the N-terminus of
The chemical shifts of most of the amino acid residues in
Aβ42 remained unchanged in the presence of 8-fold molar
excess Aβ(39−42) with the exceptions of small changes in D
and R side chains. 2D Hβ(Cβ)CO experiments optimized to
detect D side chains were collected to identify and monitor the
interaction of these side chains with Aβ(39−42). As shown in
1
Figure 4A, the H/13C signal of the D7 and D23 side-chains
showed a small shift and decreased in intensity upon addition
of Aβ(39−42) to Aβ42. The resonances for the D1 side chain
were isolated from D7 and D23 side-chain resonances and
appeared as multiple cross-peaks likely due to slow chemical
exchange. The side-chain resonance of R5 also was perturbed
upon addition of Aβ(39−42) to Aβ42 as shown by a small
upfield shift and decreased intensity of the Nε-Hε crosspeak in
15N−1H HSQC (Figure 4B). The data suggested that Aβ(39−
42) bound weakly, yet specifically to Aβ42 at positions near the
charged residues D1, R5, and D7 at the N-terminus, as well as
near D23.
To further explore the binding, we studied the interaction of
two analogues that showed weak or moderate inhibition of
Aβ42-induced toxicity (Figure 1) and increase in [Y1]Aβ42
fluorescence (Figure 3D and Supporting Information Figure
S3), Ac-VVIA and vvia, in NMR binding experiment. The
chemical shift changes of D1, D7, and D23 found upon
addition of Ac-VVIA (Supporting Information Figure S4) and
vvia (inverso-peptide) (Supporting Information Figure S5)
were small, similar to those induced by VVIA. These results
were consistent with the toxicity inhibition and intrinsic
fluorescence data, but the small magnitude of the effects
observed in the NMR experiments did not allow drawing
further conclusions regarding the binding site(s) of the
tetrapeptide derivatives on Aβ42.
8456
dx.doi.org/10.1021/jm200982p | J. Med. Chem. 2011, 54, 8451−8460

Journal of Medicinal Chemistry
Article
following general protocol: FMOC-protected, preloaded NovaSyn
TGA resin or PAL resin (0.1 mmol) was placed in a peptide synthesis
vessel, swollen in N,N-dimethylformamide (DMF), and deprotected
with 5 mL of 20% piperidine (or 4-methylpiperidine) in DMF for 20
min at room temperature. After washing with DMF thrice, a mixed
solution of 0.3 mmol FMOC-AA-OH, 0.3 mmol 2-(1H-benzotriazole-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, and 0.6 mmol
N,N-diisopropylethylamine in 4 mL of DMF was added to the reaction
vessel. The coupling reaction was performed using 40 W microwave
energy for 8 min at 50 °C. 2,4,6-Trinitrobenzenesulfonic acid test was
applied to check for remaining free amino groups.52 Coupling
efficiency was monitored by the formation of piperidine-dibenzoful-
vene or 4-methylpiperidine-dibenzofulvene using UV spectrosco-
py.53,54 Acetylation of the N-terminus of Ac-VVIA and Ac-AIVV was
performed using acetic anhydride/pyridine (1:2 v/v). After completion
of the sequence, the resin was thoroughly washed with DMF and then
with dichloromethane, dried under vacuum, and the peptide was
cleaved using a mixture of trifluoroacetic acid/1,2-ethanedithiol/H2O
(95:2.5:2.5). Peptides were precipitated by addition of cold diethyl
ether and purified by RP-HPLC. The purity of all peptides was higher
than 95% determined by analytical RP-HPLC. Peptides were further
characterized by MS and AAA. The peptide sequences, calculated
masses, and observed masses are listed in Table 2.
