Design of mechanism-based inhibitors of transthyretin amyloidosis: Studies with biphenyl ethers and new structural templates

Article abstract of DOI:10.1021/jm0700159

Transthyretin (TTR), a tetrameric thyroxine (T4) carrier protein, is associated with a variety of amyloid diseases. In this study, we explore the potential of biphenyl ethers (BPE), which are shown to interact with a high affinity to its T4 binding site t

Full text of DOI:10.1021/jm0700159

J. Med. Chem. 2007, 50, 5589-5599
5589
Design of Mechanism-Based Inhibitors of Transthyretin Amyloidosis: Studies with Biphenyl
Ethers and New Structural Templates
Sarika Gupta,†,§ Manmohan Chhibber,‡,§ Sharmistha Sinha,‡ and Avadhesha Surolia*,†
Molecular Biophysics Unit, Indian Institute of Sciences, Bangalore 560012, India, and National Institute of Immunology, Aruna Asaf Ali Marg,
New Delhi 110067, India
ReceiVed January 5, 2007
Transthyretin (TTR), a tetrameric thyroxine (T4) carrier protein, is associated with a variety of amyloid
diseases. In this study, we explore the potential of biphenyl ethers (BPE), which are shown to interact with
a high affinity to its T4 binding site thereby preventing its aggregation and fibrillogenesis. They prevent
fibrillogenesis by stabilizing the tetrameric ground state of transthyretin. Additionally, we identify two new
structural templates (2-(5-mercapto-[1,3,4]oxadiazol-2-yl)-phenol and 2,3,6-trichloro-N-(4H-[1,2,4]triazol-
3-yl) represented as compounds 11 and 12, respectively, throughout the manuscript) exhibiting the ability
to arrest TTR amyloidosis. The dissociation constants for the binding of BPEs and compound 11 and 12 to
TTR correlate with their efficacies of inhibiting amyloidosis. They also have the ability to inhibit the elongation
of intermediate fibrils as well as show nearly complete (>90%) disruption of the preformed fibrils. The
present study thus establishes biphenyl ethers and compounds 11 and 12 as very potent inhibitors of TTR
fibrillization and inducible cytotoxicity.
Introduction
agglomerate into fibers.12 TTR deposits are predominantly
extracellular in nature, while some of its variants also exhibit
tissue specificity.14 The mechanism for their tissue selectivity
and the pathway of their deposition in ViVo are as yet poorly
understood.
The quaternary structure of TTR contains two funnel shaped
thyroxine (T4) binding sites.15 Under physiological conditions,
only 10-25% of T4 in the plasma is bound to TTR.16 The
stabilization of TTR tetramer by small molecules, which bind
to the T4 pocket, is an emerging theme in a number of studies
aiming to stall amyloidogenic potential of TTR.17,18 So far, a
number of TTR amyloidosis inhibitors including both natural
and synthetic molecules that span a variety of structural classes
have met with limited success.19-38 Chemical modification and
covalent linking of molecules to TTR have also been suggested
as alternatives to these approaches.39-41
Biphenyl ether as a template to design potential inhibitors of
TTR amyloidosis has not yet been explored systematically. The
structure-activity relationship (SAR) studies have shown that
small molecule such as triclosan, could inhibit TTR aggrega-
tion.42 Triclosan, which has a biphenyl ether skeleton, resembles
T4 in its gross structure (Figure 1a). Triclosan exhibits a wide
range of pharmacological properties including antibacterial and
antimalarial activities.43 In the present report, we have synthe-
sized a series of BPE derivatives (Figure 1b and Table 1) and
investigated their ability to arrest TTR amyloidosis using a
variety of biochemical and biophysical methods. We also report
their striking ability to disrupt preformed fibers and restore the
native tetrameric state of TTR. We also report results from
screening of a library of 100 compounds to obtain some new
scaffolds for inhibiting amyloidosis. Docking studies were
performed to provide a rationale for the inhibitory potencies of
BPEs in preventing TTR amyloidosis.
Protein misfolding, misassembly, and extracellular deposition
are related to a class of diseases collectively known as
“conformational diseases”, which include Alzheimer’s disease,1
prion disease,2 dialysis-related amyloidosis,3 familial amyloid
polyneuropathy,4 and type II diabetes.5 Most of these diseases
are incurable and fatal. The proteins and peptides related to these
diseases can self-assemble into supramolecular assemblies with
a common cross-â structure. Despite a large variation in their
sequences and native structures, they adopt a similar morphology
upon fibril formation, which suggests that there is a common
mechanism underlying amyloid fibril formation.6
Transthyretin (TTRa), a tetrameric protein, transports thy-
roxine and holo retinol binding protein in plasma and cere-
brospinal fluid.7 Further, it also scavenges Aâ peptide, prevent-
ing its aggregation and thereby regulates the pathogenesis of
Alzheimer’s disease.8 TTR tetramer has a tendency to dissociate
to unfolded monomers, which aggregate together resulting in
the formation of amyloid fibers, which constitute the hallmark
of familial amyloid cardiomyopathy (FAC), senile systemic
amyloidosis (SSA, late onset), and familial amyloid polyneur-
opathy (FAP, early onset). In SSA and FAC, wild type TTR
forms amyloid deposits on the cardiac and other tissues.9,10
Approximately 100 mutants have been identified in TTR to be
involved in FAP, which affect the peripheral and autonomic
nervous system, heart,8 and the CNS.11,12 Irrespective of the
types of TTR (viz. wild type TTR or its mutants) involved in
amyloid formation, the overall mechanism of fibril formation
is similar. The common mechanism involves the dissociation
of the tetramer into non-native monomers, which eventually
* Author to whom correspondence should be addressed. Fax: +91-11-
26162125, e-mail: surolia@nii.res.in.
‡ Indian Institute of Sciences.
Results
† National Institute of Immunology.
