Enthalpy-Driven Stabilization of Transthyretin by AG10 Mimics a Naturally Occurring Genetic Variant That Protects from Transthyretin Amyloidosis

Article abstract of DOI:10.1021/acs.jmedchem.8b00817

Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a fatal disease with no available disease-modifying therapies. While pathogenic TTR mutations (TTRm) destabilize TTR tetramers, the T119M variant stabilizes TTRm and prevents disease. A comparison of potency for leading TTR stabilizers in clinic and structural features important for effective TTR stabilization is lacking. Here, we found that molecular interactions reflected in better binding enthalpy may be critical for development of TTR stabilizers with improved potency and selectivity. Our studies provide mechanistic insights into the unique binding mode of the TTR stabilizer, AG10, which could be attributed to mimicking the stabilizing T119M variant. Because of the lack of animal models for ATTR-CM, we developed an in vivo system in dogs which proved appropriate for assessing the pharmacokinetics-pharmacodynamics profile of TTR stabilizers. In addition to stabilizing TTR, we hypothesize that optimizing the binding enthalpy could have implications for designing therapeutic agents for other amyloid diseases.

Full text of DOI:10.1021/acs.jmedchem.8b00817

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Enthalpy-Driven Stabilization of Transthyretin by AG10 Mimics a
Naturally Occurring Genetic Variant That Protects from
Transthyretin Amyloidosis
Mark Miller,† Arindom Pal,† Wabel Albusairi,† Hyun Joo,† Beverly Pappas,† Md Tariqul Haque Tuhin,†
Dengpan Liang,† Raghavendra Jampala,† Fang Liu,† Jared Khan,† Marjon Faaij,† Miki Park,†
William Chan,† Isabella Graef,‡ Robert Zamboni,§ Neil Kumar,§ Jonathan Fox,§ Uma Sinha,*,§
and Mamoun Alhamadsheh*,†
†Department of Pharmaceutics & Medicinal Chemistry, University of the Pacific, Stockton, California 95211, United States
‡Department of Pathology, Stanford University, Stanford, California 94305, United States
§Eidos Therapeutics, Inc., San Francisco, California 94101, United States
S
* Supporting Information
ABSTRACT: Transthyretin (TTR) amyloid cardiomyopathy
(ATTR-CM) is a fatal disease with no available disease-
modifying therapies. While pathogenic TTR mutations
(TTRm) destabilize TTR tetramers, the T119M variant
stabilizes TTRm and prevents disease. A comparison of
potency for leading TTR stabilizers in clinic and structural
features important for effective TTR stabilization is lacking.
Here, we found that molecular interactions reflected in better
binding enthalpy may be critical for development of TTR
stabilizers with improved potency and selectivity. Our studies
provide mechanistic insights into the unique binding mode of
the TTR stabilizer, AG10, which could be attributed to
mimicking the stabilizing T119M variant. Because of the lack of
animal models for ATTR-CM, we developed an in vivo system in dogs which proved appropriate for assessing the
pharmacokinetics−pharmacodynamics profile of TTR stabilizers. In addition to stabilizing TTR, we hypothesize that optimizing
the binding enthalpy could have implications for designing therapeutic agents for other amyloid diseases.
The TTR tetramer features two largely unoccupied
thyroxine (T4)-binding sites that are formed between adjacent
monomers at the weaker dimer−dimer interface of TTR.
Tetramer dissociation into dimers, and then monomers, is the
initial and rate-limiting step in TTR amyloidogenesis.2 The
majority of TTR mutations increase the amyloidogenic
potential of TTR by lowering its thermodynamic stability
and/or decreasing the kinetic barrier for tetramer dissociation.8
While the V122I variant associated with ATTRm-CM is
kinetically destabilized, ATTR-PN is predominantly associated
with the thermodynamically destabilized V30M−TTR var-
iant.9,10
Two TTR variants, T119M and R104H, have been shown to
hyperstabilize heterotetramers composed of these variants and
either TTRwt or TTRm, preventing amyloidogenesis in
vitro.9,11 Individuals who are compound heterozygous for
both the T119M variant and the polyneuropathy associated
V30M−TTR mutation present a more benign evolution of
INTRODUCTION
■
Transthyretin (TTR) amyloidosis (ATTR) is a progressive,
fatal disease in which deposition of amyloid derived from
either mutant (TTRm) or wild-type (TTRwt) TTR causes
severe organ damage and dysfunction.1,2 Clinically, ATTR
presents as cardiomyopathy (ATTR-CM) or peripheral
polyneuropathy (ATTR-PN). ATTR-CM is an infiltrative,
restrictive cardiomyopathy characterized by progressive left
and right heart failure. Familial ATTR-CM (ATTRm-CM) is
driven by pathogenic, autosomal dominant, point mutations
resulting in amino acid substitutions that destabilize the native
TTR tetramer, prompting its dissociation.3,4 The most
prevalent mutation that causes ATTRm-CM is the V122I
variant, carried by 3.4% of African Americans, which increases
the risk of ATTR-CM several-fold in this population.5,6 In
addition, older individuals may develop ATTR derived from
wild-type TTR (ATTRwt-CM). The average life expectancy
for people with ATTR-CM is 3−5 years from diagnosis.7
Unfortunately, ATTR-CM represents one of the largest
genetically defined diseases with no approved disease-
modifying therapies.