Cell Viability Assays. The methods for evaluation of the
biological activity of the CTFs themselves and their inhibition of
Aβ42-induced toxicity were described previously.23 Briefly, PC-12 cells
were differentiated into a neuronal phenotype by incubation with
nerve growth factor (100 ng/mL) for 48 h. For initial screening of the
new analogues, the cells then were incubated with solutions of Aβ42
alone at 10 μM nominal concentration, Aβ(39−42) analogues alone at
100 μM nominal concentration, or Aβ42:Aβ(39−42) analogue
mixtures at 1:10 concentration ratio, respectively, for 24 h. Cell
viability was determined by the MTT assay using a CellTiter 96 kit
(Promega, Madison, WI). Negative controls included NaOH at the
same concentration as in the peptide solutions and medium alone. A
positive control was 1 μM staurosporine for full kill, which was used to
represent a 100% reduction in cell viability, based on which the
percentage viability of all of the experimental conditions was
calculated.
Active analogues were characterized further in dose−response
experiments. In these experiments, Aβ42 alone and Aβ42:Aβ(39−42)
analogue mixtures at 1:1, 1:2, 1:5, and 1:10, or 1:1, 1:3 and 1:10,
concentration ratios were used and cell viability was determined by
both the MTT assay and the LDH-release assay (CytoTox-ONE
Homogenous Membrane Integrity Assay kit (Promega)). At least
three independent experiments with six replicates (n ≥ 18) were
performed for each assay. The results were averaged and presented as
mean ± SEM. Dose−response assays for inhibition of staurosporine-,
α-synuclein-, or alamethicin-induced toxicity by Aβ(39−42) were
performed using a similar protocol.
Intrinsic Fluorescence. Aβ42 or its Y-substituted analogues in
the absence or presence of Aβ(39−42) or its analogues were treated
with HFIP as described previously.55 Dry, HFIP-treated peptide films
were dissolved in 60 mM NaOH at 10% of the final volume and then
diluted with 10 mM sodium phosphate, pH 7.4, to the final nominal
concentrations, Aβ42 at 5 μM and Aβ(39−42) or its analogues at 50
μM. Samples were centrifuged at 5000 g for 1 min to remove trace
amount of dust particles that could interfere with the experiment due
to light scattering. The exact concentration was determined post facto
by AAA. Fluorescence was measured using a Hitachi F4500
spectrofluorimeter (Hitachi Instruments, Rye, NH) with excitation
at 280 nm and emission in the range 290−400 nm. At least 10
measurements of ∼1 min each were taken immediately following
sample preparation. All fluorescence measurements were carried out at
22 °C with a scan rate of 240 nm/min. Slit widths used for excitation
and emission were 5 and 10 nm, respectively. The fluorescence
emission spectrum of the phosphate buffer (background intensity) was
subtracted from the emission spectrum of each sample. The area under
the curve was calculated and normalized as the fluorescence intensity
per micromole. Four independent experiments were carried out. The
Aβ42 and suggest that degradation is unlikely the reason for the
low inhibition by the acetylated analogues. The observations
that amidation of the C-terminus also lowered perturbation of
[Y1]Aβ42 fluorescence (Figure 3D) and of a chemical shift and
intensity change in the resonance of R5 (Figure 4B) provide
additional support for contribution of specific electrostatic
interactions between Aβ(39−42) and the N-terminal region of
Aβ42.
In addition to the electrostatic interactions found here,
modeling studies have suggested that the hydrophobic residues
A2 and F4 are important for interaction of Aβ with cellular
membranes and potential inhibitors.25,50 Our data also support
an amphiphilic character for the interaction between Aβ(39−
42) and the N-terminus of Aβ42. Thus, the SAR experiments
(Figure 1) show that both the hydrophobic side chain at
position 41 and the charged N-terminal amino group are
important for inhibitory activity.