§ These authors have made equal contributions.
a Abbreviations: TTR, transthyretin; BPEs, biphenyl ethers; T4, thyroxine;
SAR, structure-activity relationship; MALDI-TOF, matrix-assisted laser
desorption ionization-time of flight; Th-T, thioflavin-T; TEM, transmission
electron microscope.
Fibril Formation Assay. The stagnant fibril formation assay
at pH 4.4 showed that all the synthetic BPEs examined inhibited
TTR amyloidosis at stoichiometric concentration (Table S2 in
Supporting Information). Out of 24 designed BPEs, nine
10.1021/jm0700159 CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/19/2007

5590 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Gupta et al.
340 nm and of quantitative Congo red binding to fibril formed
by TTR in the presence and absence of inhibitors is shown in
Figure 2a. While unligated TTR exhibited maximum binding
of Congo red, it was significantly reduced in the presence of
compounds 1-12. Even 7-30 days old samples in the presence
of compounds 1-12 exhibited diminished binding of Congo
red to TTR. TTR fibrillization in the presence and absence of
compounds 1-12 was further examined by TEM. As is evident
from TEM pictures (Figure S1; see Supporting Information) the
samples containing compounds 2-12 were devoid of any fiber
or amorphous aggregates. In contrast, BPE-untreated samples
exhibit fiber and fibrillar aggregates in abundance.
Kinetics of Fibril Formation. The kinetics of fibril formation
in the presence and absence of the compounds were monitored
by linear increase in the turbidity at 340 nm for 3 h. The increase
is prominent in the absence of inhibitors, while it was
significantly reduced in the presence of the inhibitors and T4.
The inhibition was >80% by compounds 2-12 and 62% by
triclosan as compared to 75% inhibition by T4 (Figure 2b).
These results are consistent with the results of stagnant fibril
formation assay (Figure 2a). The rate of fibril formation in the
presence and absence of compounds 1-12 is given in Figure
S2 (see Supporting Information).
Thioflavin-T (Th-T) Fluorescence. Th-T fluorescence was
monitored in the presence and absence of the inhibitors at pH
4.4 at 0 and 72 h. The intensity of Th-T was high in the absence
of the compounds 2-12 and significantly less in their presence
(Figure 2c). There was no significant increase in Th-T binding
even in the samples incubated for 7-30 days in the presence
of compounds 1-12 (data not shown).
Tryptophan Fluorescence. TTR exhibits fluorescence emis-
sion maxima at 339 and 343 nm, respectively, at pH 7.2 and
4.4, indicating the presence of tryptophan residues exposed to
solvent at the surface of the protein. Conformational changes
in TTR induced upon binding of inhibitors therefore were
studied by monitoring the intrinsic fluorescence of the protein
in the presence and absence of the compounds 2-12, triclosan,
and T4 both at pH 7.2 and 4.4. A significant reduction in the
fluorescence intensity of TTR was observed in the presence of
compounds 1-4 and 6-12 at pH 7.2 (Figure 3a), which was
more pronounced at pH 4.4 (Figure 3b). This indicates a subtle
change in the exposure of tryptophan residue(s) in TTR upon
the binding of compounds 1-12, which in turn appears to be a
reflection of a change of its quaternary structure. A comparison
of the change in fluorescence intensity of TTR treated with the
compounds 1-12 show a greater reduction at pH 4.4 as
compared to that at pH 7.2, indicating that at acidic pH a greater
proportion of TTR is being driven to tetramer formation by the
binding of these compounds.
Figure 1. (a) Structures of triclosan and thyroxine, (b) Strategy for
the design of differently substituted BPE derivatives. (c) The semilog
plot of TTR (7.2 µM) stagnant acid-mediated fibrillization assay at
pH 4.4 in the presence of compounds 1-12 and thyroxine at various
concentrations over 72 h. The experiment was performed at 37 °C.
The data were fitted in to sigmoid equation as given in Supporting
Information, and the IC50 value was calculated graphically. Results are
the mean of five independent experiments with error bar.
compounds (represented as 2-10 in Table 1) inhibited the
process >90% when 3 molar equiv of them were used over
TTR (BPEs ) 21.6 µM, TTR ) 7.2 µM), except triclosan
(∼24%), and were therefore selected for a detailed study. BPEs
were found more effective as inhibitors than T4.
Out of the 100 compounds from the chemical library, 41
compounds were found to inhibit fibril formation at 100 µM
concentrations (Table S1 in Supporting Information). Of these,
only two compounds 11 and 12 were selected for further studies,
as they inhibited TTR (7.2 µM) fibrillization completely at 21.6
µM (Table 1). IC50 for compounds 2-12 are in the range of
0.47-3.5 µM, which are lower than IC50 of 7.1 µM for its
natural ligand T4 (Table 1). The dose-dependent curves for
inhibition of TTR amyloid formation by these compounds are
shown in Figure 1c. The dissociation constant Kd1 of compounds
2-12 at pH 4.4, are in nanomolar range compared to 144.0
µM of triclosan (Table 1). For compounds 2, 3, 4, 6, and 10,
the Kd2 is 37-10000-fold higher than their respective Kd1,
suggesting negative cooperativity for their binding to the second
site of TTR.
Urea Denaturation. As the rate of TTR fibrillization is
proportional to the rate of tetramer dissociation, the rate and
extent of TTR tetramer (1.8 µM) dissociation in 6.0 M urea
was monitored by evaluating the intrinsic fluorescence of TTR.