Received: May 22, 2018
© XXXX American Chemical Society
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Table 1. Comparison of the Binding Affinity, Thermodynamics, and Potency of Stabilizers to TTR in Buffer and Human
Serum
ITC parameters (kcal/mol)
%TTR occupancy in buffer
%TTR occupancy in serum
%TTR stabilization in serum
a
a
a
stabilizer
Kd (nM)
ΔG
ΔH
TΔS
(1:1 ratio)
(2:1 ratio)
(2:1 ratio)
AG10
tafamidis
diflunisal
tolcapone
1
2
3
4
4.8 1.9
4.4 1.3
407 35
20.6 3.7
90 14
258 17
251 12
1253 79
−11.34
−11.39
−8.72
−10.5
−9.61
−8.99
−9.0
−13.6
−5.0
−2.26
6.39
0.34
0.4
−0.21
2.5
79.1 1.2
49.9 3.3
28.7 0.6
71.7 2.5
58.3 0.9
38.5 0.8
29.7 0.7
17.2 0.9
98.8 2.9
49 3.3
95.4 2.9
41.5 4.6
24.2 2.3
68.4 5.1
86.7 2.3
70.4 2.2
32.1 4.9
23.2 1.1
−8.38
−10.1
−9.82
−6.49
−4.73
−2.1
16.2 3.2
71.1 2.9
75.9 3.1
63.2 2.5
43 0.6
4.27
6.0
−8.1
20.8 3.3
a
Stabilizers to TTR ratio. %TTR occupancy in buffer and human serum was determined by FPE assay. %TTR stabilization in human serum was
determined by Western blot.
Figure 1. Binding affinities and potency of stabilizers for TTR in buffer. (a) Interaction of TTR with stabilizers assessed by ITC. Thermodynamic
data (summarized in Table 1); ΔG are blue bars, ΔH are green bars, and −TΔS are red bars. (b) Fluorescence change caused by modification of
TTR in buffer (2.5 μM) by FPE probe monitored in the presence of probe alone (control DMSO) or TTR stabilizers (2.5 μM; 1:1 stabilizers to
TTR ratio). (c) Bar graph representation of percent occupancy of TTR in buffer by stabilizers in the presence of FPE probe measured after 3 h of
incubation relative to probe alone. Error bars indicate SD (n = 3). The significance of the differences were measured by one-way ANOVA followed
by Tukey’s multiple comparison test (*p ≤ 0.05; ***p ≤ 0.001).
ATTR-PN or no disease compared to kindred carrying the
V30M−TTR mutation alone.12 In addition, carriage of the
T119M rescue mutation in the absence of other destabilizing
TTR mutations has been correlated with a decreased risk of
vascular disease and increased life expectancy, as compared to
the general (noncarrier) population, by 5−10 years.13 Similar
effects have been described for the R104H−TTR variant in
Japanese individuals expressing both R104H and V30M−
TTR.14 The trans-suppressor effects of T119M and R104H are
based on different mechanisms.11 While the T119M kinetically
stabilizes the quaternary structure of TTR, the R104H variant
is thermodynamically stabilized. Therefore, the stabilizing
B
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effect of the T119M mutation is much greater than that of
R104H, which only modestly protects against TTR dissocia-
tion and aggregation in vitro.
results are summarized in Table 1. The Kd for binding of a
stabilizer to TTR is represented by the change in Gibbs free
energy of binding (ΔG), where ΔG = ΔH − TΔS. By
analyzing the thermodynamic signature of each molecule, we
can assess the relative contributions of enthalpic (ΔH;
representing the formation or breaking of chemical bonds)
and entropic forces (ΔS; associated with the amount of
disorder in a system and frequently dominated and favored by
release of bound water molecule due to hydrophobic
interactions). Despite the similar binding affinities of AG10
and tafamidis to TTR in buffer (i.e., similar ΔG values), their
binding energetics to TTR are notably different. Whereas the
binding of AG10 (ΔH = −13.60 kcal/mol and TΔS = −2.26
kcal/mol) is enthalpically driven, tafamidis binding is
approximately 50% entropic and 50% enthalpic (ΔH =
−5.00 kcal/mol and TΔS = 6.39 kcal/mol) (Figure 1a and
Table 1). The binding of tolcapone (ΔH = −10.1 kcal/mol
and TΔS = 0.4 kcal/mol) and diflunisal (ΔH = −8.38 kcal/
mol and TΔS = 0.34 kcal/mol) is entropically favorable but
mainly driven by enthalpic interactions. The unfavorable
entropic binding energy of AG10 for TTR (TΔS = −2.26 kcal/
mol) could be due to its higher polarity and/or conformational
flexibility compared to other TTR stabilizers. The themody-
namics for the binding interactions between compounds 1−4
and TTR is discussed below.