The C-terminus of Aβ42 is predicted to be shielded to a large
extent from the aqueous milieu in Aβ oligomers, a prediction
supported by multiple studies,6−8,10,51 and by the high
fluorescence of the Y residues in [Y30]Aβ42 and [Y42]Aβ42
(Figure 3C). Thus, the observation that the fluorescence of
[Y30]Aβ42 and [Y42]Aβ42 did not change significantly upon
addition of Aβ(39−42) may result from lower accessibility of
the C-terminal region of Aβ42 to the tetrapeptide. Alter-
natively, Aβ(39−42) may bind the C-terminus without causing
substantial change in Y fluorescence because the overall
hydrophobicity in the vicinity of the Y side-chain does not
change significantly. However, we did not observe any
perturbation of NMR resonances in the C-terminal region of
Aβ42 in the presence of 8-fold molar excess Aβ(39−42),
suggesting low probability of binding of the tetrapeptide in this
region.
Notably, we observed an increase in the fluorescence
emission of Aβ42 analogues and their mixtures with
tetrapeptides in wavelengths longer than the Y emission
window (Supporting Information Figure S2). This increase in
emission likely is due to light scattering and presumably reflects
promotion of Aβ42 assembly by the tetrapeptides, as we
observed previously in DLS experiments.23,24
Although further work will be required to elucidate the exact
binding mode of Aβ(39−42) to Aβ42, our data demonstrate
the specificity of this tetrapeptide as an inhibitor of Aβ42-
induced toxicity and shed light on the mechanism by which
Aβ(39−42) binds to Aβ42 and blocks its toxicity. The current
study provides structural basis for future development of
effective and stable peptidomimetic inhibitors of Aβ42
neurotoxicity as potential AD therapeutics.
EXPERIMENTAL SECTION
■
Chemicals and Reagents. 9-Fluorenylmethoxycarbonyl
(FMOC)-protected amino acids and NovaSyn TGA resin were
purchased from Novabiochem (Gibbstown, NJ). Wang and PAL resins
and all other reagents were obtained from Sigma-Aldrich (St. Louis,
MO) and were of the highest purity available. High-purity water (18.2
MΩ) was obtained using a Milli-Q system (Millipore, Bedford, MA).
Peptide Synthesis. Synthesis, purification, and characterization of
Aβ42 and Aβ42 analogues with Y substituted at positions 1, 20, 30,
and 42 and F substituted at position 10 were carried out as described
previously,34 purified using reverse-phase high-performance liquid
chromatography (RP-HPLC), and characterized by MS and amino
acid analysis (AAA).
Aβ(39−42) and its derivatives were synthesized using a Discover
microwave-assisted synthesis system (CEM, Matthews, NC) using the
8457
dx.doi.org/10.1021/jm200982p | J. Med. Chem. 2011, 54, 8451−8460

Journal of Medicinal Chemistry
Article
results were averaged and are presented as mean ± SEM of
fluorescence intensity (arbitrary units) or percentage of the
fluorescence intensity of control peptides.
C-terminal fragment; DMF, N,N-dimethylformamide; EM,
electron microscopy; FMOC, 9-fluorenylmethoxycarbonyl;
HFIP, 1,1,1,3,3,3-hexafluoroisopropanol; HSQC, heteronulear
single quantum coherence; LDH, lactate dehydrogenase; MTT,
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
MS, mass spectrometry; RP-HPLC, reverse-phase high-
performance liquid chromatography; SAR, structure−activity
relationship; ST, staurosporine; tPSA, topological polar surface
area; WT, wild-type
Electron Microscopy. Morphological examination was performed
as described briefly.56 Briefly, aliquots of each Aβ42 analogue in the
absence or presence of Aβ(39−42) or its analogues were spotted on
glow-discharged, carbon-coated Formvar grids (Electron Microscopy
Science, Hatfield, PA). The samples were the same as those used in the
fluorescence experiments. Samples were incubated for 10 min, fixed
with 5 μL 2.5% glutaraldehyde for 10 min, and stained with 5 μL of 1%
uranyl acetate for 10 min. Three to six replicates of each peptide were
analyzed using a CX 100 transmission electron microscope (JEOL,
Peabody, MA).