Compounds 1-12 exert substantial effect on the amplitude of
TTR tetramer dissociation (Figure 3c). For example at 1:3 ratio
of TTR to triclosan, 47% of the protein dissociates as compared
to the untreated control under identical conditions. In comparison,
compound 7 and T4-treated TTR under similar situation exhibits
21 and 27% dissociation, respectively. Interestingly, in the
presence of compounds 2-6 and 8-12, less than 5% of the
protein dissociates and unfolds even after 144 h, implying an
overwhelming stabilization of TTR tetramer by these compounds
(Figure 3c). These data thus allow us to infer that these
compounds act by kinetic stabilization of TTR tetramer under
Compounds buffered at pH 4.4 have also acted as potent
inhibitors of the process. Table 1 shows the stoichiometry of
binding of the compounds 1-12 to TTR tetramer. Further, in
order to rule out the presence of any fibrillar species, inhibition
of fibril formation by compounds 1-12 was tested by Congo
red binding. A comparison of the results of turbidity assay at

Biphenyl Ethers as Potential Inhibitors of TTR Amyloidosis
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5591
Table 1. Potencies of Biphenyl Ethers and Other New Structural Templates as TTR Amyloidosis Inhibitorsa
a %FF: The percent fibril formation of TTR (7.2 µM). The inhibition was tested at TTR to inhibitor ratio of 1:1 and 1:3 which corresponds to 7.2 µM
and 21.6 µM inhibitor concentrations, respectively, at pH 4.4, 37 °C. Fibril formation by TTR in the absence of inhibitors was considered as 100%. IC50
value, i.e., inhibitor concentration at which there is 50% reduction in fibril formation. Stoichiometry, the number of equivalents of inhibitor bound to one
equivalent of TTR. Results presented are mean of five different experiments. ND, not determined; *, data fitted well to a single binding site.
denaturing conditions over the amyloidogenic monomer, a
finding consistent with the rate of fibril formation at 37 °C, pH
4.4 (Figure 2a).
Glutaraldehyde Cross-Linking. To understand the mecha-
nism of inhibition of fibril formation by all the above com-
pounds, the quaternary structure of TTR in the presence and
absence of these compounds at acidic condition was character-
ized by glutaraldehyde cross-linking followed by the analysis
of the cross-linked products by SDS-PAGE. TTR at pH 7.2
migrated predominantly as a tetramer (55 kDa), whereas at pH

5592 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Gupta et al.
Figure 2. (a) Comparative bar diagram of turbidity (shaded bars) and
quantitative Congo red binding (black bars) assay to quantitate TTR
(7.2 µM) fibrillogenesis in the absence and presence of 3 equiv (21.6
µM) of compounds 1-12 and T4. Results are means of 3-5 different
observations. Congo red binding to the TTR in the presence and absence
of inhibitors after 15 days of incubation at pH 4.4 was monitored
by scanning for absorption from 400-600 nm (spectra in the inset).
Experiments were carried out at 37 °C. (b) The time course of TTR
(7.2 µM) fibril formation in the presence and absence of compounds
1-12 and T4 (21.6 µM) at 37 °C, pH 4.4. Turbidity at 340 nm
was monitored for 3 h is plotted against time. Results are mean of
three different experiments done in triplicate. Standard error was
<10%. The plot shows the rate of fibril formation in the presence and
absence of compounds 1-12 and T4 in preventing TTR fibril for-
mation is given in Supporting Information (Figure S2). (c) Thioflavin-T
fluorescence after binding to TTR (7.2 µM) in the presence and absence
of compounds 1-12 and T4 (21.6 µM) under fibrillization condition.
Fibrils were produced by acidification at pH 4.4 for 72 h at 37 °C
and monitored by the Th-T fluorescence assay. The samples were
incubated with 50 µM of Th-T for 15 min at 37 °C and excited at
450 nm, and Th-T fluorescence emissions were monitored at 480
nm. Results are means of three independent experiments done in
duplicate.
Figure 3. Conformational change in TTR induced by binding of
compounds 1-12 and T4. TTR (3.6 µM) incubated in the presence
and absence of compounds 1-12 (7.2 µM) at 25 °C (a) at pH 7.2, (b)
at pH 4.4. Change in conformation was inferred by monitoring the
intrinsic fluorescence of TTR. Samples were excited at 290 nm, and
emission was monitored in the range of 310-370 nm. Results are mean
of three different experiments. (c) Time dependence of tetramer
dissociation (fraction unfolded) of TTR in the presence and absence
of inhibitors in 6 M urea at 25 °C. TTR (1.8 µM) incubated for 1 h
with 5.4 µM of compounds 1-12 or T4 at 25 °C at pH 7.2 was used
for these studies. Urea was then added to these solutions to a final
concentration of 6 M, and the unfolding of TTR was monitored by
following the intrinsic fluorescence of the protein at 339 nm up to 144
h. Samples were excited at 290 nm. Fraction unfolded at each time
point was plotted against time. Results are mean of three different
experiments performed in duplicate.
4.4 it migrated as a monomer (Figure 4a). However, TTR
migrates predominantly as a tetramer in the presence of

Biphenyl Ethers as Potential Inhibitors of TTR Amyloidosis
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5593
compounds 1-12. Only a small amount of dimer was observed
in case of native TTR and TTR in the presence of compounds
1-12. In the absence of these inhibitors, the amount of tetramer
was decreased with time and became zero after 3 days (data
not shown). In contrast, there was no significant difference in
the tetramer concentration in the presence of these inhibitors
during 72 h to 30 days.
Mass Spectrometry. All the above observations clearly show
that compounds 1-12 prevent fibril formation by stabilizing
TTR tetramer. It can, therefore, be assumed that soluble TTR
present in the reaction mixture mostly exists as a tetramer. To
confirm these findings, MALDI-TOF of TTR samples treated
with these inhibitors for 3, 7, and 15 days subsequent to
centrifugation and filtration through 80 kDa cutoff membranes
was carried out as described in the Experimental Section. As
shown in Figure 4b, the intensity of the monomer peak gradually
decreases which completely disappear by day 7 in the absence
of the inhibitors (Figure 4b, a-c). In contrast, a peak corre-
sponding to ∼13850 Da can be observed in the presence of
inhibitor 2, shown here as a representative example, even after
15-30 days of fibrillogenesis (Figure 4b, d and e). The mass
spectra of TTR in the presence and absence of the other
compounds (viz. 1 and 3-12) are shown in Figure S3 (Sup-
porting Information).