Enthalpic Force Predicts Potency of TTR Stabilizers in
Buffer and Efficacy in Human Serum. A recent study with
diflunisal and other nonsteroidal anti-inflammatory drugs
(NSAIDs) found that ligands with favorable (i.e., larger
negative) ΔH had a proportionally higher TTR selectivity
compared to ligands with a lower influence of ΔH.16 While this
study described the correlation between enthalpic forces and
selectivity of TTR stabilizers, no correlation between binding
enthalpy of ligands and potency for stabilizing TTR or, to our
knowledge, any other multimeric proteins, has been reported
yet. To evaluate the potency of stabilizers in occupying and
stabilizing TTR in buffer, we used the fluorescence probe
exclusion (FPE) assay.18 The FPE assay uses a fluorogenic
probe (FPE probe, Supporting Information, Figure 1) that is
not fluorescent by itself, however, upon binding to the T4
binding site of TTR, it covalently modifies lysine 15 (K15),
creating a fluorescent conjugate. Ligands that bind to the T4
site of TTR will decrease FPE probe binding as observed by
lower fluorescence. A linear correlation has been reported
between the extent of fluorescence in the FPE assay and
stabilization of TTR.18 Therefore, we first used the FPE assay
to measure the potency of stabilizers for binding and stabilizing
TTR in buffer (tested at 1:1 ratio of stabilizer to TTR
tetramer; Figure 1b,c and Table 1). The order of potency of
the stabilizers for TTR in buffer was AG10 > tolcapone >
tafamidis > diflunisal.
Emerging therapies for ATTR-CM include RNA silencing
therapies and TTR stabilizers which are small molecules that
bind to the TTR T4 binding sites, thereby stabilizing TTR by
increasing the energy barrier of tetramer dissociation.3
Tafamidis and AG10 are two orally available TTR stabilizers
currently in clinical development for ATTR-CM (phase 3 and
phase 2, respectively). In addition, diflunisal and tolcapone are
two repurposed drugs that have been investigated in ATTR-
PN. We have shown earlier that the despite the similar binding
affinities (Kd) of AG10 and tafamidis to TTR (Kd = 4.8 1.9
and 4.4 1.3 nM, respectively), AG10 was more potent (in
stabilizing TTR in buffer) and more selective for binding and
stabilizing TTR in human serum.15 To account for structural
features important for effective TTR stabilization, we
performed a side-by-side comparison of the structural,
thermodynamic properties, and potency for all ATTR clinical
candidates against the crystal structures of stabilizing TTR
variants (T119M and R104H). We found that the Kd between
a stabilizer and TTR does not correlate well with either
potency or selectivity (Table 1). The mechanistic insights
gained from our studies suggest that optimizing the enthalpic
component of binding plays a crucial if not predominant role
in stabilizing TTR. This conclusion was also supported by the
synthesis and evaluation of the activity of four new AG10
analogues (compounds 1, 2, 3, and 4). Several previous studies
have shown that enthalpic forces correlated with selectivity of
ligands to target proteins including TTR.16,17 However, there
are no reports describing the role of enthalpy in enhancing the
potency of ligands in stabilizing multisubunit protein
complexes. Our data show that the high potency of AG10
could be attributed to its enthalpically driven binding,
mimicking the disease-suppressing properties of the
T119M−TTR variant in stabilizing TTR. The potency and
selectivity of AG10 for TTR was maintained when AG10 was
evaluated in beagle dogs in vivo. To the best of our knowledge,
this is the first report describing a correlation between
enthalpic binding of a ligand and enhanced potency in
stabilizing multiprotein complexes.
