Neurochemistry International 108 (2017) 481e493

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Neurochemistry International
journal homepage: www.elsevier.com/locate/nci

Inhibition of Alzheimer's amyloid-beta aggregation in-vitro by

carbenoxolone: Insight into mechanism of action
Sheetal Sharma, Bimla Nehru*, Avneet Saini
Department of Biophysics, Basic Medical Sciences Block II, Panjab University, Chandigarh 160014, India

a r t i c l e i n f o a b s t r a c t

Article history: Background: The major hallmark of Alzheimer's disease (AD) is the formation of amyloid aggregates,
Received 8 March 2017 which are formed due to improper folding of proteins leading to the aggregation of amyloid beta (Ab) 42
Received in revised form peptide. Inhibition of Ab 42 aggregation using a drug such as carbenoxolone (Cbx), which has already
9 June 2017
been stated as neuroprotective, appears to be an effective approach against AD.
Accepted 22 June 2017
Objective: The present study was designed to investigate the anti-fibrillation activity of Cbx against the
Available online 24 June 2017
Ab 42 aggregation.
Methods: The aggregation of Ab 42 peptide was observed by performing in-vitro studies and the pro-
Keywords:
Alzheimer's disease
pensity of aggregation of Ab 42 peptide was evaluated by the prediction of binding sites and amyloi-
Amyloid-beta 42 dogenic regions. The binding of Cbx in these binding sites was predicted by computational studies.
Carbenoxolone Results: Thioflavin-T (Th-T assay), congo red assay and circular dichroism (CD) analysis suggested sig-
Oligomers nificant inhibition of Ab 42 aggregation by Cbx. The propensity of aggregation of Ab 42 peptide was
Fibrils evaluated by the prediction of binding sites and amyloidogenic regions. The mechanism of anti-
Anti-fibrillation fibrillation activity of Cbx was elucidated by molecular docking and simulation studies and has been
predicted to interact with amyloidogenic residues of Ab 42 peptides as well as fibrils. Cbx also interacts
with residues involved in the stabilization of the oligomeric structure.
Conclusion: These results project Cbx as a suitable candidate for the inhibition of Ab 42 aggregation and
the therapeutic potential of Cbx against AD can further be studied using in-vivo experiments.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction as the main cause of cognitive decline and neurodegeneration in

With the increase in the life expectancy, the prevalence of
ageing related neurodegenerative diseases such as Alzheimer's
disease (AD) has increased. The ageing global population and lack
of effective treatment of AD has led to enormous social and eco-
nomic burden on the society (Nie et al., 2011; Rahimi and Kovacs,
2014). The major hallmark of AD is the formation of amyloid pla-
ques, which are formed due to improper folding of proteins leading
to the aggregation of amyloid beta (Ab) peptide. Ab peptides are
formed through the cleavage of APP (Amyloid precursor protein) by
beta and gamma-secretases. Plaque formation has been considered
Abbreviations: AD, Alzheimer's disease; Ab, Amyloid beta; Cbx, carbenoxolone;
APP, Amyloid precursor protein; Th-T, Thioflavin-T; CD, Circular dichroism; SVAU,
Sedimentation velocity analytical ultracentrifugation.
* Corresponding author.
E-mail addresses: sheetal.sh87@gmail.com (S. Sharma), bnehru@pu.ac.in
(B. Nehru), avneet@pu.ac.in (A. Saini).
AD, but in the last decade, several studies have correlated soluble
Ab 42 oligomers with the progression of AD and cognitive decline
(Zussy et al., 2013; Wang et al., 2016; Ferreira et al., 2015; Balducci
et al., 2010, 2016) and are also considered to be the key mediators of
synaptic and cognitive dysfunction (Ahmed et al., 2010; Benilova
et al., 2012; Doi et al., 2014; Jin and Selkoe, 2015; Sharma et al.,
2016).
The cleavage of APP by secretases leads to the formation of both
Ab 40 and Ab 42 peptides. Ab 42 peptides have more propensity to
form soluble oligomeric aggregates, which further lead to the for-
mation of insoluble plaques. These Ab 42 monomers can form
stable and soluble aggregates ranging from trimers to dodecamers
which can further oligomerize to form aggregates of higher order
(Bitan et al., 2003; Barghorn et al., 2005; Chen and Glabe, 2006;
Bernstein et al., 2009; Yu et al., 2009). The soluble oligomers are
stabilized by different molecular interactions between the mono-
mers. N-terminus region, hydrophobic core, hinge or turn regions
and C-terminus are the crucial regions for the aggregation of the