Inhibition of TTR-Induced Cell Cytotoxicity by Com-
pounds 2-12. Neuro2A cell culture was used to detect the
presence of any soluble toxic aggregates in the reaction mixture
used for studying the inhibition of TTR fibrillogenesis. Pre-
liminary experiments showed that mature TTR amyloid fibers
are not cytotoxic (data not shown). However, TTR solution left
under the conditions of fibrillogenesis induces toxicity to
Neuro2A cells in a concentration-dependent manner in 72 h
(Figure 4c). Figure 4c shows the TTR-induced ∼41% cytotox-
icity under the fibrillogenesis conditions. However, preincuba-
tion of TTR (25 µM) with the compounds 2-12 (50 µM)
exhibited viability in the range of 86-99.5%. Compounds 2-12
at 50 µM by themselves had no effect on the survival of
Neuro2A cells in culture, demonstrating their nontoxic nature
at the concentrations used (Figure 4c).
Molecular Docking. To validate and understand the basis
of the inhibitory activities of these closely related BPEs in TTR
amyloidosis, docking studies were performed. The crystal-
lographic structure of the TTR-T4 complex44 showed its
hormone-binding sites, which are composed of three symmetry-
related hydrophobic small depressions, termed halogen-binding
pockets. The molecular docking of all these inhibitors with TTR
shows their binding to the T4 binding site with binding of one
ring to P3 and the other to P1 pocket. Interactions with the
residues lining the pocket are mainly through hydrophobic and
electrostatic interactions. Figure 5 shows the overlap of several
of the MOE-docked BPEs and compound 11 and 12 in the
thyroxine binding pocket of TTR complexed with T4. Triclosan
and other biphenyl ethers with R1 ) Cl bind deep in the P3
pocket of TTR while the dihalogenated phenyl ring is positioned
in the outer P1 pocket of the binding cavity, viz. the two chlorine
atoms of the B-ring of tricolsan are accommodated in P1 and
P1′ pockets, respectively (Figure 5). Van der Waals interactions
between both the chloride substituent of the ligand and the side
chains of Leu17, Ala108, Thr119, and Val121 from the adjacent
TTR subunit contribute to the stabilization of the tetramer. The
atoms of BPEs that are involved in extensive hydrophobic
interactions with Ala108, Leu110, Ser117, Thr118, and Thr119
of the monomer AA′ and CC′ appear to contribute significantly
to the overall stability of the tetrameric structure. Thus two
Figure 4. a: Oligomeric status of TTR in the presence and absence
of compounds 1-12 at pH 4.4 after 15 days of incubation under
fibrillization conditions. The reaction mixtures were neutralized and
the samples cross-linked with gluteraldehyde. They were then elecro-
phoresed on 12% SDS-PAGE. TTR complexes with compounds 1-12
migrated mainly as the tetramer. N, is TTR at -20 °C; M, molecular
wt standards. b: Stability of TTR treated with compounds 1-12. Mass
spectra of TTR at (a) 0 h, (b) 72 h, (c) 7 days at pH 4.4, (d) native
TTR, and (e) in presence of inhibitor 2 after 7 days. The mass spectra
of TTR with compounds 3-12 at 15 days are given in Figure S3 in
Supporting Information. c: Efficacies of compounds 2-12 in protecting
Neuro2a cells against cytotoxicity of TTR. The graph shows the %
viability of cells treated with TTR (25 µM) alone (black bar), at 2
equiv of compound 2-12 (50 µM) with respect to TTR concentration
(hatched bars), and compounds alone (open bars). Dose-dependent curve
of TTR-induced cytotoxicity to Neuro2a cells is given in the inset.
Results are mean of three different experiments conducted in triplicate.

5594 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Gupta et al.
Figure 5. Results of the docking of compounds 2-12 on the TTR tetramer. Molecular docking with the TTR tetramer was done with each of the
inhibitors using MOE-2005 software. There are two symmetrically equivalent positions for ligands in each tetramer of TTR. For the sake of clarity,
only one of the symmetry equivalent positions of the binding site for the T4 together with other inhibitors docked therein is shown. (a) The overlap
structure of docked compounds 2-12 to TTR and thyroxine (orange) bound to TTR (PDB code 2ROX). (b) Blown up version of the thyroxine
binding site along with compounds 2-6, 10, and 12 overlapped with T4. Color codes are T4 (orange), 2 (cyan), 3 (red), 4 (light green), 5 (blue),
6 (purple), 10 (pink), and 12 (peach). (c) Overlap of the mode of binding of the best inhibitor (compound 6; shown in purple) with T4 (orange) at
the T4 binding site of TTR. The interactions between TTR and the inhibitors in each of the minimum energy structures were evaluated using
Clus-Pro online software.46,47 The side chains of protein residues that interact with a given ligand are shown. Residues involved in interactions
within e4.5 Å have only been shown.
molecules of these BPEs bind to the two hormone-binding
pockets in an antiparallel orientation. In case of R1 ) H, the
unsubstituted ring binds to P3 pocket and R2 and R3 substituted
ring binds to the outer pocket to stabilize the structure (Figure
5). In the case of compounds 11 and 12, the benzene ring binds
deep in the P3 pocket making hydrophobic interactions, while
heteroatoms N and O of the other ring are involved mainly in
salt bridges with the residues lining the P1 pocket (Figure 5).
Fibril Disruption. The strong affinity of these compounds
for TTR and the remarkable stability of the resulting complex
prompted us to investigate their ability to inhibit TTR fibril
elongation. Their ability to disrupt preformed fibers was also
examined. BPEs examined were not only able to inhibit the
elongation of early fibrils but also exhibited disruption of the
mature fibrils in a dose-dependent manner. Moreover, these
compounds also disrupted the formation of various intermediates

Biphenyl Ethers as Potential Inhibitors of TTR Amyloidosis
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5595
Native-PAGE analysis of supernatants of above samples after
1 month clearly shows presence of abundant soluble protein
compared to the untreated control (Figure 6b). Further, the effect
of the compounds 2-12 on the ultrastructural properties of the
TTR fiber was examined by TEM. Figure 6d shows the presence
of abundant fibers (8-20 nm wide and 200-600 µm long) and
fibrillar aggregates as compared to the control samples (Figure
6c). The fibrils with width of 14.4-21.6 µM were disrupted
into fragments of 2-5 nm width and 20-100 µm long after
the second dose of compounds 2-12. Figure 6e and 6f shows
a representative picture of the fibers disrupted by compounds
2-12 subsequent to their second and third doses, respectively.