REFERENCES
■
Solution-State NMR. Uniformly isotopically labeled Aβ42 ([15N]
or [13C/15N]) were purchased from rPeptide (Athens, GA) and
treated with HFIP to disaggregate pre-existing aggregates as described
previously.55 The peptide was dissolved in 20 mM potassium
phosphate, pH 7.2, at nominal concentration 1 mg/mL and then
sonicated for 1 min. Aβ(39−42) was dissolved in 10 mM NaOH at 2
mg/mL and sonicated for 1 min. Then, Aβ42 and Aβ(39−42) were
mixed slowly to final concentrations 32 and 256 μM, respectively (1:8
concentration ratio). A control 15N-Aβ42 sample at 32 μM was
prepared by adding the same proportion of buffer and NaOH in the
absence of Aβ(39−42) to the Aβ42 stock solution to match the
solvent concentration and pH. 2D 15N−1H HSQC NMR spectra of
freshly prepared 15N-labeled Aβ42 samples in the absence or presence
of Aβ(39−42) were collected at 4 °C using a 600 MHz Bruker Avance
II spectrometer equipped with a cryoprobe. The acquisition time was
∼37 min for each HSQC spectrum. The average intensity percentage
of the five strongest, unequivocally assigned peaks in Aβ42 (Y10, V18,
A21, I32, and L34) was used to calculate the relative Aβ42 monomer
concentration. Hβ(Cβ)CO experiments were collected to support
aspartate assignments in Aβ42 and to monitor Aβ42 and Aβ(39−42)
interactions.
(1) Walsh, D. M.; Selkoe, D. J. Aβ oligomersa decade of discovery.
J. Neurochem. 2007, 101, 1172−1184.
(2) Kirkitadze, M. D.; Bitan, G.; Teplow, D. B. Paradigm shifts in
Alzheimer’s disease and other neurodegenerative disorders: the
emerging role of oligomeric assemblies. J. Neurosci. Res. 2002, 69,
567−577.
(3) Hardy, J.; Selkoe, D. J. The amyloid hypothesis of Alzheimer’s
disease: progress and problems on the road to therapeutics. Science
2002, 297, 353−356.
(4) Ferreira, S. T.; Vieira, M. N.; De Felice, F. G. Soluble protein
oligomers as emerging toxins in Alzheimer’s and other amyloid
diseases. IUBMB Life 2007, 59, 332−345.
(5) Dahlgren, K. N.; Manelli, A. M.; Stine, W. B. Jr.; Baker, L. K.;
Krafft, G. A.; LaDu, M. J. Oligomeric and fibrillar species of amyloid-β
peptides differentially affect neuronal viability. J. Biol. Chem. 2002, 277,
32046−32053.
(6) Bitan, G.; Kirkitadze, M. D.; Lomakin, A.; Vollers, S. S.; Benedek,
G. B.; Teplow, D. B. Amyloid β-protein (Aβ) assembly: Aβ40 and
Aβ42 oligomerize through distinct pathways. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 330−335.
(7) Bernstein, S. L.; Dupuis, N. F.; Lazo, N. D.; Wyttenbach, T.;
Condron, M. M.; Bitan, G.; Teplow, D. B.; Shea, J.-E.; Ruotolo, B. T.;
Robinson, C. V.; Bowers, M. T. Amyloid-β protein oligomerization
and the importance of tetramers and dodecamers in the aetiology of
Alzheimer’s disease. Nature Chem. 2009, 1, 326−331.
ASSOCIATED CONTENT
■
S
* Supporting Information
Dose−response inhibition of Aβ42-induced toxicity by Aβ(39−
42) analogues, change in intrinsic fluorescence of each Y-
substituted Aβ42 analogue upon addition of Aβ(39−42) and
derivatives, fluorescence change of [Y1]Aβ42 upon addition of
each Aβ(39−42) derivative, and chemical shift of Aβ42 in
solution-state, 2D-NMR in the absence and presence of Ac-
VVIA or vvia. This material is available free of charge via the
Internet at http://pubs.acs.org.