http://dx.doi.org/10.1016/j.neuint.2017.06.011
0197-0186/© 2017 Elsevier Ltd. All rights reserved.
482 S. Sharma et al. / Neurochemistry International 108 (2017) 481e493
monomers (Nie et al., 2011; Maji et al., 2009). These monomers
contain hydrophobic C-terminal residues which are oriented to-
wards the center of the oligomer. The interaction between Phe19
and Leu34 is common between Ab 42 oligomers and fibrils and is
stabilized by solvent-accessible turns at His13-Gln15, Gly25-Gly29
and Gly37-Gly38. In Ab 42 fibrils, residues 1e17 may be unstruc-
tured, with residues 18e42 forming a b-turn-b fold. Molecular
contacts have been reported within the monomer unit of Ab 42
fibrils between Phe19 and Gly38 and between Met35 and Ala42. In
both Ab 40 and Ab 42, the turn conformation is stabilized by hy-
drophobic interactions and by a salt bridge between Asp23 and
Lys28 (Ahmed et al., 2010).
The formation of these monomers is a physiologically relevant
event but their aggregation is pathogenic. So, the inhibition of this
aggregation might emerge as a therapeutic strategy to halt the
progression of AD. In the present study, we have explored the anti-
fibrillation activity of Carbenoxolone (Cbx) against amyloid beta
aggregation, which has already been established as a neuro-
protective agent. Carbenoxolone (Fig. 1), an 18-glycyrrhetinic acid
derivative extracted from liquorice root, has been used for the
treatment of peptic ulcers for a long time (Campisi et al., 1985) and
it also has neuroprotective and nootropic effects (Hellmich et al.,
2013). It crosses blood-brain barrier and improves memory
consolidation in rats and verbal fluency in aging humans by
inhibiting the activity of 11b-hydroxysteroid dehydrogenase type 1
(11b-HSD1), which converts inactive cortisone to active cortisol in
the brain (Jahn et al., 2003; Khorasani et al., 2009; Sandeep et al.,
2004; Thakur and Nehru, 2015). Cbx is often associated with in-
duction of heat shock proteins (Nagayama et al., 2001; Thakur and
Nehru, 2014) and blocking of gap junctions (Tovar et al., 2009;
Gareri et al., 2004).
Cbx is widely reviewed for its various pharmaceutical properties
but its role in the inhibition of amyloid beta aggregation is not
studied previously. In light of various neuroprotective properties of
Cbx in the brain, the current study aims to explore another aspect of
neuroprotection by investigating the mechanism of anti-fibrillation
activity of Cbx on the in-vitro amyloid beta 42 aggregation. Our
results will help in the better understanding of the binding in-
teractions of Cbx with Ab 42 aggregates and also, will aid in the
designing of novel Ab 42 aggregation inhibitors.
2. Material and methods
2.1. Chemicals
All the chemicals used in the present study were of analytical
grade and were obtained from various chemical companies. Ab
1e42 peptide was obtained from Anaspec; USA. Carbenoxolone
disodium, Thioflavin-T and congo red were procured from Sigma-
Fig. 1. Structure of carbenoxolone.
Aldrich; USA.
2.2. Preparation of amyloid beta 1e42 oligomers
Ab1e42 oligomers were prepared by the method described by
Lioudyno et al. (2012). Briefly, 1 mg Ab1e42 peptide was dissolved
in 100 ml of 100 mM NaOH and incubated for 25 min, followed by
the addition of 900 ml 10 mM sodium phosphate buffer. These
samples were subsequently kept in closed eppendorf tubes at room
temperature for further aggregation.
2.3. Amyloid beta soluble content determination
SVAU (Sedimentation Velocity Analytical Ultracentrifugation)
was also used to determine the amount of soluble aggregates in
solution after sedimentation of Ab 42 fibrils (Lashuel et al., 2002).
To determine the amount of unsedimented Ab 42 in solution, two
radial scans were collected at 3000 rpm (only fibrils are sedi-
mentable at this speed) and 20,000e30,000 rpm (soluble high
molecular weight oligomers of Ab 42 sediment but not monomer).
2.4. Tricine-SDS-PAGE
The aggregation of Ab 1e42 peptide was determined by Tricine-
SDS-polyacrylamide gel electrophoresis on 16% gels as described
(Scha€gger, 2006). Briefly, 40 mg of the aggregated peptide at two
time intervals was loaded into two lanes and the gels were run at
4
C using tris-tricine running buffer initially at 15 mA and then at
25 mA. The gels were run for 3e4 h, fixed with fixing solution and
then stained with 0.025% coomassie dye for protein visualization.
2.5. Thioflavin T (Th T) fluorescence assay
Thioflavin T binding assay was performed by combining 50 ml of
100 mM Ab 42 solutions (previously incubated at 37
C in the
absence and presence of drugs) to 450 ml solution of 10 mM ThT in
10 mM phosphate, pH 7.4, and 100 mM KCl. Fluorescence mea-
surements were recorded in a spectrofluorometer at 25
C using a
1-cm pathlength quartz cell. The excitation and emission wave-
lengths were set at 450 (slit width 4 nm) and 490 nm (slit width
8 nm) respectively (Lashuel et al., 2002).
2.6. Congo red binding assay for fibril formation
Congo red was prepared as described previously Klunk et al.
(1999) and diluted to 10 mM in 50 mM Tris-HCl, 150 mM NaCl, pH
7.4. Congo red binding was carried out by combining 50 ml of
100 mM Ab 1e42 solutions with 1 150 ml of 10 mM Congo red so-
lution mixed and incubated for 5e10 min. The binding was deter-
mined by using the UVevisible spectra (200e700 nm) collected on
a Beckman UVevisible spectrophotometer in a mini-cuvette with a
1-cm pathlength.
2.7. Circular dichroism spectroscopy
CD spectroscopy (JASCO Polarimeter, Japan) was used to observe
the structural changes during the aggregation of Ab 42 peptide as
described by Nichols et al. (2015). After incubating the Ab 42
peptide solutions (55 mM) in the presence or absence of the drug
(100 mM), each solution was subjected to spectroscopic studies and
each spectra was an average of four scans. The spectra were
collected in mdeg form with wavelength range of 190e260 nm,
using 2-mm path length cell. Secondary structure estimates were
done by the analysis of CD data using online servers such as CAPITO
and BeStSel. CAPITO (CD Analysis and Plotting Tool) is a web server-
 S. Sharma et al. / Neurochemistry International 108 (2017) 481e493
based tool and predicts the secondary structure elements via
extraction of information from a calculated set of basis spectra and
a matching-based approach (Wiedemann et al., 2013). BeStSel (Beta
Structure Selection) is used for the secondary structure estimation
that takes into account the twist of b-structures and can reliably
distinguish parallel and antiparallel b-sheets and accurately esti-
mates the secondary structure for a broad range of proteins
(Micsonai et al., 2015).
2.8. Binding site prediction
3D structure files of Alzheimer's Ab 42 peptide (PDBID: 1IYT)