Discussion
There is much evidence to show that conformational changes
are sufficient for the conversion of a number of normally soluble
human proteins into amyloid fibrils, the hallmark of human
amyloid diseases.6 Presently, there is no general strategy for
treating human amyloid diseases. The most promising approach
includes stabilization of the native state of these proteins, which
is best exemplified by TTR amyloidosis. The earlier reports have
demonstrated that native state kinetic stabilization is a viable
therapeutic approach to prevent TTR amyloidosis.45 In this
study, we systematically screened a chemical library of 100
compounds and used structure-based design of substituted BPEs
to construct small molecule inhibitors against TTR-associated
amyloidosis. The site of amyloidosis in ViVo in humans is not
known. Therefore, we have evaluated these small molecule-
based inhibitors of TTR amyloidosis under different conditions
to demonstrate kinetic stabilization independent of experimental
conditions. T4, a known inhibitor and natural ligand of TTR,
was used for comparison in these screening studies. Screening
of a chemical library and biphenyl ether derivatives, described
in this report, yielded some of the most potent inhibitors of acid-
mediated TTR fibril formation (see Table S1 in Supporting
Information).
The experimental results (Figures 1-6 and Table 1) dem-
onstrate that several biphenyl ether derivatives are excellent
inhibitors of TTR (7.2 µM) fibril formation. Most of them have
shown ∼ 100% inhibition of amyloidosis at 21.6 µM concentra-
tion when tested against 7.2 µM of TTR. The entire structure
of 2, 3, and 6 appears to be important for their efficacy.
Correlation of their structure with activity show that the R1 )
Cl substituted phenyl ring is essential for inhibition as can be
discerned from the poor activity of R1 ) NO2 substituted BPE
(Table S2 in Supporting Information). Compound 10, an
analogue of 2, lacking both chlorine atoms, is less effective,
indicating the importance of van der Waals interactions for their
binding to TTR, a prerequisite for tetramer stabilization.
Substitution at R2 ) OH by OCH3 in compound 7 decreases
the activity. In contrast, activity was increased in compound 6,
indicating that substitution at R3 position is valuable. Indeed,
inhibitors 2, 3, and 6, which are analogues of triclosan, also
exhibited high potency signifying that carbonyl, carboxylic, and
alkyl halide substitutions at R3 position improve their efficacy
over triclosan. Thus, the Cl group at the R1 position and the
carbonyl group at the R3 position of these biphenyl derivatives
are important for inhibition of TTR fibrillation. The number of
active R2- and R3-substituted BPEs (2-7, Table 1) suggest that
additional manipulation of BPE at these positions could yield
inhibitors that are even more potent.
Figure 6. a: Th-T fluorescence of TTR fibers after disruption by
compounds 2-12. The TTR fiber formed at 24, 48, and 72 h were
incubated with three doses (7.2 µM each) of compounds 2-12 for 15
days. The disruptions of preformed TTR fiber by these compounds
were monitored by Th-T fluorescence by incubating samples with 50
µM of Th-T for 15 min at 37 °C. The samples were excited at 450 nm,
and emissions were monitored at 480 nm. Results are mean of three
different experiments executed in duplicates. b: Native-PAGE analysis
of samples of TTR fibers disruption after 1 month of incubation with
compounds 2-12 at 37 °C. TTR fibers incubated with compounds
2-12 for 15 days were centrifuged, and supernatant was loaded on
gel to see the amount of soluble protein present. c-f: Transmission
electron micrograph of control sample showing fibrillar aggregates (1:2
diluted, 4.2 K) (c), control sample showing full length fibers (1:100
diluted, 8.2 K) (d), fibers incubated with compounds 2-12 for 2 days
were clearly disrupted (16.5 K) (e), magnified view of disrupted fibers
(87 K) (f).
formed during TTR fibrilization. While single doses of inhibitors
were adequate to prevent fibril elongation, multiple doses were
required for the disruption of the preformed fibers. Disruption
started after the second dose. The change in turbidity with time
in the presence and the absence of compounds 2-12 is shown
in Figure S4 (see Supporting Information). The disruption of
fibrils formed at 24, 48, and 72 h, viz after three dosages of
these compounds, was also confirmed by Th-T fluorescence
assay (Figure 6a). While the fluorescence intensity of Th-T in
control samples increased with time, no enhancement in intensity
was observed in the samples incubated for 15 days with
compounds 2-12. All these compounds disrupt preformed TTR
fibers as well. Compounds 4, 5, 6, 9, 10, and 12 were found
more effective as fibril disrupters under the conditions used.
Of the 100 compounds from the chemical library, 41
compounds inhibited the process. Of these, two compounds (11
and 12) were found to be the most potent. The compounds thus

5596 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Gupta et al.
identified from the chemical library are heterocyclic compounds.
The high potency and the significant differences in their
molecular scaffolds as compared to the existing core structures
(biphenyl ether, biphenyl amine, biphenyl, etc.), makes them
promising leads for the design of potent TTR-specific amyloi-
dosis inhibitors. Fibrillization of TTR with added compounds
1-12 buffered at pH 4.4 was similar, suggesting the affinity of
inhibitors for TTR is greater than the propensity of TTR tetramer
dissociation and misfolding. The IC50 determined for compounds
2, 3, and 6 are lowest values observed so far for the inhibition
of TTR fibrillization. The low IC50 values (less than 1 equiv of
TTR) for all these ligands clearly indicate that substantial
inhibition of amyloidogenesis does not require kinetic stabiliza-
tion of every TTR tetramer by the binding of a small molecule.
