Inhibition of Amyloid Beta 1-42 Peptide Aggregation
Emma Jenkins, Qiuming Wang, and Jie Zheng*
Department of Chemical and Biomolecular Engineering
The University of Akron
Akron, Ohio 44325
Abstract
Alzheimer’s disease (AD) is a chronic neurodegenerative disease that affects an estimated 5.2
million Americans and is the seventh-leading cause of death. A neuropathological hallmark of
AD is the formation of insoluble amyloid plaques in the human brain caused by the misfolding
and self-assembly of the 39- to 43- residue-long amyloid beta (Aβ) peptides. It is believed that
soluble Aβ oligomers, rather than monomers (initial species) or insoluble mature fibrils (final
species), are major toxic agents responsible for synaptic dysfunction in the brains of patients
with AD. Inhibition of the formation of these toxic oligomers of Aβ has emerged as an approach
to developing medications for AD. Due to highly hydrophobic nature of Aβ and characteristic
cross-β structure in Aβ oligomers/fibrils, the promising Aβ inhibitors should have the following
chemical structures/groups to interfere with Aβ aggregation: short sequences (less than 14
residues or groups) for uncomplicated synthesis and characterization and aromatic groups end
groups. In this work, we examined the efficacy of small molecular inhibitors against prevention
of formation of highly ordered aggregates using fluorescence spectrometry and atomic force
microscopy (AFM). The results indicate that a rigid end group is beneficial for inhibition, rather
than a flexible carbon chain tail. Two of the inhibitors tested showed high inhibition potential
when used at a concentration ratio of 5:1 (inhibitor: Aβ). However, the effectiveness greatly
decreased when the concentration ratio was decreased to 3:1; therefore, it is recommended that
future cell toxicity testing be done at the higher concentration.
Background
Amyloid fibril formation plays a role in at least 25 different diseases, including Alzheimer’s
disease, type 2 diabetes and Parkinson’s disease (1-6). The amyloid aggregates associated with
AD are comprised of 39 to 43 amino acids (7-17). The toxic species believed to play a key role
in AD is the intermediate species of Aβ 1-42. Aβ1-42 is formed from the cleavage of the
transmembrane amyloid precursor protein (APP) by β- and γ-secretases. APP is also cleaved in a
healthy cellular process; however, this is done by α- and γ-secretases, as shown in Fig. 1 (18).
The mechanism of Aβ morphology changes is debated. The different species include Aβ
oligomers, fibers and plaques. Aβ plaques are extracellular fibrillar deposits. Soluble oligomers
have been observed prior to fibril formation, leading researchers to believe they are an
intermediate species; however, recent research suggests that oligomers are not obligate
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intermediates in the fibrillization pathway, and are, rather, an alternate pathway that may or may
not lead to fiber formation (19).
Figure 1. The process by which cleavage of APP results in Aβ 1-42 (Beta-amyloid) if done by βand γ-secretases or is a normal cellular process if done by α- and γ-secretases (18).
There are a few current therapeutic strategies for AD, but the benefit from them are often small
or unapparent (20). Patients with neurodegenerative diseases have overstimulation of the Nmethyl-D-aspartate (NMDA) receptor by glutamate. One current medication to combat this is
Memantime, which is an uncompetitive NMDA-receptor antagonist (21). Another therapeutic
method is to administer the medication called Donepezil. This is a cholinesterase inhibitor that
works by blocking acetylcholinesterase and butylcholinesterase, which are enzymes responsible
for hydrolyzing acetylcholine (22). There are some side effects from these medications;
however, the primary motivation for finding a new treatment method is simply from the lack of
effectiveness of current medications.
Due to an increased need to fight against AD, it is critical to find effective pharmaceutical
treatments for this disease. There are two possible approaches that various researchers are
2
considering: Aβ aggregation inhibition or enzyme control. For enzyme control, the goal is to
control the concentrations of by β- and α-secretases to inhibit the process from occurring. The
effects of limiting β-secretase are not fully understood because its other functions in the body are
not yet known; thus, this option was considered inferior. As for aggregation inhibition, possible
inhibitors include short residue peptides, nanoparticles, Aβ antibodies or small molecular
inhibitors. The focus of this work did not deal with short residue peptides; however, they have
shown in previous research to be viable candidates for inhibition (23). Previous research has yet
to find effective nanoparticles with significant inhibition. Another option is the use of Aβ
antibodies, which have shown to be quite effective at lab scale testing (24-26). Unfortunately,
the testing on humans was cancelled in phase II of the clinical trials (27). It was found that
certain antibodies had significant inhibition in some patients; however, other patients had
adverse reactions to the same antibodies, resulting in death. Previous work shows the potential
for small molecular inhibitors and is the focus of this research (12, 28-37). There are some
conflicting studies regarding optimal small molecular inhibitor chemical structure. Reinke, et. al
found that effective inhibitors were comprised of phenyl end groups, with a hydroxyl substitution
on one end group and had a linker region that was slightly flexible (8-16Å, 2-3 freely rotating
carbons) (12). However, Zhou, et. al found that flexible hydrophobic tails provided better
inhibition than rigid aromatic end groups (38). The aim of this research was to find effective Aβ
inhibitors to prevent the aggregation of toxic Aβ oligomers associated with AD.