and Ab 42 fibrils (PDBID: 2BEG) were retrieved from Protein
Database PDB. Both 1IYT and 2BEG contain 10 models of NMR-
determined structures (Crescenzi et al., 2002; Luhrs et al., 2005).
The prediction of pockets and voids present in Ab 42 peptide
was done with RaptorX Binding and CASTp (Computed Atlas of
Surface Topography of proteins). RaptorX Binding is a web-based
server and predicts the binding sites of a protein sequence based
upon the predicted 3D model by RaptorX. For binding site pre-
dictions, a protein structure is built from the top-ranked template
chosen from alignment of the target sequence and the structures in
the template library. In cases where multiple domains are found, a
structure is built and the binding site predictions are made for each
domain (Ka €llberg et al., 2012). CASTp is an online resource that aims
to provide a detailed quantitative characterization of concave re-
gions in the three dimensional structure of the protein. These
include interior cavities and surface pockets of proteins that are
frequently associated with binding events. CASTp uses four letter
PDB file as input. Probe radius of 1.4 Å (default value) is used for
binding site prediction of Ab 42 peptide. It uses the weighted
Delaunay triangulation and the alpha complex for shape mea-
surements (Dundas et al., 2006).
2.9. Prediction of the amyloidogenic region
The regions susceptible for the amyloid formation were pre-
dicted by using Waltz, Fold amyloid and Aggrescan softwares.
Waltz was developed with the main aim of prediction of amyloi-
dogenic regions which were the main starting points for research
related to amyloid formation. Here the protein FASTA sequence was
submitted online and the result was generated. It uses a position-
specific scoring matrix to determine amyloid-forming sequences
and allows users to identify and better distinguish between amy-
loid sequences and amorphous beta-sheet aggregates (Maurer-
Stroh et al., 2010). Fold amyloid requires PDBid or FASTA format
of the Ab 42 peptide. It uses the expected probability of hydrogen
bonds formation and expected packing density of residues as the
basis for the prediction of the amyloidogenic regions in a protein
sequence (Garbuzynskiy et al., 2010). Aggrescan server uses the
protein FASTA sequence and is based on an aggregation-propensity
scale for natural amino acids derived from in vivo experiments and
on the assumption that short and specific sequence stretches
modulate protein aggregation (Conchillo-Sole  et al., 2007).
2.10. Docking
Cbx structure was obtained from Pubchem (CID636402) and
converted to PDB file using OpenBabel 2.4.0 (O'Boyle et al., 2011).
Autodock Vina was used for docking experiments (Trott and Olson,
2010) and the coordinates for NMR solution structures of Ab1e42
peptide and fibril were obtained from pdb (id: 1IYT and 2BEG). The
peptides and ligand input files were generated in pdbqt format
with the help of Autodock tools (Morris et al., 2009). The gasteiger
charges were assigned and polar hydrogens were added to the
483
receptor and ligand. The receptor was kept rigid during the docking
procedure. The grid boxes of 60  48  66 point size for Ab 42
peptide and 48  40  40 point size for Ab 42 fibril were used with
a spacing 0.375 Å, enclosing the whole molecule. The Vina
parameter “exhaustiveness” was set to the value of 10 and
computation was done with a computer consisting of four pro-
cessors. 90 different poses for the ligand were generated with Ab 42
peptide as well as with Ab 42 fibril. Docked poses with lowest free
energy change and catalytically favorable interactions were
considered for further analysis. After docking, each structure was
visually inspected to ensure that the molecules were actually
docked into the expected binding sites and interacted with pre-
dicted amyloidogenic residues. All structures were saved as PDB
files and visualized in Discovery Studio 4.0. Hydrogen bonds, hy-
drophobic interactions and distances for various interactions were
calculated.
2.11. Molecular dynamics (MD) simulation
The most stable Ab 42 peptide, Ab 42 peptide-Cbx complex, Ab
fibril and Ab fibril-Cbx complex obtained from the docking studies
were subjected to 10 ns MD simulations. All simulations were
performed using GROMACS 4.5.5 software package together with
the GROMOS96 force field (Berendsen et al., 1981, 1984). The
protein-ligand assembly was solvated using spc216 explicit water
model (Miyamoto and Kollman, 1992) and the whole system was
neutralized with the addition of positive ions (Naþ) by replacing
the water molecules. The system was then subjected to energy
minimization by steepest descent method with 500 maximum
numbers of steps to remove steric conflicts between water and
peptide and was equilibrated for 100 ps under an isothermal-
isobaric ensemble and isochoric-isothermal ensemble respec-
tively. Electrostatic interactions were calculated using the particle
mesh Ewald (PME) method (Darden et al., 1993). Temperature
(300 K) and pressure (1 atm) were controlled by the V-rescale
thermostat and Berendsen barostat, respectively (Berendsen et al.,
1984; Bussi et al., 2007). Newton's motion equations were inte-
grated by leapfrog algorithm with a 2 fs time step. LINCS algorithm
was employed to constrain all bond lengths (Hess et al., 1997).
Initial velocities were assigned according to a Maxwell distribution
(Essmann et al., 1995). MD simulations were run for 10 ns and the
trajectory was updated after every 2 ps. The snapshots of simula-
tion trajectories were visualized using visual molecular dynamics
(VMD) software (Humphrey et al., 1996). Secondary structure
analysis was done by using 2Struc server, which uses defined sec-
ondary structure of proteins (DSSP) for analysis (Klose et al., 2010).
2.12. ADMET prediction
ADMET analysis consists of the absorption, distribution, meta-
bolism, excretion and toxicity analysis of a compound in the human
body. The pharmacokinetic profile of a compound predicted by the
ADMET analysis helps in the assessment of the pharmacological
activity of the compound as a drug. The admetSAR (ADMET struc-
tureeactivity relationship) database has been used in the present
study as ADMET prediction tool. It is an open source, text and
structure searchable, and continually updated database that col-
lects, curates and manages available ADMET-associated properties
data from the published literature (Cheng et al., 2012).
2.13. Statistical analysis
The results were presented as mean ± standard deviation. The
statistical analysis was done by performing one-way ANOVA. Sig-
nificance was determined as p  0.05.
484 S. Sharma et al. / Neurochemistry International 108 (2017) 481e493