RESULTS
■
Determination of Binding Affinities and Thermody-
namics of Interactions Between Stabilizers and TTR. We
used isothermal titration calorimetry (ITC) to determine the
binding affinities (Kd) and the mechanisms underlying
molecular interactions of all TTR stabilizers in clinical
development (i.e., AG10, tafamidis, diflunisal, and tolcapone)
and AG10 analogues 1, 2, 3, and 4. Most of the reported TTR
ligands bind to the two identical T4 binding sites of TTR with
strong negative cooperativity, and therefore the binding of the
first ligand will dominate the total binding energy as well as the
stabilizing effect. While some differences in cooperativity can
be observed in the ITC thermograms, these differences will
have minor influences on the binding energy as well as the
stabilizing effect. Therefore, the Kd values reported in Table 1
were based on data fitted to an independent single-site binding
model.16 The binding affinities of AG10 and tafamidis to TTR
in buffer (Kd = 4.8 1.9 and 4.4 1.3 nM, respectively) were
4-fold higher than tolcapone (Kd = 20.6 3.7 nM) and ∼100-
fold higher than diflunisal (Kd = 407 35 nM). The Kd values
for compounds 1−4 ranged from 90 to 1250 nM, and the
We then employed the FPE and Western blot assays to
evaluate the efficacy (representing both potency and
selectivity) of stabilizers (10 μM) in occupying and stabilizing
TTR in human serum (TTR concentration 5 μM) (Table 1).
The Western blot assay measures the amount of intact TTR
tetramer after 72 h of acid treatment in the presence and
absence of stabilizers. The order of efficacy of the stabilizers in
human serum was similar to what we observed for the potency
with TTR in buffer (AG10 > tolcapone > tafamidis >
diflunisal; Table 1). The potency and efficacy of diflunisal was
the lowest which is predicted based on its significantly lower
binding affinity to TTR (Kd = 407 35 nM) compared to all
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Figure 2. Efficacy of stabilizers in occupying and stabilizing TTR in human serum. (a) Representative Western blot image for the stabilization of
TTR in human serum subjected to acid-mediated (pH 4.0) denaturation in the presence of AG10 (10 μM) and other stabilizers tested at their
estimated mean clinical Cmax at steady state when administered at the doses indicated: diflunisal (250 mg bid, 200 μM), tafamidis (80 mg qd, 20
μM), tolcapone (100 mg tid, 20 μM). (b) Bar graph representation of stabilization data obtained from Western blot experiments. Error bars
indicate SD (n = 3). (c) Fluorescence change caused by modification of TTR in human serum by FPE probe monitored in the presence of probe
alone (control DMSO), AG10 (10 μM), or TTR stabilizers (at their estimated mean clinical steady state Cmax). (d) Bar graph representation of
percent occupancy of TTR in human serum by stabilizers in the presence of FPE probe measured after 3 h of incubation relative to probe alone.
Error bars indicate SD (n = 4). The significance of the differences were measured by One-Way ANOVA followed by Tukey’s multiple comparison
test (n.s., not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).
other stabilizers (20−80-fold lower affinity than other
stabilizers). Surprisingly, there was no correlation between
the Kd values of the three other stabilizers and their potency
and efficacy in occupying and stabilizing TTR in both buffer
and human serum. For example, the potency and efficacy of
tolcapone was higher than that of tafamidis despite the fact
that the binding affinity of tolcapone to TTR (Kd = 20.6 3.7
nM) is slightly lower than the binding affinity of tafamidis to
TTR (Kd = 4.4 1.3 nM). Interestingly, both potency and
efficacy of these stabilizers for TTR in buffer and serum
correlated very well (R2 = 0.98) with their binding enthalpy
(ΔH = −13.6, −10.1, and −5.0 kcal/mol; for AG10, tolcapone,
and tafamidis, respectively). This data indicate that the
enthalpically driven binding of AG10 and tolcapone to TTR
(discussed in detail below) is the primary driver of their
efficient stabilization of TTR compared to other stabilizers.
We then used the Western blot assay to compare the efficacy
of AG10 (10 μM) to other stabilizers at their reported mean
maximum plasma concentrations in human (Cmax of 20 μM for
80 mg tafamidis qd, 200 μM for diflunisal 250 mg bid, and 20
μM for tolcapone 100 mg dose tid). AG10 at 10 μM
completely stabilized TTR in human serum (%TTR
Supporting Information, Figure 2) and tafamidis (pKa =
3.73) are higher than that for diflunisal (pKa = 2.94).
Therefore, the percentage ionization of the carboxylic acid
groups of stabilizers might vary at pH 4. This could affect the
strength of the electrostatic interaction between the carboxylic
acid groups and the ε-amino groups of lysine 15 (K15) and
K15′ at the top of the T4 binding sites which could affect the
potency of the stabilizer. To address this concern, we
performed the Western blot assay using urea buffer (pH
7.4).19 The TTR stabilization data of in urea buffer
(Supporting Information, Figure 3) is similar to the data
obtained from Western blot in acidic pH and from the FPE
assay at physiological pH. Consistent with the Western blot
TTR stabilization assay data, T4 binding site occupancy by 10
μM AG10 in the FPE assay was essentially complete (%TTR
occupancy 96.6 2.1%) and higher than all stabilizers at their
reported clinical Cmax. The target occupancy for tolcapone at
20 μM (%TTR occupancy 86 3.2%) was higher than those
of tafamidis and diflunisal (%TTR occupancy ∼65% at 20 and
200 μM, respectively) (Figure 2c,d).