(8) Bitan, G.; Vollers, S. S.; Teplow, D. B. Elucidation of primary
structure elements controlling early amyloid β-protein oligomerization.
J. Biol. Chem. 2003, 278, 34882−34889.
(9) Lazo, N. D.; Grant, M. A.; Condron, M. C.; Rigby, A. C.; Teplow,
D. B. On the nucleation of amyloid β-protein monomer folding.
Protein Sci. 2005, 14, 1581−1596.
(10) Urbanc, B.; Cruz, L.; Yun, S.; Buldyrev, S. V.; Bitan, G.; Teplow,
D. B.; Stanley, H. E. In silico study of amyloid β-protein folding and
oligomerization. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17345−17350.
(11) Tjernberg, L. O.; Naslund, J.; Lindqvist, F.; Johansson, J.;
̈
AUTHOR INFORMATION
Karlstrom, A. R.; Thyberg, J.; Terenius, L.; Nordstedt, C. Arrest of β-
amyloid fibril formation by a pentapeptide ligand. J. Biol. Chem. 1996,
271, 8545−8548.
■
Corresponding Author
*Phone: (310) 206-2082. Fax: (310) 206-1700. E-mail:
gbitan@mednet.ucla.edu.
(12) Soto, C.; Kindy, M. S.; Baumann, M.; Frangione, B. Inhibition of
Alzheimer’s amyloidosis by peptides that prevent β-sheet
conformation. Biochem. Biophys. Res. Commun. 1996, 226, 672−680.
(13) Lowe, T. L.; Strzelec, A.; Kiessling, L. L.; Murphy, R. M.
Structure−function relationships for inhibitors of β-amyloid toxicity
containing the recognition sequence KLVFF. Biochemistry 2001, 40,
7882−7889.
(14) Findeis, M. A.; Musso, G. M.; Arico-Muendel, C. C.; Benjamin,
H. W.; Hundal, A. M.; Lee, J. J.; Chin, J.; Kelley, M.; Wakefield, J.;
Hayward, N. J.; Molineaux, S. M. Modified-peptide inhibitors of
amyloid β-peptide polymerization. Biochemistry 1999, 38, 6791−6800.
(15) Hughes, E.; Burke, R. M.; Doig, A. J. Inhibition of toxicity in the
β-amyloid peptide fragment β-(25−35) using N-methylated
derivativesa general strategy to prevent amyloid formation. J. Biol.
Chem. 2000, 275, 25109−25115.
Present Address
⊥Department of Biology, Whittier College, 13406 East
Philadelphia Street, Whittier, CA 90608.
ACKNOWLEDGMENTS
■
We thank Dr. David Teplow for the use of his spectro-
fluorometer and plate reader and Drs. Brigita Urbanc, Raz
Jelinek, Farid Rahimi, Inna Solomonov, and Panchanan Maiti
for critical reading of the manuscript and helpful discussions.
The work was supported by grant AG027818 from the NIH/
NIA.
(16) Gordon, D. J.; Sciarretta, K. L.; Meredith, S. C. Inhibition of β-
amyloid(40) fibrillogenesis and disassembly of β-amyloid(40) fibrils by
short β-amyloid congeners containing N-methyl amino acids at
alternate residues. Biochemistry 2001, 40, 8237−8245.
ABBREVIATIONS USED
■
AAA, amino acid analysis; Aβ, amyloid β-protein; AD,
Alzheimer’s disease; CHC, central hydrophobic cluster; CTF,
8458
dx.doi.org/10.1021/jm200982p | J. Med. Chem. 2011, 54, 8451−8460

Journal of Medicinal Chemistry
Article
(17) Zhang, S.; Iwata, K.; Lachenmann, M. J.; Peng, J. W.; Li, S.;
Stimson, E. R.; Lu, Y.; Felix, A. M.; Maggio, J. E.; Lee, J. P. The
Alzheimer’s peptide Aβ adopts a collapsed coil structure in water. J.