Misfolded monomeric TTR aggregates into amyloid fibrils via
a straightforward self-association mechanism where all forward
steps in the pathway are favorable, whereas the rate of
aggregation is dependent on the concentration of the misfolded
TTR monomer.46 Since the concentration of monomer depends
on dissociation of the tetramer, controlling the energetics of
tetramer dissociation will allow significant control over the
amyloidogenic monomer concentration dictating the rate of TTR
amyloidogenesis. Thus, tight binding of a small inhibitory
molecule at substoichiometric concentrations (less than 1 equiv)
may be sufficient to reduce the concentration of amyloidogenic
monomeric TTR in the serum, thereby preventing the disease.
This was evident by the kinetics of fibril formation in the
presence and absence of inhibitors. A significant decrease in
the rate of fibril formation by compounds 2-12 compared to
the control was observed. The time course of TTR fibrillogenesis
shows that it lacks a lag phase and is not seedable, which is in
agreement with earlier reports.47,45
observed under denaturing conditions, implying pH-independent
stabilization of the protein in the presence of compounds 1-12.
It has been reported earlier that the dissociation of the tetramer
is a critical and rate-determining step in TTR related fibrillo-
genesis.48 The ability of these inhibitors to impose kinetic
stability on tetrameric TTR can be best evaluated by the rate of
tetramer dissociation. Tetramer dissociation leads to the unfold-
ing of the monomer in the presence of 6.0 M urea at 25 °C. To
understand the mechanism of inhibition of fibril formation,
denaturation kinetics of TTR in 6.0 M urea was carried out.
The TTR tetramer does not denature in urea; however, dis-
sociation to monomer is required for urea-induced tertiary
structural changes which can be detected by monitoring the
changes in intrinsic tryptophan fluorescence.49,17 The TTR
tetramer dissociation is drastically slowed down in the presence
of compounds 1-12, compared to the control. However, the
decrease was less for T4 and triclosan, clearly indicating that
the efficacies of the compounds 2-12 in increasing the energy
barrier for tetramer dissociation is responsible for the stabiliza-
tion of the tetramer of TTR.
Several reports indicate that inhibition of fibril formation can
lead to accumulation of soluble prefibrillar oligomeric inter-
mediates, the most cytotoxic species.50,51 In the present study,
cross-linking experiments with glutaraldehyde clearly show that
even under the conditions conducive for fibrillization, TTR
exists mostly as a tetramer in the presence of compounds 1-12
and T4 (Figure 4a). In contrast, no tetramer was observed in
unligated TTR, ruling out the possibility of the formation of
the prefibrillar cytotoxic species in the presence of compounds
2-12 during fibril formation. The stability of TTR-inhibitor
complex was determined under denaturing condition by extend-
ing the incubation time to 7-15 days. An analysis of MALDI-
TOF data shows remarkable stability of TTR by BPEs compared
to that observed in the presence of T4 (Figure 4b). Even after
30 days of incubation, TTR retains its tetrameric structure in
the presence of 3 equiv of compounds 2-12 under conditions,
in which the absence of these compounds would have led to
fibrilization within 72 h.
An increasing appreciation of soluble oligomeric intermediate
as the major cytotoxic species in amyloidosis prompted us to
test the effect of these compounds on TTR-induced cytotoxicity
in Neuro2A cells. The cell cytotoxicity assay shows that TTR-
induced cytotoxicity was inhibited by the compounds 2-12 that
stabilize the TTR tetramer (Figure 4c). Interestingly, these
inhibitors are not cytotoxic at the concentration tested and add
to the potential of these BPEs as inhibitors of fibril formation
and the consequent pathogenesis of the disease.
An analysis of TTR-ligand docking studies indicates that
the BPEs establish optimum interactions within the hormone
binding site (Figure 5). Binding of BPEs to TTR are dominated
by hydrophobic and electrostatic interactions with residues 15,
17, 108, 110, 117, 119, and 121. In spite of their common
structural features, considerable differences were observed in
the mode of binding to the T4 binding site. While the halogen
binding pockets in TTR provide primarily a hydrophobic
surface, conformational changes of its side chains facilitate
additional hydrogen bonding interactions. The ligand-induced
conformational changes of TTR not only allow energetically
favorable interactions between ligand and the protein but also
stabilize the nonamyloidogenic tetramer of TTR against pH-
mediated dissociation by the formation of inter-subunit hydrogen
bonds.
The dissociation constant for all these compounds at pH 4.4
correlates well with their efficacy. The strong negatively
cooperative binding of compounds 2, 3, 4, 6, and 10 suggests
that the binding of ligand to one site is sufficient to stabilize
the tetramer to prevent amyloidosis. Kds are low enough in case
of 7, 9, 11, and 12 to saturate both binding sites in TTR at 3.6
µM, i.e., equivalent to the physiological concentration (3.6 µM),
ensuring that there is enough inhibitor to saturate both the
binding sites. These biphenyl ether derivatives are better
inhibitors as compared to T4 at lower concentrations. The
inhibition of fibrillogenesis by compounds were further evalu-
ated by Congo red binding and Thioflavin-T fluorescence
(Figure 2a and 2c), as both bind specifically with protein fibers.
Significant decrease in the amount of Congo red binding in the
presence of inhibitors indicates absence of fibril formation
compared to the unligated TTR. Similarly, binding of Th-T to
TTR fibril and thus fluorescence was also decreased in the
presence of compounds 1-12, providing further support to our
explanation about the inhibition of TTR amyloidosis by these
compounds.