Binding Interactions Between AG10 and S117/S117′
of TTR Mimic Molecular Interactions within the
Disease-Protective T119M Mutation. We investigated the
correlation between binding enthalpy and TTR stabilization by
comparing reported cocrystal structures of stabilizers with
stabilization: 95.4
4.8%); the other compounds stabilized
∼50−75% of tetrameric TTR at their reported clinical Cmax
(Figure 2a,b). The pKa values for AG10 (pKa = 4.13;
D
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Figure 3. Crystal structures highlighting similar interactions caused by the T119M mutation and binding of AG10 to TTR. (a) Quaternary
structure of AG10 bound to V122I−TTR (PDB 4HIQ)15 shown as a ribbon representation with monomers colored individually. Close-up views of
one of the two identical T4 binding sites with different colored ribbons for the two monomers of the tetramer composing the binding site. Key
hydrogen bonds between the pyrazole ring of AG10 and S117/117′ are highlighted by dashed lines. (b) Crystal structure of the stabilizing
T119M−TTR variant (PDB 1FHN)22 with dashed lines highlighting key interactions between the hydroxyl groups of S117 and S117′. (c) Crystal
structure of TTRwt (PDB 3CFM).26 (d) Crystal structure of thermodynamically stabilized R104H−TTR (PDB 1X7T).27
TTR against the crystal structures of stabilizing TTR variants
(T119M and R104H). We hypothesized that this could allow
us to identify functional groups of amino acids within the T4
binding sites of TTR which are important for binding and
stabilization of TTR. The carboxylic acid moieties of AG10,
tafamidis, diflunisal, and the hydroxyl group on tolcapone all
participate in electrostatic interactions with the ε-amino groups
of lysine 15 (K15) and K15′ at the top of the T4 binding
sites.15,20,21 The enthalpically driven binding of AG10 and
tolcapone to TTR is driven by additional hydrogen bonds that
both molecules form within the T4 binding site. The carbonyl
group of tolcapone forms one hydrogen bond with hydroxyl
side chain of T119 of TTRwt (distance ∼2.6 Å; ideal distance
for a hydrogen bond is <3 Å). The longer distance between the
carbonyl group of tolcapone and hydroxyl side chain of T119′
on the adjacent monomer (distance ∼7.6 Å) preclude the
formation of a second hydrogen bond.20 Interestingly, this
interaction is weaker between tolcapone and V122I−TTR
(distance between the carbonyl group of tolcapone and
hydroxyl side chains of T119 and T119′ of V122I−TTR are
∼5.5 and ∼9.6 Å, respectively), which could explain the lower
binding affinity (Kd = 56 nM) and potency of tolcapone
toward V122I−TTR compared to TTRwt.20 In the case of
tafamidis, there is no hydrogen bonding at the base of the T4
pocket; instead, the chlorine atoms of the 3,5-dichloro ring are
also placed into halogen binding pocket (HBP) 3 and 3′,
where they interact with TTR through predominantly
hydrophobic interactions. In addition to electrostatic inter-
actions between the carboxylic acid moiety of AG10 and K15/
K15′, AG10 also forms two hydrogen bonds with the hydroxyl
side chain serine 117 (S117) and S117′ (distance ∼2.8 Å) of
adjacent monomers in the low dielectric macromolecular
interior of the T4 binding site (Figure 3a). These additional
hydrogen bonds are likely to be responsible for the driving
force for the dominant enthalpic binding of AG10 to TTR.
Remarkably, similar hydrogen bonds have been reported
within the inner cavity of the kinetically stabilizing trans-
suppressor T119M−TTR variant (Figure 3b).22,23
The two S117 side chain hydroxyl groups of monomers A
and B in T119M variant TTR form direct hydrogen bonds
with a distance of 2.8 Å, which are not observed in TTRwt
(distance between the two S117 residues ∼6.0 Å) (Figure 3c).