Struct. Biol. 2000, 130, 130−141.
(35) Maji, S. K.; Ogorzalek Loo, R. R.; Inayathullah, M.; Spring, S.
M.; Vollers, S. S.; Condron, M. M.; Bitan, G.; Loo, J. A.; Teplow, D. B.
Amino acid position-specific contributions to amyloid β-protein
oligomerization. J. Biol. Chem. 2009, 284, 23580−23591.
(36) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; 2nd ed.;
Kluwer Academic/Plenum Publishers: New York, 1999; p 698.
(37) Zagorski, M. G.; Yang, J.; Shao, H.; Ma, K.; Zeng, H.; Hong, A.
Methodological and chemical factors affecting amyloid β peptide
amyloidogenicity. Methods Enzymol. 1999, 309, 189−204.
(38) Stine, W. B. Jr.; Dahlgren, K. N.; Krafft, G. A.; LaDu, M. J. In
vitro characterization of conditions for amyloid-β peptide
oligomerization and fibrillogenesis. J. Biol. Chem. 2003, 278, 11612−
11622.
(18) Hochdorffer, K.; Marz-Berberich, J.; Nagel-Steger, L.; Epple, M.;
̈
̈
Meyer-Zaika, W.; Horn, A. H.; Sticht, H.; Sinha, S.; Bitan, G.;
Schrader, T. Rational design of β-sheet ligands against Aβ42-induced
toxicity. J. Am. Chem. Soc. 2011, 133, 4348−4358.
(19) Hetenyi, C.; Szabo, Z.; Klement, T.; Datki, Z.; Kortvelyesi, T.;
Zarandi, M.; Penke, B. Pentapeptide amides interfere with the
aggregation of β-amyloid peptide of Alzheimer’s disease. Biochem.
Biophys. Res. Commun. 2002, 292, 931−936.
(20) Szegedi, V.; Fulop, L.; Farkas, T.; Rozsa, E.; Robotka, H.; Kis,
̈
̈
Z.; Penke, Z.; Horvath, S.; Molnar, Z.; Datki, Z.; Soos, K.; Toldi, J.;
Budai, D.; Zarandi, M.; Penke, B. Pentapeptides derived from Aβ1−42
protect neurons from the modulatory effect of Aβ fibrilsan in vitro
and in vivo electrophysiological study. Neurobiol. Dis. 2005, 18, 499−
508.
(39) Yan, Y.; Wang, C. Aβ42 is more rigid than Aβ40 at the C
terminus: implications for Aβ aggregation and toxicity. J. Mol. Biol.
2006, 364, 853−862.
(40) Gessel, M. M.; Wu, C.; Li, H.; Bitan, G.; Shea, J. E.; Bowers, M.
T. Unpublished results.
(21) Pratim Bose, P.; Chatterjee, U.; Nerelius, C.; Govender, T.;
Norstrom, T.; Gogoll, A.; Sandegren, A.; Gothelid, E.; Johansson, J.;
Arvidsson, P. I. Poly-N-methylated amyloid β-peptide (Aβ) C-terminal
fragments reduce Aβ toxicity in vitro and in Drosophila melanogaster. J.
Med. Chem. 2009, 52, 8002−8009.
(22) Rahimi, A. F.; Shanmugam, A.; Bitan, G. Structure−function
relationships of pre-fibrillar protein assemblies in Alzheimer’s disease
and related disorders. Curr. Alzheimer Res. 2008, 5, 319−341.
(23) Fradinger, E. A.; Monien, B. H.; Urbanc, B.; Lomakin, A.; Tan,
M.; Li, H.; Spring, S. M.; Condron, M. M.; Cruz, L.; Xie, C. W.;
Benedek, G. B.; Bitan, G. C-Terminal peptides coassemble into Aβ42
oligomers and protect neurons against Aβ42-induced neurotoxicity.
Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 14175−14180.