3. Results and discussion

The formation of soluble Amyloid beta 42 aggregates is

considered as the major cause in the progression of AD (Ferreira
et al., 2015; Selkoe et al., 2016) and several studies have been
performed in the past to inhibit the aggregation using different
biological and chemical agents (Doig and Derreumaux, 2015). In the
present study, we tested the ability of Cbx as an amyloid beta 42
aggregation inhibitor. To do so, first the formation of soluble Ab 42
aggregates was characterized by SVAU analysis and separation of
these aggregates on Tris-tricine SDS-PAGE was done. The alter-
ations in the process of Ab 42 aggregation in the presence of Cbx
were explored by performing in-vitro studies such as Th-T assay,
congo red assay and CD analysis. To elucidate the process of Ab 42
aggregation, the binding sites present in the Ab 42 peptide were
predicted using online servers such as CASTp and RaptorX binding.
Further, the amyloidogenic residues responsible for the aggregation
of Ab 42 peptide were predicted using Waltz, Fold amyloid and
Aggrescan online servers. Also, docking studies of Cbx with both Ab
42 peptide and fibril were performed to predict the binding of Cbx
with these residues. To consider Cbx as a potential drug candidate,
its efficacy was also predicted by ADMET analysis using admetSAR
server.

3.1. Characterization of amyloid beta 1e42 aggregation

Sedimentation velocity analytical ultracentrifugation (SVAU)
was performed on the Ab 42 solutions and it was found that most of
the Ab 42 was present in the form of soluble aggregates even after 4
days of aggregation (Fig. 2). To further examine the state of ag-
gregation, the aggregated Ab 42 solution was then run on 16%
native PAGE (Fig. 3). The aggregation of Ab 42 peptide depends
upon several factors such as pH, concentration, temperature and
time of incubation after solubilization in the aqueous medium. Ab
42 peptide has very high tendency to form aggregates and it starts
aggregating as soon as it gets an aqueous media (Haass and Selkoe,
2007). After four days of incubation, different bands between
43 kDa and 14.3 kDa were observed in addition to the band below
14.3 kDa. As reported in some previous studies (Maji et al., 2009;
Lee et al., 2015), the band below 14.3 kDa may represent the di-
mers and the bands between 14.3 and 43 kDa may represent the
trimers, tetramers, pentamers, hexamers, heptamers, octamers,
Fig. 3. Separation of Ab 1e42 aggregates on 16% Tris tricine-PAGE. Amyloid beta 1e42
peptides (1 mg/ml) were incubated for four days in 4 days in sodium phosphate buffer
(pH 7.4, 10 mM) and samples were run on 16% polyacrylamide gel. Molecular mass
standards are indicated in the lane on the right.
nonamers and decamers. The presence of low molecular weight
oligomers confirmed the presence of soluble content in the Ab 42
aggregated solutions as shown by SVAU analysis. These soluble low
molecular weight aggregates of Ab 42 are considered as the major
cause of AD. Ab 42 oligomers are reported to inhibit long-term
potentiation, cause neurodegeneration and lead to cognitive
decline in AD patients (Selkoe et al., 2016; Haass and Selkoe, 2007;
Hardy and Selkoe, 2002; Selkoe et al., 2012). Therefore, the inhi-
bition of the process of aggregation of these Ab 42 aggregates may
be considered as a therapeutic measure to prevent AD.
3.2. Effect of carbenoxolone on Ab 42 peptide aggregation

Ab 42 peptide solutions (100 mM) were incubated at 37
C for 4

Fig. 2. Sedimentation velocity profiles of a sample of Ab 1e42 (100 mM) after incu-
bation for 4 days in sodium phosphate buffer (pH 7.4, 10 mM). Values are expressed as
mean ± SD; n ¼ 4.
days in the absence and presence of Cbx at equimolar concentra-
tions. In order to confirm the presence of aggregates, we performed
the thioflavin T fluorescence assay and congo red assay with these
Ab 42 peptide solutions. Th-T specifically binds to amyloid aggre-
gates (LeVine, 1999) and its fluorescence is enhanced after binding
(Alam et al., 2016; Siddiqi et al., 2016). All the Ab 42 peptide solu-
tions except the freshly prepared solutions showed enhanced
fluorescence (Fig. 4), but the fluorescence was found to be signifi-
cantly reduced in the Cbx treated solutions (in both 50 and 100 mM
concentrations). Cbx showed 22.54% and 47.16% inhibition of Ab 42
peptide aggregation at 50 and 100 mM concentrations respectively.
Congo red also binds to the amyloid aggregates (Ramshini et al.,
2015) and an increase in the absorbance was observed along with
a red shift of the spectral maximum (Nusrat et al., 2016). This in-
crease in the absorbance was present in the aggregated Ab 42 so-
lutions (orange line) as compared to congo red alone solution (blue
line). There was no change in the absorbance in the freshly pre-
pared Ab 42 peptide solution (yellow line) and in the solutions
having Ab 42 aggregated in the presence of Cbx (grey line) (Fig. 5a
 S. Sharma et al. / Neurochemistry International 108 (2017) 481e493 485

Fig. 4. Extent of aggregate formation in the presence and absence of carbenoxolone. The bars represent the extent of Ab 42 aggregate formation based on a quantitative Thioflavin T
binding assay in the absence and presence of Cbx at 50 mM and 100 mM concentrations. Values are expressed as mean ± SD; n ¼ 4. *, p  0.05, compared to Ab 42 non-aggregated
group; #, p  0.05, compared to Ab 42 aggregated group.