To understand the conformational states of the TTR in the
presence and absence of inhibitors, intrinsic tryptophan fluo-
rescence of the protein was monitored. Reduction in intensity
at pH 7.2 in the presence of compounds 1-12 indicates
interaction of these compounds leading to conformational change
in protein (Figure 3a). In contrast, drastic reduction in the
intensity was observed in TTR at pH 4.4 (Figure 3b). The
emission maxima of TTR under fibrilogenesis conditions (pH
4.4) showed 3 nm red shift (343 nm) compared to TTR at pH
7.2, where emission maxima was at 340 nm, signifying a subtle
conformational change at pH 4.4. In the presence of inhibitors,
however, decreased quenching of TTR fluorescence was
Besides, inhibiting the fibril formation, these compounds are
also able to prevent the elongation of small prefibrillar species

Biphenyl Ethers as Potential Inhibitors of TTR Amyloidosis
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5597
inhibitor ratio of 1:1 and 1:3, respectively, in 1% DMSO in a total
volume of 0.5 mL. The experiments were conducted at 25 °C. After
1 h, the samples were diluted with 0.5 mL of 200 mM sodium
acetate buffer pH 4.4 containing 100 mM KCl and 1 mM EDTA,
vortexed, and incubated at 37 °C for 72 h. In another assay,
inhibitors were buffered in the same pH 4.4 buffer, and TTR was
added directly to this solution and incubated at 37 °C for 72 h to
assess the affinity of these inhibitors for TTR under fibrillation
condition. The IC50 of inhibition of these inhibitors were studied
by incubating the TTR (7.2 µM) with 0.5-50 µM of inhibitors,
and the IC50 values were calculated graphically as provided in
Supporting Information. The stoichiometry of the binding of the
inhibitors was determined by HPLC as described by Green et al.,23
Dissociation constant of binding of these compounds to TTR was
determined by fluorescence titration, and data were fitted into the
Adair equation for two binding sites as given in Supporting
Information. All experiments were done in triplicate.
Time Course of Fibril Formation. The kinetics of inhibition
was studied by taking 7.2 µM TTR and 21.6 µM of inhibitors in a
96-microwell plate. The change in turbidity was monitored at 340
nm in the microplate reader using Magellan software. Details are
given in Supporting Information. All experiments were performed
three times in triplicates. Change in absorbance (mean of three
different experiments) at 340 nm was plotted against time in the
presence and absence of inhibitors. Initial rate of fibril formation
was calculated from the value of the slope by linear fitting of the
change in absorbance at 340 nm as a function of time.
Congo Red Binding. The amount of bound Congo red was
estimated as reported earlier53 using the equation, moles of Congo
red bound/L of amyloid suspension ) A540(nm)/25295 - A477(nm)/
46306.
Thioflavin-T Fluorescence. The thioflavin-T (ThT) binding
assays were performed in a Jobin Yvon Fluoromax spectrofluo-
rimeter using an excitation slit width of 2 nm and emission slit
width of 5 nm as reported earlier.55 In brief, samples were incubated
with 50 µM of Th-T for 15 min at 37 °C in the dark. The samples
were excited at 450 nm, and emissions were monitored at 480 nm.
The inner filter effect was corrected by using the following equation,
(Figure 6). Fibril formation is completed within 72-80 h in
case of TTR, and compounds 2-12 disrupt the fibers within
this time limit, signifying the presence of structural motifs for
the binding of these compounds.
Conclusion
The results presented above establish that the biphenyl ether
template provides the shape and size complementarity to the
TTR binding pocket, and carbonyl/carboxylic and chloride
groups at position R3 and R1, respectively, are necessary for
potentiating the interactions. The high potency of some of these
compounds compared to any of the inhibitors described so far
can be explained by their structural complementarity to the T4
binding region of TTR as well as the solubility and stability of
the complexes. Moreover, binding of compounds 2-12 has an
overall stabilizing effect on the TTR quaternary structure that
surpasses the ability of other inhibitors. These molecules are
able to prevent cytotoxicity induced by TTR in a neuronal cell
culture. Further, they inhibit the fibril elongation at any step of
the process as well as disrupt the preformed fibrils. The present
study also shows that compounds 11 and 12 could be promising
new structural templates for the design of potent inhibitors as
therapeutic agents against TTR amyloidosis.
All of the compounds studied here act by raising the energy
barrier for dissociation by stabilizing the tetrameric ground state
of TTR. The kinetic stabilization of the TTR tetramer is the
most feasible strategy, since the identity of the species of the
TTR amyloidogenesis pathway that induces toxicity still remains
unknown. Although binding of these compounds to any amy-
loidogenic TTR mutants has not been tested, there are reports
showing little or no structural changes in tetrameric conforma-
tion and T4 binding site in most of the TTR mutants. Hence, it
can be assumed that molecules, which bind to wild type TTR,
may also bind to these mutants. Therefore, a therapeutic strategy
based on the development and administration of small molecules
inhibitors could be explored for the prophylaxis of asymptomatic
gene carriers. TTR has been shown to play an important role
in keeping Aâ in soluble form.8 Hence, the increased stabiliza-
tion of TTR by these compounds might also prevent the
progression of other neurodegenerative disorders.
Fc ) F × antilog[(Aex + Aem)/2]
where Fc is the corrected fluorescence and F is the measured one,
Aex and Aem are the absorbances of the reaction solution at the
excitation and emission wavelengths, respectively. All experiments
were done in triplicate.
Intrinsic Fluorescence of TTR. For monitoring the intrinsic
fluorescence of TTR, 0.2 mL of 3.6 µM TTR solutions was
incubated with 10.8 µM concentration of a given inhibitor at pH
7.2 and 4.4. Samples were excited at 290 nm, and emission was
recorded between 310 and 370 nm. All the experiments were
performed at least three times and typically in duplicates.
Urea Denaturation. TTR (1.8 µM) was incubated with 5.4 µM
inhibitors at 37 °C for 30 min in 400 µL of 50 mM phosphate
buffer containing 1 mM EDTA, 1 mM DTT and 100 mM KCl.