(24) Li, H.; Monien, B. H.; Lomakin, A.; Zemel, R.; Fradinger, E. A.;
Tan, M.; Spring, S. M.; Urbanc, B.; Xie, C. W.; Benedek, G. B.; Bitan,
G. Mechanistic investigation of the inhibition of Aβ42 assembly and
neurotoxicity by Aβ42 C-terminal fragments. Biochemistry 2010, 49,
6358−6364.
(25) Urbanc, B.; Betnel, M.; Cruz, L.; Li, H.; Fradinger, E. A.;
Monien, B. H.; Bitan, G. Structural basis for Aβ(1−42) toxicity
inhibition by Aβ C-terminal fragments: discrete molecular dynamics
study. J. Mol. Biol. 2011, 410, 316−328.
(26) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Experimental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv. Drug
Delivery Rev. 2001, 46, 3−26.
(27) Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.; Ward,
K. W.; Kopple, K. D. Molecular properties that influence the oral
bioavailability of drug candidates. J. Med. Chem. 2002, 45, 2615−2623.
(28) Bertrand, R.; Solary, E.; O’Connor, P.; Kohn, K. W.; Pommier,
Y. Induction of a common pathway of apoptosis by staurosporine. Exp.
Cell Res. 1994, 211, 314−321.
(29) Arispe, N.; Diaz, J. C.; Simakova, O. Aβ ion channels. Prospects
for treating Alzheimer’s disease with Aβ channel blockers. Biochim.
Biophys. Acta 2007, 1768, 1952−1965.
(30) Sokolov, Y.; Kozak, J. A.; Kayed, R.; Chanturiya, A.; Glabe, C.;
Hall, J. E. Soluble amyloid oligomers increase bilayer conductance by
altering dielectric structure. J. Gen. Physiol. 2006, 128, 637−647.
(31) Fringeli, U. P.; Fringeli, M. Pore formation in lipid membranes
by alamethicin. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 3852−3856.
(32) Auluck, P. K.; Caraveo, G.; Lindquist, S. α-Synuclein: membrane
interactions and toxicity in Parkinson’s disease. Annu. Rev. Cell Dev.
Biol. 2010, 26, 211−233.
(33) Condron, M. M.; Monien, B. H.; Bitan, G. Synthesis and
purification of highly hydrophobic peptides derived from the C-
terminus of amyloid β-protein. Open Biotechnol. J. 2008, 2, 87−93.
(34) Maji, S. K.; Amsden, J. J.; Rothschild, K. J.; Condron, M. M.;
Teplow, D. B. Conformational dynamics of amyloid β-protein
assembly probed using intrinsic fluorescence. Biochemistry 2005, 44,
13365−13376.
(41) Janssen, J. C.; Beck, J. A.; Campbell, T. A.; Dickinson, A.; Fox,
N. C.; Harvey, R. J.; Houlden, H.; Rossor, M. N.; Collinge, J. Early
onset familial Alzheimer’s diseasemutation frequency in 31 families.
Neurology 2003, 60, 235−239.
(42) Wakutani, Y.; Watanabe, K.; Adachi, Y.; Wada-Isoe, K.;
Urakami, K.; Ninomiya, H.; Saido, T. C.; Hashimoto, T.; Iwatsubo,
T.; Nakashima, K. Novel amyloid precursor protein gene missense
mutation (D678N) in probable familial Alzheimer’s disease. J. Neurol.
Neurosurg. Psychiatry 2004, 75, 1039−1042.
(43) Ono, K.; Condron, M. M.; Teplow, D. B. Effects of the English
(H6R) and Tottori (D7N) familial Alzheimer disease mutations on
amyloid β-protein assembly and toxicity. J. Biol. Chem. 2010, 285,
23186−23197.
(44) Qahwash, I.; Weiland, K. L.; Lu, Y. F.; Sarver, R. W.; Kletzien, R.
F.; Yan, R. Q. Identification of a mutant amyloid peptide that
predominantly forms neurotoxic protofibrillar aggregates. J. Biol. Chem.