aggregated (for 4 days; blue line) solutions (Fig. 6). The spectra
exhibited a minimum at 216 nm and a maximum at 197 nm, which
indicates typical conversion of Ab 42 monomers and small aggre-
gates into b-sheet-rich aggregates. These results were in accor-
dance with the previous reports about the transition from
monomeric to the aggregated state of Ab 42 peptide (Lee et al.,
2015). When these peptide solutions were incubated with Cbx
(grey line), the transitions were observed but the extent was much
less than that of aggregated solutions. This observation suggested
that Cbx altered the secondary structural changes during Ab 42
assembly. The CD spectra was analyzed by using online servers such
as CAPITO and BeStSel to confirm the presence of ordered struc-
tures (a-helix and b-strand) in different Ab 42 solutions. The CD
spectra of Ab 42 peptide aggregation in the presence of Cbx
(100 mM) showed an increase in the helical content from 6 to 18%
and a decrease in the b-sheet content from 49 to 30% as compared
to the Ab 42 aggregated solutions. These results indicate that Cbx
inhibits the Ab 42 peptide aggregation by checking the b-sheet

Fig. 5. Spectral characteristics of congo red and Ab 1e42 aggregates. The absorbance
spectra of congo red and Ab 1e42 aggregates in the presence and absence of carbe-
noxolone (100 mM) in (a) 200e700 nm region and (b) 390e590 nm region. Each
absorbance spectra was an average of four scans.

and b). So, these amyloid specific assays suggested the inhibition of
Ab 42 aggregation in the presence of Cbx.
To further investigate the efficacy of Cbx as an amyloid aggre-
gation inhibitor, CD was used to monitor the formation of Ab 42
aggregates and secondary structure variations in the absence and Fig. 6. CD spectra of Ab 1e42 aggregates in the presence and absence of carbenox-
presence of Cbx. Ab 42 peptide showed substantial secondary olone. The CD analysis was performed using 100 mM Ab 1e42 solutions and these
structural changes between freshly prepared (orange line) and solutions were incubated for four days in the absence or presence of Cbx (100 mM).
Each absorbance spectra was an average of four scans.
486 S. Sharma et al. / Neurochemistry International 108 (2017) 481e493

formation. 3.4. Docking study of carbenoxolone with Ab 42 peptide and Ab 42

As these in-vitro studies suggested inhibition of Ab 42 peptide fibril
aggregation by Cbx, in-silico binding analysis of Cbx with Ab 42
peptide as well as fibril was carried out to understand the mech- The in-silico analysis of Ab 42 peptide revealed the position of
anism of action. The prediction of binding sites and amyloidogenic amyloidogenic residues and hydrophobic cavities. To cross validate
regions of Ab 42 peptide was done and ADMET analysis was also whether Cbx interacts with any of these residues, Cbx (CID636402)
performed to predict the suitability of Cbx as a potential drug was docked with the Ab 42 monomer (PDBID: 1IYT) using autodock
candidate. vina. This in-silico binding analysis revealed that Cbx forms mo-
lecular interactions with the amino acid residues of hydrophobic
core of Ab 42 peptide.
3.3. Binding site prediction and propensity of aggregation of Ab 42
The maximum binding affinity of Cbx and Ab 42 peptide was
peptide
predicted to be 6.8 kcal/mol (Table 1) and Cbx was predicted to
interact with Arg5, Ser8, Val12, Gln15, Lys16 and Phe20 residues of
Structural pockets on the protein surfaces and binding cavities
Ab 42 peptide as shown in the Fig. 8a and b. This conformation was
in the interior of the protein mainly represent the binding and
stabilized by a strong hydrogen bond (dO…H ¼ 1.43 Å, dO…O ¼ 2.40 Å
active sites of proteins. The prediction of these sites helps in the
and/OHO ¼ 131.2
) between the hydroxyl moiety of carboxyl group
identification of surface accessible regions (pockets) and interior
(donors) of Cbx and carbonyl oxygen (acceptor) of the backbone of
inaccessible cavities of the protein (Binkowski et al., 2005). Also,
residue Arg5 of the peptide. Hydrogen bonds with the donor-
the amino acid residues present inside these sites may be associ-
acceptor distances of 2.2e2.5 Å have been categorized as strong
ated with the binding of the drug (Singh et al., 2013). CASTp server
and mostly covalent (Jeffrey, 1997). As the cavity predictions by
predicted the cavities for Ab 42 peptide (PDBID: 1IYT) and revealed
CASTp have shown the presence of Arg5 residue inside pocket 1,
the presence of three pockets 1 (Asp1, Ala2, Arg5 and His6), 2
this hydrogen bond formation may prevent the cavity formation.
(Val24, Lys28, Ile31 and Met35) and 3 (Lys28, Ile31, Ile32 and
Also, the hydrophobic interaction of Cbx with Lys16 and Val12
Met35). RaptorX binding server predicted the presence of four
residues, which were predicted to be present in the amyloidogenic
pockets as pocket 1 (Gly9, Val12, His13, Lys16, Leu17 and Phe20), 2
region of Ab 42 peptide, may interfere with the folding of the
(Asp7, Ser8, Glu11, Val12 and Lys16), 3 (Gly25 and Lys28) and 4
peptide to form aggregates.
(Asp1, Phe4, Arg5, Ser8, Gly9 and Val12).
Cbx was also docked with Ab 42 fibril (PDBID: 2BEG) using
Prediction of the amyloidogenic regions in the polypeptide
autodock vina and the maximum binding affinity was predicted to
chains is also very important because such regions are responsible
be 7.6 kcal/mol (Table 2). In-silico binding analysis revealed that
for amyloid formation and aggregation. Also, in some previous
Cbx formed strong molecular interactions with the C-terminus and
studies, various regions in the Ab 42 peptide (Fig. 7) such as N-
the hydrophobic core of the Ab 42 fibril and these may have
terminus, hydrophobic core, hinge or turn regions and C-terminus,
interfered with the stabilization of the fibrillar structure as evi-
are reported to contribute differently in the process of Ab 42 pep-
denced in Fig. 9a and b. Ab 42 fibril structure consisted of five Ab 42
tide aggregation (Nie et al., 2011). His13-Lys16 region assists in the
peptide chains (A, B, C, D and E) folded in to a fibrillar structure. Cbx
conversion of Ab 42 peptide secondary structure from soluble and
was found to interact with residues Leu17, Val18, Phe19, Phe20,
unordered a-helix to stable b-sheet rich conformation, which
Ala21, Val36 and Val 40 of chain A and with residues Ala21, Val36
further propagates the aggregation of monomers. The nucleation of
and Val40 of chain B through various non-covalent interactions.
this aggregation by b-hairpin structure is considered as the rate
Amine group (donor) in the backbone of residue Phe19 of chain A of
limiting step (Ahmed et al., 2010). Beta-strand structure is created
the fibril formed a strong hydrogen bond (dN…H ¼ 1.42 Å, dN…
by the formation of hinge or turn regions and is stabilized by a salt

