Synthesis and evaluation of ferrocenoyl pentapeptide (Fc-KLVFF) as an inhibitor of Alzheimer's Aβ1-42 fibril formation in vitro

Article abstract of DOI:10.1016/j.bmcl.2011.07.111

Aggregation and fibril formation of β-amyloid peptides (Aβ) is the key event in the pathogenesis of Alzheimer's disease. Many efforts have been made on the development of effective inhibitors to prevent Aβ fibril formation or disassemble the preformed Aβ fibrils. Peptide inhibitors with sequences homologous to the hydrophobic segments of Aβ can alter the aggregation pathway of Aβ, together with decrease of the cell toxicity. In this study, the conjugate of ferrocenoyl (Fc) with pentapeptide KLVFF (Fc-KLVFF), was synthesized by HBTU/HOBt protocol in solution. The inhibitory effect of Fc-KLVFF on Aβ_1-42 fibril formation was evaluated by thioflavin T fluorescence assay, and confirmed by atomic force microscopy (AFM) and transmission electron microscopy (TEM) analyses. Fc-KLVFF shows high inhibitory effect towards the fibril formation of Aβ_1-42. Additionally, the attachment of ferrocenoyl moiety onto peptides allows us to investigate the interaction between the inhibitor and Aβ_1-42 in real-time by electrochemical method. As expected, tethering of ferrocenoyl moiety onto pentapeptide shows improved lipophilicity and significant resistance towards proteolytic degradation compared to its parent peptide.

Full text of DOI:10.1016/j.bmcl.2011.07.111

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5818–5821
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of ferrocenoyl pentapeptide (Fc-KLVFF)
as an inhibitor of Alzheimer’s Ab1–42 fibril formation in vitro
Chuan-Wan Wei a,b, Yong Peng a, Lin Zhang a, Qiyu Huang a, Ming Cheng a, You-Nian Liu a, , Juan Li a,
⇑
⇑
a College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, PR China
b College of Chemistry and Chemical Engineering, University of South China, Hengyang, Hunan 421001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Aggregation and fibril formation of b-amyloid peptides (Ab) is the key event in the pathogenesis of Alz-
heimer’s disease. Many efforts have been made on the development of effective inhibitors to prevent Ab
fibril formation or disassemble the preformed Ab fibrils. Peptide inhibitors with sequences homologous
to the hydrophobic segments of Ab can alter the aggregation pathway of Ab, together with decrease of the
cell toxicity. In this study, the conjugate of ferrocenoyl (Fc) with pentapeptide KLVFF (Fc-KLVFF), was syn-
thesized by HBTU/HOBt protocol in solution. The inhibitory effect of Fc-KLVFF on Ab1–42 fibril formation
was evaluated by thioflavin T fluorescence assay, and confirmed by atomic force microscopy (AFM) and
transmission electron microscopy (TEM) analyses. Fc-KLVFF shows high inhibitory effect towards the
fibril formation of Ab1–42. Additionally, the attachment of ferrocenoyl moiety onto peptides allows us
to investigate the interaction between the inhibitor and Ab1–42 in real-time by electrochemical method.
As expected, tethering of ferrocenoyl moiety onto pentapeptide shows improved lipophilicity and signif-
icant resistance towards proteolytic degradation compared to its parent peptide.
Received 15 March 2011
Revised 14 July 2011
Accepted 28 July 2011
Available online 2 August 2011
Keywords:
b-amyloid aggregation
Inhibitor
Ferrocene
KLVFF
Ó 2011 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is the most common cause of demen-
tia and there is so far no clinically accepted treatment to cure it
or to halt its progression. In the past two decades, tremendous re-
search efforts have been made to understand the pathogenesis of
AD, thus providing a platform to develop effective pharmacologi-
cal treatments. Although the precise mechanisms of neurodegen-
eration in AD are not yet fully discovered, several pieces of
evidence indicate that the conformational changes along with
the aggregation and fibril formation of beta-amyloid (Ab) might
play a pivotal role in the pathogenesis of AD. The Ab peptide, a
39–43 residue amphipathic peptide, is formed after sequential
and its derivatives3 were found to inhibit or reduce the aggregation
and toxicity of Ab in vitro. Especially, in recent years, many
modified peptides or peptidomimetics derived from the parent
Ab have been synthesized as inhibitors towards Ab aggregation.4
Among the peptide inhibitors, KLVFF, which contains the hydro-
phobic core of Ab1–42 and can interact with the corresponding res-
idues of Ab via self-cognition, shows high inhibitory effect for Ab
fibril formation and disassembles the preformed Ab fibrils.5 Thus,
KLVFF sequence was widely employed as a leading compound for
the development of inhibitory agents for Alzheimer’s disease.5,6
However, the inherent properties of KLVFF, such as poor stability
against enzymatic degradation and low lipophilicity, hinder its
application as an inhibitor for Ab fibril formation. Lots of
approaches were proposed to improve such properties of KLVFF.
For example, Findeis et al. added a bulky group to the N-terminus
of the peptide motif, and designed the peptide analogues cholyl-
LVFFA-OH (PPI-368), which showed significantly improved activity
in inhibiting aggregation of Ab in vitro.7
cleavage of the amyloid precursor protein (APP), by b- and
c-
secretase. Understanding the aggregation process and developing
agents that can inhibit Ab fibril formation or disassemble the pre-
formed toxic Ab fibrils is at the forefront of treatment for those
suffering from Alzheimer’s disease. A lot of progress has recently
been made in the design of inhibitors for Ab fibril formation and/
or misfolding.
To date, lots of structurally different compounds have been
identified to prevent Ab aggregation or disassemble the preformed
fibrils. For example, some small organic molecules, such as the dye
Congo red,1 apomorphine derivatives,2 the antibiotic rifampicin
Ferrocene and its derivatives exhibit manifold medicinal
properties such as antimalarial,8–10 antitumor11,12 and antibacte-
rial properties of ferrocenyl compounds,13,14 due to their biological
activity, low cytotoxicity, high lipophilicity and unique
electrochemical behavior.15,16 In particular, when incorporated
into a drug, ferrocene moiety could yield interesting results. For in-
stance, Metzler-Nolte and co-workers synthesized ferrocene-
[Leu(5)]-enkephalin conjugate and found that the introduction of
the ferrocene moiety enhanced the uptake into cells and the
⇑
Corresponding authors. Tel.: +86 731 8883 6964; fax: +86 731 8887 9616 (Y.-
N.L.).
E-mail addresses: liuyounian@csu.edu.cn (Y.-N. Liu), juanli@csu.edu.cn (J. Li).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.111