These unique hydrogen bonds lead to closer contacts between
the two dimers (∼4.8 Å) within the TTR tetramer and
highlight the potential importance of these hydrogen bonds in
the antiamyloidosis and disease-protective effects of the
T119M variant on the TTR tetramers. The role of S117 in
stabilizing TTR has been also suggested by the binding of
flavonoids that are capable of forming a single hydrogen bond
with one S117.24,25 Interestingly, the distance between the
S117 and S117′ residues in the thermodynamically stabilized
R104H variant, which does not involve kinetic stabilization of
the tetrameric TTR, is similar to that of TTRwt (average
dimerdimer distance is ∼5.6 Å, Figure 3c,d).26,27 The lack of
hydrogen bonding between the hydroxyl groups of S117 and
S117′ in the R104H variant (which is a less potent trans-
suppressor mutant than T119M) highlights the importance of
these hydrogen bonds in the antiamyloidogenic and disease
suppressing effects of kinetically stabilizing the TTR tetramer
in the T119M variant. By forming two direct hydrogen bonds
with S117 and S117′ in the TTR tetramer, AG10 creates a
similar electrostatic bridge as is found in the protective T119M
variant. This data is supported by the analysis of 40 reported
crystal structures which highlighted the closer dimerdimer
contacts in the crystal structures of both T119M−TTR
(distance ∼4.8 Å) and AG10V122I−TTR (distance ∼4.66
Å) compared to TTRwt or TTRm (distance ∼5.5 Å)
(Supporting Information, Table 1). It is important to note
that other known TTR stabilizers do not interact with S117/
S117′ of TTR.
E
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a
Scheme 1. Synthesis of AG10 Analogues 1, 2, 3, and 4
a
(a) 5a, (i) acetylacetone, DBU, benzene, rt, 3 days, (ii) hydrazine hydrate, ethanol, 90 °C, 4 h, (iii) NaOH, MeOH/water, 50 °C, 14 h; (b) 5b, (i)
acetylacetone, DBU, benzene, rt, 3 days, (ii) hydrazine hydrate, ethanol, 90 °C, 4 h; (c) (i) NaH, MeI, DMF, rt, 12 h, (ii) NaOH, MeOH/water, 50
°C, 14 h; (d) 5b, (i) 3,5-heptanedione, DBU, benzene, rt, 3 days, (ii) hydrazine hydrate, ethanol, 90 °C, 4 h; (e) NaOH, MeOH/water, 50 °C, 14
h.
Characterization of Key Functional Groups of AG10
Important for TTR Stabilization. To investigate the
enthalpic contribution of each of the functional groups of
AG10 on TTR binding and stabilization, we synthesized and
tested four AG10 analogues (compounds 1, 2, 3, and 4;
Scheme 1) and evaluated their ability to bind and stabilize
TTR (Figure 4). AG10 binds TTR with unfavorable entropy
(TΔS = −2.26 kcal/mol). The fluorine atom of AG10 is placed
into HBP1 of TTR, and therefore we hypothesized that the
entropic binding of AG10 to TTR could be optimized by
replacing the fluorine atom of AG10 with an iodine
(compound 1). Modeling studies suggest the iodine of 1 fits
in HBP1 of TTR (where the iodine of T4 binds) which could
improve the entropic binding by displacing more water
molecules from HBP1 (Figure 4a). Compound 1 displayed
significantly lower binding affinity (Kd = 90 14 nM) to TTR
We synthesized a methyl-ester analogue of AG10 (compound
2, Figure 4a) to test the effect of modifying the two salt bridges
that AG10 forms at the periphery of the T4-binding site.
Compound 2 displayed significantly lower affinity (Kd = 258
17 nM) to TTR in buffer compared to AG10 (Kd = 4.8 1.9
nM), which could be explained by the lower strength of
potential hydrogen bonds between the ester group of 2 and
K15/K15′ (ΔH = −6.49 kcal/mol) compared to the salt
bridge in AG10 (Figure 4b). Compound 2 also displayed
reduced potency for TTR in buffer and human serum
compared to AG10 and compound 1 (Figure 4c−e and
Table 1).
The 3,5-dimethyl-1H-pyrazole ring of AG10 sits deep within
the inner cavity of the T4-binding site and forms two hydrogen
bonds with the S117 and S117′ of adjacent subunits.15 By
blocking these interactions, we can effectively observe their
enthalpic contribution using ITC and the FPE assay,
respectively. Therefore, we synthesized compound 3 which
has an N-methyl pyrazole. The N-methyl group would restrict
the pyrazole ring of 3 to form only one hydrogen bond with
one of the adjacent TTR subunits (Figure 4a). We also
synthesized compound 4 where the dimethyl pyrazole of AG10
was replaced with diethyl pyrazole. Modeling studies suggested
that the bulk of the diethyl groups would prevent the
molecules for reaching deep in the T4-binding site, thereby
decreasing its ability to potentially form any hydrogen bonds
with S117/S117′ (Figure 4a). As predicted by modeling, both
3 (Kd = 251 12 nM) and 4 (Kd = 1253 79 nM) showed
greatly reduced binding affinity to TTR in buffer. This reduced
affinity was translated into a significant decrease in potency for
TTR in buffer and human serum, especially for compound 4.