O ¼ 2.42 Å and/NHO ¼ 134.5 ) with the OH moiety of carboxyl group
bridge between Lys28, Asp23 and Glu22 facilitating the close
(acceptor) of Cbx. The latter also interacted via CH…O interaction
proximity of hydrophobic segments (hydrophobic core and C-ter-
(dH…O ¼ 2.18 Å) with Val18 (Ca) residue of chain A of the fibril. CH…
minus). These hydrophobic segments of Ab 42 play a crucial role in
O interactions with the donor-acceptor distances less than 3.5 Å
the process of aggregation (Ahmed et al., 2010; Chimon et al.,
have been categorized as strong interactions (Horowitz and Trievel,
2007). So, to know the presence of these residues in the amyloi-
2012). Formation of such strong hydrogen bonds together with
dogenic regions, the prediction of amyloidogenic regions was per-
CH…O interaction and hydrophobic interactions are the major
formed in the Ab 42 peptide using three different online servers.
guiding force behind the structural stability of cbx-Ab 42 fibril
Waltz server predicted the residues 16e21 and 37e42 while Fold
complex.
amyloid server predicted the residues 16e21 and 32e36 as the
Phe19 plays a crucial role in the formation of oligomeric and
amyloidogenic regions in the Ab 42 peptide. These predictions were
further validated by Aggrescan server which predicted residues
16e23 and 29e42. As the predicted amyloidogenic regions lie in Table 1
the hydrophobic core and C-terminus of the Ab 42 peptide, further Binding affinity of carbenoxolone with Ab 42 peptide. Binding affinities of 9 best
the docking studies were performed to evaluate the interactions suited binding modes are given in kcal/mol and distance from best mode is given by
rmsd l.b. (lower bound) and rmsd u.b. (upper bound).
between Cbx and the residues present in these regions.
Mode Affinity Distance from best mode
(kcal/mol)
rmsd l.b. rmsd u.b.

1. 6.8 0.000 0.000

2. 6.7 4.549 13.057
3. 6.7 5.322 12.856
4. 6.6 4.559 7.576
5. 6.2 14.297 18.238
6. 6.1 14.265 19.092
7. 6.0 13.788 20.582
Fig. 7. Detailed regions of Ab 42 peptide. N-terminus comprises of residues 1e11,
8. 5.9 14.211 18.800
16e20 residues form hydrophobic segment, hinge regions consist of residues 22e23
9. 5.8 5.795 9.916
and 26e28 while 40e42 residues form c-terminus.
 S. Sharma et al. / Neurochemistry International 108 (2017) 481e493 487

Fig. 8. Docked view of carbenoxolone with residues Arg5, Lys16 and Val12 of Ab 42 peptide (2a) and 2D view of the interactions (2b). Cbx formed a conventional hydrogen bonding
with Arg5 residue of Ab 42 peptide at a distance of 2.4 Å (Fluorescent green color). Hydrophobic interactions of Val12 and Lys16 residues are shown in light pink color and van der
Waals interactions of Ser8, Gln15 and Phe20 are shown in light green color. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

Table 2 et al., 2010). As reported by some previous studies, the close

Binding affinity of carbenoxolone with Ab 42 fibril. packing of Phe19 with Ile32, Leu34, Val36, Gly38 and Gln15 with
Mode Affinity Distance from best mode Val36, His13 and Val40 stabilizes the Ab 42 aggregates (Ahmed
(kcal/mol)
rmsd l.b. rmsd u.b.
et al., 2010; Luhrs et al., 2005). Also three consecutive repeats of
GxxxG motif including Gly33, Leu34, Met35, Val36 and Gly37 form
1. 7.6 0.000 0.000
ridges and grooves on amyloid surface, which facilitate the amyloid
2. 7.5 20.401 25.278
3. 7.5 20.366 25.142 aggregation (Eskici et al., 2013; Liu et al., 2005). As we incubated
4. 7.0 22.257 26.256 the Ab 42 peptide for 4 days, the amyloidogenic and hydrophobic
5. 7.0 21.278 24.870 residues might have formed these interactions to produce soluble
6. 7.0 18.562 21.269 aggregates of Ab 42 peptide as shown by SVAU analysis and Tricine
7. 6.9 22.585 24.785
8. 6.9 23.216 27.361
SDS-PAGE. To inhibit this aggregation process, first the drug must fit
9. 6.8 20.583 25.730 into a pocket in the Ab 42 peptide as well as fibril and second, must
alter the interactions of closely packed residues. As shown in the
present study, Cbx has been predicted to form various interactions
fibrillar aggregates of Ab 42 peptide and also forms side chain with residues present inside the pockets and also with the closely
packing with Leu34 and molecular contacts with Gly38 (Ahmed packed residues. These interactions might have resulted in the
488 S. Sharma et al. / Neurochemistry International 108 (2017) 481e493