2003, 278, 23187−23195.
(45) He, W. L.; Barrow, C. J. The Aβ 3-pyroglutamyl and 11-
pyroglutamyl peptides found in senile plaque have greater β-sheet
forming and aggregation propensities in vitro than full-length Aβ.
Biochemistry 1999, 38, 10871−10877.
(46) Schilling, S.; Lauber, T.; Schaupp, M.; Manhart, S.; Scheel, E.;
Bohm, G.; Demuth, H. U. On the seeding and oligomerization of
pGlu-amyloid peptides (in vitro). Biochemistry 2006, 45, 12393−
12399.
(47) Russo, C.; Violani, E.; Salis, S.; Venezia, V.; Dolcini, V.;
Damonte, G.; Benatti, U.; D’Arrigo, C.; Patrone, E.; Carlo, P.;
Schettini, G. Pyroglutamate-modified amyloid β-peptides Aβ
N3(pE)-strongly affect cultured neuron and astrocyte survival. J.
Neurochem. 2002, 82, 1480−1489.
(48) Wirths, O.; Breyhan, H.; Cynis, H.; Schilling, S.; Demuth, H. U.;
Bayer, T. A. Intraneuronal pyroglutamate-Aβ 3−42 triggers
neurodegeneration and lethal neurological deficits in a transgenic
mouse model. Acta Neuropathol. 2009, 118, 487−496.
(49) Hwang, C. S.; Shemorry, A.; Varshavsky, A. N-Terminal
acetylation of cellular proteins creates specific degradation signals.
Science 2010, 327, 973−977.
(50) Urbanc, B.; Betnel, M.; Cruz, L.; Bitan, G.; Teplow, D. B.
Elucidation of amyloid β-protein oligomerization mechanisms: discrete
molecular dynamics study. J. Am. Chem. Soc. 2010, 132, 4266−4280.
(51) Zhao, W. Q.; Toolan, D.; Hepler, R. W.; Wolfe, A. L.; Yu, Y.;
Price, E.; Uebele, V. N.; Schachter, J. B.; Reynolds, I. J.; Renger, J. J.;
McCampbell, A.; Ray, W. J. High throughput monitoring of amyloid-
β42 assembly into soluble oligomers achieved by sensitive
conformation state-dependent immunoassays. J. Alzheimer's Dis.
2011, 25, 655−669.
(52) Chan, W. C.; White, P. D. Fmoc Solid-Phase Peptide Synthesis: A
Practical Approach; Oxford University Press: New York, 2000; p xxiv,
346.
8459
dx.doi.org/10.1021/jm200982p | J. Med. Chem. 2011, 54, 8451−8460

Journal of Medicinal Chemistry
Article
(53) Gude, M.; Ryf, J.; White, P. D. An accurate method for the
quantitiation of Fmoc-derivatized solid phase support. Lett. Pept. Sci.
2002, 9, 203−206.
(54) Varady, L.; Rajur, S. B.; Nicewonger, R. B.; Guo, M.; Ditto, L.
Fast and quantitative high-performance liquid chromatography
method for the determination of 9-fluorenylmethoxycarbonyl release
from solid-phase synthesis resins. J. Chromatogr., A 2000, 869, 171−
179.
(55) Rahimi, F.; Maiti, P.; Bitan, G. Photo-induced cross-linking of
unmodified proteins (PICUP) applied to amyloidogenic peptides. J.
Vis. Exp. 2009, http://www.jove.com/index/details.stp?id=1071.
(56) Li, H.; Monien, B. H.; Fradinger, E. A.; Urbanc, B.; Bitan, G.
Biophysical characterization of Aβ42 C-terminal fragments: inhibitors
of Aβ42 neurotoxicity. Biochemistry 2010, 49, 1259−1267.
8460
dx.doi.org/10.1021/jm200982p | J. Med. Chem. 2011, 54, 8451−8460