C.-W. Wei et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5818–5821
5819
permeation coefficient of [Leu(5)]-enkephalin.17 Jaouen and
co-workers attached the ferrocene moiety into an anticancer drug
tamoxifen.18
In order to combine the lipophilic nature, proteolytic stability
and the unique electrochemical behavior of ferrocenoyl moiety
and inhibitory characteristic of the pentapeptide KLVFF, we
designed a ferrocenoyl attached peptide Fc-Lys(Z)-Leu-Val-Phe-
Phe-OMe (Fc-KLVFF) (see Fig. 1). The inhibitory effects of the
ferrocenoyl conjugate on Ab1–42 aggregation were investigated by
thioflavin T (ThT) fluorescence assay, and confirmed by AFM,
TEM analyses and electrochemical method in vitro.
The ferrocenoyl attached pentapeptide Fc-KLVFF, corresponding
to the residues 16–20 of Ab, was synthesized using standard Boc
protecting group and activated by HBTU/HOBt strategy in solution
following the reported procedures.19,20 The identity of the ferroce-
noyl peptide was confirmed by ESI-MS, NMR, UV–vis and FT-IR.
The proton and carbon chemical shifts of NMR for Fc-KLVFF were
assigned by analysis of COSY, HSQC, 13C and HMBC spectra (for de-
tails see Supplementary data).
The binding of ThT dye to amyloid is specific and generates a
shift in the emission spectrum of ThT and a fluorescence enhance-
ment proportional to the amount of amyloid.21 Thus, ThT fluores-
cence assay was utilized to quantify the inhibitory effect of the
compounds on Ab1–42 fibril formation in vitro. All the measure-
ments were carried out under the same experimental conditions
as described in Supplementary data. The kinetics of fibril formation
of Ab1–42 was examined by ThT fluorescence in the absence or pres-
ence of each inhibitor for 12 days at 32 °C. The inhibitory effect of
inhibitors on Ab1–42 fibril formation is presented in Figure 2.
Figure 2. Kinetics of KLVFF and Fc-KLVFF inhibition on Ab1–42 fibril formation using
ThT fluorescence assay. The freshly solubilized 10
l
M Ab1–42 was incubated at 32 °C
M Fc-KLVFF (j) in a
alone (h), or in the presence of 40 M KLVFF (O) and 40
l
l
mixed solvent of DMSO and water (3:1, V/V) for the indicated times. Fibril
formation was quantified by measuring fluorescence intensity. Data were expressed
as percentage of maximum fibril formation (fluorescence intensity). Each experi-
ment was repeated three times (n = 3). Error bars represent the standard deviation
(SD) of the fluorescence measurement.
As depicted in Figure 2, the freshly solubilized 10 lM Ab1–42,
when incubated alone at 32 °C, shows a gradual increase in fluores-
cence signal. The fluorescence intensity reaches its plateau at the
sixth day of incubation. When Ab1–42 is incubated in the presence
of 40 lM of KLVFF, the fluorescence intensity increases and reaches
its maximum at the fourth day. However, the maximum fluores-
cence intensity of Ab1–42 in the presence of KLVFF is much lower
than that of Ab1–42 alone. After that, a slight decrease of the fluores-
cence intensity in the presence of KLVFF was observed. Fc-KLVFF
exhibits similar ThT fluorescence pattern as that of KLVFF. Both
KLVFF and Fc-KLVFF reveals significantly inhibitory effects, result-
ing in 89% and 93% reduction of the fibril formation, respectively.
Our results point to robust inhibitory effects of Fc-KLVFF toward
Ab1À42 fibril formation. In addition, we tested the dose–response
relationship to the inhibitory effect. As shown in Figure 3, the
inhibitory effect enhances rapidly with increasing dosage of inhib-
itors. The IC50 values (the molar ratio of inhibitor to Ab1À42 with
50% maximum effect) for Fc-KLVFF and KLVFF are 0.18 and 0.33,
respectively. The results clearly indicate that both Fc-KLVFF and
KLVFF have excellent inhibitory effects on Ab1–42 fibril formation.
Figure 3. Dose–response inhibition of inhibitors on Ab1–42 fibril formation. The
freshly solubilized 10 lM Ab1–42 was incubated at 32 °C in the presence of KLVFF(j)
and Fc-KLVFF (h) of difference concentrations in a mixed solvent of DMSO and
water (3:1, v/v) for four days. Fibril formation was quantified by measuring
fluorescence intensity.
Figure 1. Structures of KLVFF and Fc-KLVFF.