Fig. 9. Docked view of carbenoxolone with residues Val18, Phe19, Ala21, Val36, Val40 of chain A and Val40 of chain B of Ab 42 fibril (3a) and 2D view of the interactions (3b). Cbx
formed a conventional hydrogen bonding with Phe19 residue of Ab 42 fibril at a distance of 2.4 Å (Fluorescent green color). Hydrophobic interactions of Ala21, Val36 and Val40
residues are shown in light pink color and van der Waals interactions of Leu17, Phe20, Ala21 and Val36 are shown in light green color. Cbx also formed a C-H bonding with Val18
residue at a distance of 3.2 Å (tea green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

alteration of the close packing existed in the aggregates and might
have led to the inhibition of Ab 42 peptide aggregation and also
might have disturbed the Ab 42 fibril stability.
3.5. Molecular dynamics (MD) simulations of Ab 42 peptide and
fibril in the presence and absence of Cbx
MD simulations were performed to understand the molecular
basis of the interactions between Cbx and Ab 42 peptide and fibril.
The simulation results suggested that Cbx interacts favorably with
some of the residues (Phe4, Arg5, His6, Tyr10, Val12, His14, Gln15,
Lys16, Val18, Phe19 and Ala30) of Ab 42 peptide as shown in Fig. 10.
Cbx forms hydrogen bonding with Arg5, Gln15 and Phe4 and also,
hydrophobic interactions with some residues of the peptide. The
secondary structure content of Ab 42 peptide was also measured in
the presence and absence of Cbx. There was a decrease in the
percentage secondary structure content (alpha-helix and beta-
sheet) in the Cbx bound Ab 42 peptide after 10 ns simulation
(Table 3 and Fig. S1). The torsion angles of residues of Ab 42 peptide
after 10 ns MD simulations are given in Table S1. The unstructured
peptide content was higher in the Cbx bound Ab 42 peptide as
compared to the unbound Ab 42 peptide. Cbx interacts with Ab 42
peptide through hydrophobic interactions and hydrogen bonding
that might be responsible for unstructured conformations of the
peptide and also, inhibition of peptide folding.
Cbx interacted preferentially with some residues (Leu17, Val18,
Phe19, Phe20, Ala21, Asp23, Lys 28, Leu34, Val36 and Val40 of chain
A and Ile32, Leu34 and Val40 of chain B) of Ab 42 fibril as shown in
 S. Sharma et al. / Neurochemistry International 108 (2017) 481e493 489

Fig. 10. MD simulation trajectories of Cbx bound to Ab 42 peptide at time t ¼ 0, t ¼ 2ns, t ¼ 5ns, t ¼ 8ns and t ¼ 10ns. Cbx is shown as yellow stick. Hydrophobic and hydrophilic
regions are shown as brown and blue shade in the solid ribbon, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

Table 3
Secondary structure analysis of Cbx bound Ab 42 peptide and fibril after 10 ns simulation.

Mode 0 ns 10 ns

Alpha-helix (%) Beta-sheet (%) Others (%) Alpha-helix (%) Beta-sheet (%) Others (%)

Unbound Ab 42 peptide 76.19 0.0 23.80 49.88 14.52 35.59

Cbx bound Ab 42 peptide 64.28 0.0 35.71 18.92 9.28 61.78
Unbound Ab fibril 0.0 76.92 23.07 0.0 64.61 35.38
Cbx bound Ab fibril 0.0 76.15 23.84 0.0 56.15 43.84

Fig. 11. Cbx forms hydrogen bonding with Phe19 and Asp23 and presence and absence of Cbx. There was a decrease in the per-
also, hydrophobic interactions with some residues of the fibril. The centage secondary structure content (beta-sheet) in the Cbx bound
secondary structure content of Ab 42 fibril was also measured in the Ab 42 fibril after 10 ns simulation (Table 3 and Fig. S2). The torsion