In recent studies, using Thioflavin T (ThT) as a fluorescent marker of Aβ is a common method to
determine inhibitor efficacy. One proposed mechanism involves the micelles formed by the
hydrophilic head and hydrophobic tail of ThT, shown in Fig. 2. The binding of the micelles to
amyloid fibrils amplifies the emission fluorescence, meaning the higher the Aβ fibril
concentration, the higher the fluorescence intensity (39). Although ThT is widely used in Aβ
inhibition research, the difficulties with it are also widely known, but not fully understood. It has
been found in previous research that for some inhibitors the ThT signal will remain low even
when AFM shows the presence of aggregation (29, 40). One possible theory for this
phenomenon is that the inhibitor will quench the sample. This means that an electron from the
inhibitor will become excited and will not return to the ground state, being absorbed by another
species in the solution. This results in a lower result from the ThT assay. Therefore, the
application of AFM in combination with fluorescence is favored in order to validate the results.
Figure 2. ThT chemical structure.
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Project Objectives
The objective of this project was to find a small molecular inhibitor that successfully inhibits Aβ
aggregation. The seven inhibitors that were tested, shown in Fig. 3, have many of the necessary
characteristics for inhibition described in literature, including some with rigid, aromatic end
groups and some with more flexible hydrophobic tails. An additional benefit is that these
potential inhibitors present a holistic therapeutic method, being made mainly of Chinese herbs.
Figure 3a. Chemical Structures for Inhibitors SM-1, SM-2, SM-3 and SM-4 (left to right)
Figure 3b. Chemical Structures for Inhibitors
Gu-1, Gu-2 and Gu-3 (top to bottom and left to right)
Methodology
Aβ1-42 Preparation
Aβ1-42 (American Peptide Inc., Sunnyvale, CA) was purchased in lyophilized form and stored at
-20°C. A homogeneous solution of Aβ in the unstructured monomer conformation that is free of
seeds is desired for testing purposes. To obtain this, Aβ was dissolved in 100% 1,1,1,3,3,34
hexafluoro-2-propanol (HFIP) and allowed to sit for 2 hours, sonicated for 30 minutes, and
centrifuged for 30 min at 4 oC and 14000 rpm. The HFIP is used to break hydrogen bonds in any
of the peptides that are not in the monomer conformation. Sonication is used to remove any
preexisting aggregates or seeds (41). After this mixing and separating process, only about 75% of
the top Aβ solution was removed, to avoid getting any of the aggregates or seeds in the solution.
This portion was then frozen with liquid nitrogen and dried with a freeze drier. The Aβ samples
were stored at -80°C until use.
PBS Preparation
The phosphate buffered saline (PBS) solution was prepared by dissolving dry powder in 1 liter
de-ionized water. This yields a 0.01 M PBS solution, containing 0.138 M NaCl and 0.0027 M
KCl, with a pH of 7.4 at 25°C. Before being combined with the ThT, the PBS was filtered using
a 0.45 μm pore size filter.
ThT Preparation
A fresh concentrated solution (1000 mM) of ThT-PBS was made weekly. Small aliquots of this
solution were stored at 20°C and one was thawed daily to prepare the fresh ThT-PBS solution at
a concentration of 20 μM that was used in the fluorescence samples. This method was suggested
in literature to help lessen the sporadic behavior of ThT (42, 43).
Sample Preparation
First, 10 μL of dimethyl sulfoxide (DMSO) were added to 0.1 mg of freeze-dried Aβ1-42. This
was stirred on a vortex stirrer for approximately 5 seconds, sonicated for 1 minute, and allowed
to sit for 5 minutes. After sitting, the Aβ-DMSO mixture was added to 1 mL of the filtered PBS
(10 mM aqueous, 37°C) for the control solutions. This was a resulting concentration of 20 μM
Aβ. For the first round of inhibitor solutions, the Aβ-DMSO mixture was added to a mixture of
1 mL filtered PBS and 2 μL of inhibitor-DMSO. The inhibitor-DMSO mixture was prepared by
adding DMSO to the selected inhibitor until a concentration of 50 mM was reached. The
resulting concentrations of the Aβ-inhibitor solutions were 20 μM Aβ and 100 μM inhibitor.