5820
C.-W. Wei et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5818–5821
Figure 4. AFM images of 10
l
M Ab1–42 incubated at 32 °C for 7 days in the absence (a) or in the presence of 40
lM Fc-KLVFF (b), and 40 lM KLVFF (c) in a mixed solvent of
DMSO and water (3:1, v/v). The scale of each image is 2 Â 2
lm. The cross-sectional contours showed the representative aggregates identified by the line.
Furthermore, a control experiment was performed to test the
amyloid-like fibril formation of Fc-KLVFF alone using ThT fluores-
cence assay. A 40 lM Fc-KLVFF or KLVFF was incubated alone for
7 days at 32 °C, respectively. No obvious ThT fluorescence was ob-
served in either solution (data no shown), which suggests that nei-
ther KLVFF nor Fc-KLVFF undergoes amyloid-like fibril formation in
solution.
AFM and TEM were employed to confirm the inhibitory effect of
Fc-KLVFF on Ab fibril formation. As revealed by Figure 4a, in the
absence of inhibitor, Ab1–42 formed visible amyloid-like, 6–10 nm
the interaction between Ab and Fc-KLVFF according to the redox-
active properties of ferrocenoyl conjugate. The electrochemical
behavior of Fc-KLVFF was investigated by cyclic voltammetry
(CV). Fc-KLVFF exhibits a reversible electrochemical one-electron
redox, with the ratio of oxidative to reductive peak currents of
close to unity. The cathodic and anodic peak potentials were
observed at 0.546 and 0.624 V versus Ag/AgCl, respectively. In
addition, the kinetics of Ab1–42 fibril formation in the presence of
Fc-KLVFF was calculated by conducting differential pulse voltam-
metry (DPV). As shown in Figure 6, the oxidation current decreases
sharply in a very short time when Fc-KLVFF is incubated with
Ab1–42, and reaches its plateau after 6 h. The trend is consistent
with that of ThT fluorescence assay, which further confirms that
the Fc-KLVFF is capable of interacting with Ab1–42 instantaneously.
in diameter (or height) fibrils. In contrast, in the presence of 40 lM
Fc-KLVFF, only a few insoluble globular aggregates were observed
but no protofibrils or any kind of fibrillar materials were shown
(cf. Fig. 4b). We also found that the incubation of Ab1–42 in the pres-
ence of 40
lM KLVFF yields amorphous aggregates, but the height of
In addition, a 40 lM Fc-KLVFF was incubated alone under the same
the amorphous deposit is lower than that in the presence of
Fc-KLVFF (cf. the cross-sectional contours of representative aggre-
gates were given below the AFM images in Figure 4). The AFM results
are in striking agreement with the results reported by Etienne et al.
who used modified KLVFF (AMY-1 and AMY-2) as inhibitors.22 In our
work, as shown in Figure 5, Fc-KLVFF and KLVFF are able to inhibit Ab
1–42 fibrillization even after 5 months of incubation at room temper-
ature. TEM images of Ab1–42 incubated in the presence of Fc-KLVFF at
room temperature for 5 months displayed no signs of fibril
formation but rather only globular, non-fibrillar protein aggregates
(Fig. 5a). In the same condition, Ab1–42 incubated with KLVFF also
displays no identifiable fibrils but only amorphous aggregates
(Fig. 5b). The AFM and TEM analytical results are in good agreement
with ThT fluorescence.
experimental conditions as a control experiment. It was found that
only 7.6% decrease in the anodic current of Fc-KLVFF occurred after
24 h of incubation in the mixed solvents, probably due to the slight
aggregation of Fc-KLVFF. Consequently, the decrease of peak
current of Fc-KLVFF in the presence of Ab can be mainly attributed
to the association of the Fc-KLVFF with Ab1–42 monomer and/or
oligomer in solution to form insoluble adducts or the attachment
of the Fc-KLVFF to Ab1–42 aggregates, but not the self-aggregation
of Fc-KLVFF.
The major drawback for peptides as drugs in central nervous
system diseases is their rapid degradation by proteolytic enzymes
and poor membrane permeability. For example, the use of cholyl-
LVFFA-OH as a therapeutic agent is restricted by its propensity to
be cleared up almost completely after the hepatic first pass.23
One of our aims of modifying of KLVFF with ferrocenoyl moiety
is to improve its proteolytic stability in blood stream and increase
To obtain further evidence for the interaction of the Fc-KLVFF
with the Ab1–42, we utilized the electrochemical method to study
Figure 5. TEM images of 10 lM Ab1–42 incubated for 5 months at room temperature in the presence of 40 lM Fc-KLVFF (a), and 40 lM KLVFF (b).