The order of potency for stabilizing TTR was similar in both
buffer and serum (1 > 2 > 3 > 4; Figure 4c−e and Table 1). As
in buffer compared to AG10 (Kd = 4.8
1.9 nM). ITC
analysis showed that while the entropic interaction of 1 with
TTR was more favorable compared to AG10 (TΔS = −0.21
and −2.26 kcal/mol, respectively), there was a significant drop
in the enthalpic contribution to the binding (ΔH = −9.82 and
−13.6 kcal/mol, respectively) (Figure 4b). As suggested by
modeling, the decrease in binding enthalpy could be explained
by the decrease in strength of the salt bridge between the
carboxylic acid moiety of 1 and K15/K15′ (distance ∼4.7 Å
compared to ∼2.8 Å for AG10). Compound 1 also displayed
reduced potency for TTR in buffer (58.3 0.98%) and human
serum (75.9 3.1%) compared to AG10 (Figure 4c−e and
Table 1).
The carboxylic acid moiety of AG10 forms two salt bridges
directly with the ε-amino groups of K15 and K15′ at the
periphery of the T4-binding site, which serves to close the T4
pocket around AG10 and partially shield it from the solvent.
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Figure 4. Hydrogen bonds between the pyrazole ring of AG10 and S117/S117′ of TTR are important for effective binding to TTR. (a) Chemical
structures and in silico docking study of synthesized AG10 analogues 1, 2, 3, and 4. Co-crystal structure of AG10 bound to TTR used for the
docking experiment. 1 is the iodo-analogue of AG10. 2 is the methyl-ester form of AG10 that cannot form salt bridge with K15/15′. 3 is the
methyl-pyrazole form of AG10 that can potentially form only one hydrogen bond with either K15 or K15′. 4 is the diethyl-pyrazole analogue of
AG10 which affects both hydrogen bonds with S117/S117′. (b) Interaction of TTR with analogues assessed by ITC. Thermodynamic data; ΔG are
blue bars, ΔH are green bars, and −TΔS are red bars. (c) Fluorescence change caused by modification of TTR in buffer (2.5 μM) by FPE probe
monitored in the presence of probe alone (control DMSO) or TTR stabilizers (2.5 μM; 1:1 stabilizers to TTR ratio). (d) Bar graph representation
of percent occupancy of TTR in buffer by stabilizers in the presence of FPE probe measured after 3 h of incubation relative to probe alone. Error
bars indicate SD (n = 3). (e) Bar graph representation of Western blot data for the stabilization of TTR in human serum by analogues (10 μM; 2:1
stabilizers to TTR ratio). Error bars indicate SD (n = 4). The significance of the differences were measured by one-way ANOVA followed by
Tukey’s multiple comparison test (n.s., not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).
we observed with the clinical ATTR stabilizers, the potency of
AG10 and compounds 1, 2, 3, and 4 in occupying and
stabilizing TTR correlated very well (R2 = 0.98) with the
binding enthalpy of these molecules (ΔH = −13.6, −9.82,
−6.49, −4.73, and −2.1 kcal/mol, respectively). Interestingly,
despite the similar binding affinities of 2 and 3, their potency
was significantly different (Table 1). The higher potency of 2
compared to 3 could be explained by its favorable enthalpic
binding (ΔH = −6.49 and −4.73 kcal/mol, respectively)
(Figure 4b). This observation is similar to the data obtained
for AG10 and tafamidis (i.e., similar Kd values but significantly
different potency) (Table 1). These results highlight the
crucial role played by the pyrazole ring and the importance of
the hydrogen bonds it forms with the two TTR dimers,
mimicking the interactions in the protective T119M−TTR
mutation and enhancing the kinetic stability of the TTR
tetramer.