After the most successful candidates were found, testing was done at decreased inhibitor
concentrations (20 μM Aβ and 60 μM inhibitor). After mixing, all solutions were incubated at
37°C, without agitation. Fluorescence samples of each solution were taken approximately every
two hours the first day after being mixed and once or twice a day the following two days. The
samples contained 2 mL of the 20 μM ThT-PBS solution and 10 μL of the Aβ-inhibitor-PBS
solution. The samples sat for 10 minutes before testing, under foil, to give the ThT and Aβ time
to interact. AFM samples were prepared using the same solutions: 10 μL for 0 hour-1 day
samples and 20 μL for samples after 1 day. The solution was put on a freshly pealed mica
surface, sat for 1-2 minutes, rinsed with de-ionized water, air dried and sat for 1 day before AFM
testing.
5
It is important to note that the concentration of Aβ used in these solutions is much higher than
the concentration that is present in Alzheimer’s patients. The actual concentration is less than 1
μM, as compared to the 20 μM used in this testing. Likewise, the inhibitor concentration may
need to be lower to be safe in the human body.
Results and Discussion
Inhibition behavior of SM group small molecular inhibitors on Aβ aggregation
The fiber generation processes of SM-1, SM-2, SM-3 and SM-4, with a 5:1 concentration ratio
(inhibitor: Aβ), were monitored by fluorescence spectroscopy and are graphed in Fig. 4. The
more Aβ fibers in the sample, the more binding with ThT will occur, resulting in increased
fluorescence intensity. By comparing the intensity of the control experiment (20 μM Aβ
incubated at 37°C) to that of the inhibitors, it is clear that within 50 hours incubation time, all
four of these inhibitors greatly decrease the Aβ aggregation, at this concentration. The inhibitor
efficacy from greatest to least is SM-1, SM-2, SM-3, SM-4, according to the fluorescence data.
The repeat experiments produced data nearly the same as in the first experiments, providing
validation for the use of ThT fluorescence.
The AFM images for the control are shown in Fig. 5. Aβ proto-fibers, approximately 2 nm high
and 800 nm long, form quickly, as shown in the image after 4 hours of incubation. The
aggregation increases dramatically, and after 8 days of incubation, very large fibril aggregates
are apparent. These images support the inhibition performance described above when comparing
to the AFM images for the SM samples in Fig. 6-8. Although SM-3 and SM-4 do not inhibit Aβ
aggregation as well as SM-1 and SM-2, they still greatly decrease the aggregation found in the
control samples. After 10 hours incubation time, the SM-1 and SM-2 samples look quite similar
and showing mostly Aβ oligomers. The 1 day samples for these two also look similar, showing
few, small proto-fibrils. In addition, it took numerous scans on the AFM to find any protofibrils. A difference can be noticed after 2 days incubation time; here, SM-1 shows worse
inhibition than SM-2, with a few globular deposits as opposed to the single proto-fibril in the
SM-2 sample. After 8 days of incubation, both SM-1 and SM-2 have globular deposits. It was
clear early on that SM-3 and SM-4 were outperformed by the first two inhibitors. SM-3 forms
many large fiber-like aggregates at 5.6 hours, worsening at 8.6 hours. SM-4 forms much smaller
fiber aggregates at 8 hours incubation time.
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Figure 4. Fluorescence intensity vs. incubation time at 37°C for inhibitors SM-1, SM-2,
SM-3, SM-4, the control with no inhibitor and the repeat experiments for them
Figure 5. Control AFM images at 0h, 4h, 7.5h, 1 day, and 8 days incubation time (5X5 μm)
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Figure 6. SM-1 AFM images at 10 hours, 1 day, 2 days
and 8 days incubation time (scans at 5X5 μm)
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Figure 7. SM-2 AFM images at 10 hours, 1 day, 2 days and
8 days incubation time (scans at 5X5 μm)
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SM-3:5.6 h
SM-3:8.6 h
SM-4:8 h
Figure 8. SM-3 AFM images at 5.6 and 8.6 hours incubation time
and SM-4 at 8 hours (scans at 2X2 μm)
Inhibition behavior of dilute SM-1 and SM-2 on Aβ aggregation
Fig. 9 illustrates that SM-1 and SM-2 still have inhibition potential at the diluted concentration of
3:1 (inhibitor: Aβ); however, the effectiveness is greatly decreased when compared to the 5:1
concentration ratio samples. According to the fluorescence data, the performance of the diluted
SM-1 and SM-2 are very similar, with SM-2 performing better from 0-20 hours and SM-1
performing better following that. The AFM images (Fig. 10 & 11) between the two are
comparable as well. The first samples (at 3.3 and 6.3 hours) are somewhat indistinguishable.