Fig. 11. MD simulation trajectories of Cbx bound to Ab 42 fibril at time t ¼ 0, t ¼ 2ns, t ¼ 5ns, t ¼ 8ns and t ¼ 10ns. Cbx is shown as yellow stick. Hydrophobic and hydrophilic
regions are shown as brown and blue shade in the solid ribbon, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
490 S. Sharma et al. / Neurochemistry International 108 (2017) 481e493
angles of residues of Ab 42 fibril after 10 ns MD simulations are
given in Table S2. The unstructured aggregate content was higher in
the Cbx bound Ab 42 fibril as compared to the unbound Ab 42 fibril.
The stability of the beta-sheet in Ab 42 fibril has been reported to be
dependent upon the intermolecular salt bridge between Asp23-
Lys28 (Luhrs et al., 2005). Cbx interacted with Ab 42 fibril
through hydrophobic interactions and hydrogen bonding. Cbx
interacted with Asp23 residue of chain A of Ab 42 fibril by forming
the hydrogen bonding (Fig. 11) disrupting the salt bridge between
Asp23-Lys28 residues. This might have led to the disturbance in the
backbone hydrogen bonding and might have resulted in the for-
mation of unstructured aggregates. Cbx interacted with Ab 42
peptide as well as with the Ab 42 fibril through hydrophobic in-
teractions and hydrogen bonding, which might have led to the
formation of unstructured conformations. The monomeric subunit
of the Ab 42 oligomer and fibril have some common interactions
such as contacts between Phe19-Leu34, His13-Gln15, Asp23-Lys28,
Gly25-Gly29 and Gly37-Gly38 (Ahmed et al., 2010). So, these in-
teractions might have prevented the formation of Ab 42 oligomeric
intermediates. Thus, Cbx was able to prevent the formation of toxic
Ab 42 species by inhibiting the aggregation of the Ab 42 monomers
and by forming the unstructured fibrillar aggregates.
Root-mean-squared-deviations (RMSD) from the starting sym-
metric model were calculated as a function of time for both Ab 42
peptide and fibril. Models with lower RMSD values were consid-
ered to be more stable (Shafrir et al., 2010). Cbx bound Ab 42
peptide was less stable as it had higher RMSD (1.14 nm) as
compared to the unbound Ab 42 peptide (0.94 nm) after 10 ns
simulation (Fig. 12a). Cbx bound Ab 42 fibril was less stable as it had
higher RMSD (0.38 nm) as compared to the unbound Ab 42 fibril
(0.26 nm) after 10 ns simulation (Fig. 12b). Root-mean-squared-
fluctuation (RMSF) values were also calculated for each residue to
determine the stability of the model. Segments with high RMSF
values are considered to be more dynamic and less stable (Shafrir
et al., 2010). Cbx bound Ab 42 peptide was found to be more dy-
namic having higher RMSF values as compared to the unbound Ab
42 peptide after 10 ns simulation (Fig. 13a). The RMS fluctuations of
the chains (chain A and B) of Ab 42 fibril interacting with Cbx were
observed. Residues present in the region 28e42 and Asp23 of chain
A and region 23e42 of chain B were found to have high RMSF
values in the Cbx bound Ab 42 fibril (Fig. 13b and c). These highly
dynamic residues might have initiated the formation of unordered
structures leading to the formation of unstructured aggregates.
Fig. 12. RMSD values of backbone for unbound and Cbx bound Ab 42 peptide (a) and fibril (b).
Solvent accessible surface area (SASA) was also observed for
both Ab 42 peptide and fibril. There was an increase in the SASA for
Cbx bound Ab 42 peptide (26.90e20.61 nm2) as compared to un-
bound Ab 42 peptide (26.32e19.42 nm2) (Fig. S3a). SASA for Cbx
bound Ab 42 fibril (51.02e45.28 nm2) was also high after 10 ns
simulation as compared to unbound Ab 42 fibril (51.71e45.25 nm2)
(Fig. S3b). The increase in the SASA signifies the decrease in the
hydrophobicity of the peptide as well as the fibril, which means a
decrease in the beta-sheet content. Beta-sheet promotes the pro-
tection of hydrophobic side chains from solvent exposure, which
leads to a decrease in the SASA (Granata et al., 2015). The increase in
the Cbx bound Ab 42 peptide and fibril SASA might have led to the
decrease in the beta-sheet content, which was also confirmed by
the secondary structure analysis.
After 10 ns MD simulation, the unbound Ab 42 peptide was
having higher secondary structure content and was more stable in
its folded state. But Cbx binding to the peptide resulted in a
decrease in the secondary structure content and also made the
folded state unstable and unstructured. Similarly, Cbx binding
decreased the secondary structure content in the Ab 42 fibril and
formed the unstructured aggregates. These unstructured confor-
mations are considered to be less toxic and are not involved in the
formation of toxic oligomeric species (Du et al., 2015). Thus, Cbx
was able to inhibit on-going pathway of Ab 42 aggregation and also
was able to destabilize the pre-formed fibrils leading to the for-
mation of less toxic aggregates.
3.6. ADMET analysis of carbenoxolone
ADMET analysis of Cbx (using admetSAR server) predicted that
Human Intestinal Absorption (HIA) and Blood-Brain Barrier (BBB)
permeability scores were 0.96 and 0.80 respectively. These scores
indicated that Cbx is readily absorbed from the intestine and also
crosses the blood-brain barrier. Cbx was also predicted to be sub-
strate and non-inhibitor for p-glycoprotein. Cbx was predicted as
nonsubstrate and non-inhibitor of CYP450 microsomal enzyme
which revealed that it will not hamper the biotransformation of
drugs metabolized by CYP450 enzyme. In terms of mutagenic
properties, Cbx was predicted to be non-AMES toxic and non-
carcinogenic and LD50 was predicted to be 2.39 g/kg (oral) and
92 mg/kg (i.p.) for rats. So, the predicted ADMET properties project
Cbx as a suitable drug candidate for aggregation inhibition of Ab 42
peptide.
 S. Sharma et al. / Neurochemistry International 108 (2017) 481e493
Fig. 13. RMSF values of residues for unbound and Cbx bound Ab 42 peptide (a) and fibril (b and c).
4. Conclusion
The inhibition of Ab aggregation is the inhibition of a protein-
protein interaction between monomers, alteration of proper
folding of the monomers and destabilization of the pre-formed
aggregates. Based on the observations from in-vitro and in-silico
studies, Cbx has altered these interactions by forming strong in-
teractions with the residues present in the amyloidogenic regions
of Ab 42 monomers as well as fibrils. Cbx inhibits the aggregation of
Ab 42 peptide and destabilizes the Ab 42 aggregates. Hydrogen
bonding and hydrophobic interactions are the major bases for the
stabilization of Ab 42-Cbx complex and are responsible for the anti-
fibrillation activity of Cbx. Also, ADMET analysis has predicted Cbx
as a suitable drug candidate. So these observations strongly project
Cbx as an Ab 42 peptide aggregation inhibitor candidate, but
further in-vivo studies are required to explore the therapeutic po-
tential against AD.
Grant information
Financial assistance from University Grants Commission (UGC),
Govt. of India is gratefully acknowledged.
Conflict of interest
The authors declare no conflict of interest.
491
Acknowledgments
Financial assistance from UGC, New Delhi, India is greatly
acknowledged.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.neuint.2017.06.011.
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