C.-W. Wei et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5818–5821
5821
degradation. In particular, the attachment of ferrocenoyl moiety
onto peptides allows us to investigate the aggregation pathway
of Ab1–42 in situ by electrochemical method. Nevertheless, the
synthesized ferrocenoyl KLVFF shows poor solubility in water. In
order to solve the problem, we are trying to synthesize the ferroce-
noyl peptide with inclusion of some hydrophilic amino acid
residues on the C-terminus, and planning to use this novel ferroce-
noyl peptide to control neuronal toxicity from Ab species as part of
the future work in cellular system.
Acknowledgments
We thank Dr. Feimeng Zhou (California State University, Los
Angeles) for helpful discussions in electrochemistry. We are
grateful to the National Natural Science Foundation of China
(Nos. 20876179, 20904066 and 21076232) for financial support.
Figure 6. Inhibition of Fc-KLVFF towards Ab1–42 fibril formation detecting by using
DPV method. The 40 M Fc-KLVFF was incubated in the absence (j), and in the
presence of freshly solubilized 10 M Ab1–42 (h) in a mixed solvent of DMSO and
l
Supplementary data
l
water (3:1, v/v) at 32 °C for the indicated times. The experiment was repeated three
times (n = 3). Error bars represent the standard deviation (SD) of the current.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.07.111.
References and notes
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its lipophilicity. Proteolytic stability of KLVFF and Fc-KLVFF were
determined by reverse-phase high-performance liquid chromatog-
raphy (RP-HPLC). The experimental results showed that the
modification with ferrocenoyl moiety increases the resistance of
the compound to proteolysis. After 3 h incubation with proteolytic
enzyme at 37 °C, only 18% of KLVFF was detected, while 58% of the
Fc-KLVFF survived. After 24 h incubation, KLVFF was almost totally
decomposed, while 48% of Fc-KLVFF still remained intact. The
results are consistent with the experimental hypothesis, that is,
the ferrocenoyl tethered KLVFF exhibits remarkable resistance to
proteolytic degradation in comparison with its parent peptide
KLVFF.
Lipophilicity is another important factor of drug permeability
and plays a dominant role in toxicity predictions.24 Here, the
octanol/water partition coefficient (log P value) was used to de-
scribe the lipophilicity of the inhibitors. To determine the lipophil-
icity of KLVFF and Fc-KLVFF, log kw values of all compounds were
determined by RP-HPLC and then converted to log P values by
comparison to reference compounds25 (cf. Table 1). The experi-
mental results demonstrated that the substitution of a Boc group
by a more lipophilic ferrocenoyl resulted in an increase in the
lipophilic value of Fc-KLVFF compared to KLVFF itself.
In this Letter, aiming at improving the inhibitory effect, lipo-
philicity and stability towards proteolytic degradation of KLVFF, a
potential inhibitor for Ab aggregation, we tethered a redox and
lipophilic ferrocenoyl moiety to pentapeptide KLVFF. The synthe-
sized Fc-KLVFF showed high inhibitory effect upon Ab fibril forma-
tion, good lipophilicity and high resistance to proteolytic