After 26 hours of incubation, SM-1 forms a small amount of long, thin fibers and SM-2 forms a
small amount of shorter, more globular aggregates. By comparing the AFM images to those for
the control experiment (Fig. 14), it is not clear if there is any inhibition after 1 day of incubation.
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Figure 9. Fluorescence intensity vs. incubation time at 37°C for inhibitors SM-1 and SM2 at a concentration ratio of 3:1 (inhibitor:Aβ), and the control experiment
Figure 10. SM-1 (3:1 concentration ratio of inhibitor: Aβ) AFM
images at 3.3 h, 6.3 h and 26 h incubation time (scans at 5X5 μm)
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Figure 11. SM-2 (3:1 concentration ratio of inhibitor: Aβ) AFM
images at 3.3 h, 6.3 h and 26 h incubation time (scans at 5X5 μm)
Inhibition behavior of Gu group on Aβ aggregation
The fiber generation processes of Gu-1, Gu-2 and Gu-3, with a 5:1 concentration ratio (inhibitor:
Aβ), were monitored by fluorescence spectroscopy and are graphed in Fig.12. The fluorescence
data indicate that the Gu samples do slightly inhibit Aβ aggregation, as compared to the control
experiments; however, they do not do so as effectively as the SM inhibitors. These graphs
suggest that Gu-3 is the most successful out of this grouping. The intensity of Gu-2 goes below
that of Gu-3 around 30 hours incubation time; however, Gu-2 has great variance in
measurements, and thus, the data may not be reliable.
The AFM images, Fig. 13, present similar results. Inhibitors Gu-1 and Gu-2 can be eliminated
very quickly, by looking at the 9.3 hour samples. Although they are different types, both
inhibitors allow a great deal of aggregation; Gu-1 permits globular aggregation and Gu-2 permits
long proto-fibril aggregates. Gu-3 is superior to the first 2 inhibitors, with no aggregation at 9.2
hours. Small aggregation starts after 1 day and increases to large fibril aggregates at 30.2 hours,
producing much less desirable results than SM-1 and SM-2.
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Figure 12. Fluorescence intensity vs. incubation time at 37°C for inhibitors Gu-1, Gu-2,
Gu-3, at a concentration ratio of 5:1 (inhibitor: Aβ) and the control with no inhibitor
13
Figure 13. AFM images: Gu-1, Gu-2 and Gu-3 at 9.3 h
and Gu-3 at 30 h (scans at 5X5 μm)
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Conclusion
In comparison to the SM samples, all of the Gu samples have low inhibition potential. This
validates the results of Reinke, et al., that specify that a rigid end group is beneficial for
inhibition, rather than a flexible tail as described by Zhou, et al. (12, 38). The inhibitors with the
most inhibition potential are SM-1 and SM-2, with the best performance belonging to SM-1.
The only difference between SM-1 and SM-2 is presence of a double bond in the cyclopentane
epoxide in SM-2. According to Reinke, et. al, end phenyl groups are preferred, which may lead
one to think the presence of the double bond in the ring would increase inhibitor effectiveness;
however, they did not test simple aromatic end groups or epoxides, only phenyl groups and
carbon chains (flexible tails). This could be an explanation as to why the anticipated result was
not found. Additionally, the performance of SM-1 and SM-2 are extremely close and the
differences may be due to experimental error. SM-1 and SM-2 inhibit better than SM-3 and SM4 due to the left side phenyl end groups on both of them as compared to the cyclohexane group
with no double bonds, which is the end group for SM-3 and SM-4. A similar justification
explains why Gu-3 outperforms Gu-1 and Gu-2. Gu-1 has one flexible carbon chain end group
and Gu-2 has a cyclohexane epoxide with only one double bond, as compared to Gu-3 with two
phenyl end groups.
Future work should consist of cell toxicity testing for SM-1 and SM-2 at the most effective
concentration, which was a ratio of 5:1 (inhibitor: Aβ).
Acknowledgements
This research would not have been possibly without the help of Qiuming Wang and Dr. Jie
Zheng at the University of Akron. I would like to thank Qiuming for testing the AFM samples
and helpful guidance in the project. I would also like to thank the Ohio Space Grant Consortium
for the funding.
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