After 30 min, 600 µL of 10 M urea stock in the same buffer was
added to bring the final concentration of urea to 6 M. Tryptophan
fluorescence was measured in the range of 310-370 nm up to 144
h as described above and corrected for inner filter effect. The results
are the mean of three different experiments done in duplicates
Glutaraldehyde Cross-Linking. The quaternary structural changes
of TTR at pH 4.4 in the presence and absence of all these inhibitors
was monitored by glutaraldehyde cross-linking and SDS-PAGE.56
The cross-linked samples were run on 12.5% SDS-PAGE. TTR
incubated at pH 7.5 was used as a control. The gels were stained
with Coomassie brilliant blue and analyzed by Bio-Rad GS-710
imaging densitometer.
Experimental Section
Preparation of Inhibitors. The chemical library was purchased
from Chemical Diversity Labs Inc, San Diego, CA. Synthesis of
substituted BPE has recently been reported by our group.52 Triclosan
was obtained from Kumar Chemicals, Bangalore, India. The
schemes used for the synthesis of the BPEs are provided in the
Supporting Information. Stock solutions (10 mM) of the compounds
used were prepared in DMSO. All compounds diluted from the
stock solution were soluble at 50-100 µm concentrations in
aqueous buffer used for assessing their inhibitory potencies in assays
to monitor TTR amyloidosis.
Expression and Purification of WT-TTR. TTR cloned in
pMMHa vector was a kind gift of Dr. P. Raghu from National
Institute of Nutrition, Hyderabad, India. Protein expression and
purification was performed as reported earlier.53 Protein concentra-
tions were measured by absorbane at 280 nm. Protein purity was
assessed by SDS-PAGE, and its mass was confirmed by electro-
spray ionization mass spectrometry (ESI-MS).
Stagnant Acid-Mediated TTR Aggregation Assay. The ef-
ficacy of compounds 1-12 was determined by stagnant acid-
mediated turbidity assay at 340 nm using Tecan GENios microplate
reader. Details of the assay are provided as Supporting Information.
The solution of TTR tetramer (7.2 µM; 0.4 mg/mL) in 5 mM
sodium phosphate, 100 mM KCl, 1 mM EDTA, pH 7, were
incubated with inhibitor at 7.2 and 21.6 µM, viz, TTR tetramer to
Mass Spectrometry. Aliquots of the samples of TTR kept for
assaying fibril formation in the absence (0 h sample) and presence
of compounds 1-12 at 3-15 days were centrifuged at 15 000 rpm
for 30 min and then filtered through 80 kDa cutt-off membranes.
Subsequently, 1 µL of the filtrate was mixed with 1 µL of matrix,

5598 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Gupta et al.
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structural quest for insights into amyloid and prion diseases. Structure
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3-(4-hydroxy-3,5-dimethoxy-phenyl)prop-2-enoic acid in ethanol
containing 0.5% trifluoroacetic acid and loaded on to the target
plate. Mass spectra were recorded using Brucker MALDI-TOF
instrument. TTR frozen at -20 °C, pH 7.4, was used as the positive
control.
Cell Culture and Cytotoxicity Inhibition Assay. The effect of
TTR and compounds 2-12 on adherent human neuro2a cell line
was monitored as described earlier.57 Experimental details are
provided in the Supporting Information. All the assays were carried
out twice in triplicates. The results were calculated as (% viability)/
(% cytotoxicity).
Molecular Docking. The MOE-2005 (Molecular Operating
Environment) software was used to perform docking. The inbuilt
algorithms were followed. Before docking, the coordinates of the
biphenyl ethers were energy minimized using the force field
MMFF94. Prior to docking studies all the water molecules have
been removed from TTR (PDB code 1e4h) structure. Molecular
docking with the TTR tetramer was done with each of the
mentioned compounds. From a cluster of 100 docked structures,
the one with the minimum energy was considered for further studies.
Further, we evaluated the interactions between TTR and the
inhibitors in each of the minimum energy structures using LPC-
CSU online software.58 We overlapped these docked structures of
compounds 1-12 with the crystal structure of T4 bound to human
TTR (PDB code 2ROX) to validate the accuracy of docking
experiments.
Fibril Disruption Assay. A 14.4 µM concentration of TTR was
incubated with 100 mM sodium acetate pH 4.4 containing 1 mM
EDTA, 0.1M KCl for 0, 1 3, 6, 12, 24, 48, and 72 h at 37 °C.
After indicated time points, the fibers (7.2 µM) were incubated
with the compounds 2-12 (14.4 µM) in PBS at 37 °C. The
disruption was followed for 7 days by turbidity measurements at
340 nm and Th-T fluorescence. A second dose of the inhibitor (7.2
µM) was added after 24 h of incubation, and samples were
incubated and monitored further for 5-6 days. After 15-20 days
of incubation, samples were examined by transmission electron
microscopy (TEM). All experiments were done thrice in triplicates
each time.
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Transmission Electron Microscopy. The samples were vortexed
and immediately absorbed to glow discharged carbon-coated 200
mesh copper grids. Negative staining was done by 3% uranyl
acetate. Details of the method used are given in the Supporting
Information. The grids were visualized with a FEI TECNAI G2 at
120 kV. The picture was captured using Mega View III camera
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Acknowledgment. Authors thank Dr. Ira Surolia for the
discussions. This work was supported by a grant from the
Department of Biotechnology (DBT), Govt. of India, to A.S.
A.S. holds a J. C. Bose Fellowship of Department of Science
and Technology, Govt. of India. Manmohan Chhibber is a
postdoctoral fellow of DBT.
(22) Green, N. S.; Foss, T. R.; Kelly, J. W. Genistein, a natural product
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haideen, N. N.; Purkey, H. E.; Nichols, C.; Chiang, K. P.; Walkup,
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Supporting Information Available: The experimental section,
schemes of biphenyl ethers synthesis, TEM showing different fibril
morphologies, mass spectra of TTR inhibitor complexes, inhibition
of fibril elongation and fibril disruption (turbidity assay). Tables
containing results of all the compounds tested and purity data of
synthesized compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
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