Examining the Effect of Enthalpy on the Selectivity
of AG10 to TTR. To examine the role of enthalpy on the
selectivity of AG10 for TTR over other abundant serum
proteins, we tested the concentration−effect relationship of
AG10 and tafamidis in the FPE assay in whole human serum
(Supporting Information, Figure 4a,b). We tested AG10 and
tafamidis because their binding affinities for TTR in buffer is
very similar (Kd = 4.8 1.9 and 4.4 1.3 nM, respectively)
but their thermodynamics for binding TTR, especially the
enthalpy component, is significantly different. Therefore, the
data obtained in serum would largely reflect selectivity. AG10
displayed a progressive concentration-dependent occupancy,
with complete occupancy achieved at AG10 concentrations
≥10 μM. Even at substoichiometric concentrations, AG10 was
able to occupy and stabilize the majority of TTR (at 5 μM,
TTR occupancy of 69.2% by FPE, 74.5% stabilization by
Western blot). In contrast, there was a more modest increment
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Figure 5. AG10 has high selectivity for binding TTR over albumin or other abundant human serum proteins. (a) Gel filtration and dialysis assay
comparing AG10 and tafamidis (each at 30 μM) incubated with purified human serum albumin (600 μM). The concentration of tafamidis bound
to albumin after gel filtration (i.e., dialysis time 0 h) was normalized to 100%. Error bars indicate SD (n = 3). (b) 24 h time-course for dialysis of
AG10 (10 μM) incubated with purified human TTR (5 μM). Error bars indicate SD (n = 3). (c) Fluorescence change due to modification of
purified human TTR (5 μM) by FPE probe monitored for 6 h in the presence of probe alone (black circles), probe plus albumin (600 μM) (black
triangles), probe plus all [fibrinogen (5 μM), albumin (600 μM), IgG (70 μM), transferrin (25 μM)] (gray triangles), probe and AG10 (10 μM)
(red squares) or probe and AG10 plus albumin (green diamonds), and probe and AG10 plus all [fibrinogen (5 μM), albumin (600 μM) IgG (70
μM), transferrin (25 μM)] (blue circles). (d) %TTR occupancy in buffer by AG10 in the presence of FPE probe and other serum proteins
measured after 3 h of incubation relative to probe alone. (e,f) Same experiment described for AG10 was performed for tafamidis. Error bars indicate
SD (n = 3). The significance of the differences were measured by One-Way ANOVA followed by Tukey’s multiple comparison test (n.s., not
significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).
in either occupancy or stabilizing activity of tafamidis at
concentrations above 20 μM (Supporting Information, Figure
4c,d). A good correlation (R2 = 1.0) between TTR occupancy
(by FPE) and TTR stabilization (by Western blot) was
observed when the activity of AG10 was evaluated (Supporting
Information, Figure 5a,b). For tafamidis, there was a good
correlation (R2 = 0.87) for concentrations up to 10 μM,
however, at higher concentrations, there was a plateau in the
FPE assay (Supporting Information, Figure 5c,d).
The selectivity of AG10 and tafamidis for TTR was further
investigated by repeating these assays in buffer in the presence
or absence of purified serum proteins. AG10 or tafamidis (30
μM) were preincubated with purified human serum albumin
(at its physiological concentration of 600 μM) and then
subjected to gel filtration followed by dialysis. At time 0
(immediately after gel filtration), less AG10 was bound to
albumin compared to tafamidis (18.3 0.98 vs 24.1 1.1 μM;
Figure 5a). Following dialysis vs buffer for 24 h, the
concentration of AG10 bound to HSA was lower than that
for tafamidis (7.8 0.1 vs 18.8 2.1 μM). These data indicate
that AG10 has a lower binding affinity for albumin compared
to tafamidis. In parallel, the binding of AG10 to TTR was also
investigated in this gel filtration/dialysis assay. AG10 (10 μM)
was preincubated with an equimolar ratio TTR (5 μM of
tetrameric TTR, representing 10 μM of TTR T4 binding
sites). The dissociation of AG10 from TTR was slow for the
first six hours (AG10−TTR molar ratio of ∼1.2:1) and
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Figure 6. Activity of AG10 and tafamidis in the FPE and Western blot assays performed with pooled dog serum. (a) Fluorescence change caused by
modification of dog TTR in commercially available beagle dog serum by FPE probe monitored in the presence of probe alone (control DMSO,
black circles), AG10 (10 μM) or tafamidis (10 μM). (b) Percent occupancy of dog TTR in dog serum by AG10 and tafamidis in the presence of
FPE probe measured after 3 h of incubation relative to probe alone. Error bars indicate SD (n = 4). (c) Western blot image for the stabilization of
TTR in pooled dog serum against acid-mediated denaturation in the presence of AG10 (10 μM) and tafamidis (10 μM). Serum samples were
incubated with DMSO or test compounds in acetate buffer (pH 4.0) for the desired time period (0 and 72 h) before cross-linking and
immunoblotting. (d) Bar graph representation of stabilization data obtained from Western blot experiments. Error bars indicate SD (n = 3). The
significance of the differences were measured by One-Way ANOVA followed by Tukey’s multiple comparison test (n.s., not significant; *p ≤ 0.05;
**p ≤ 0.01; ***p ≤ 0.001)
maintained a 1:1 molar ratio over a 24 h incubation (Figure
5b).
activity of TTR stabilizers in occupying and stabilizing TTR is
commonly assessed ex vivo in blood samples obtained from
patients before and after dosing of the stabilizer. To explore
the in vivo activity of AG10, this same approach was used in
the healthy beagle dog. The healthy beagle dog was chosen as
an experimental model for several reasons. All amino acids in
the T4 binding sites of TTR, where AG10 and other stabilizers
bind, are conserved between dog and human.28 We also tested
the concentration of TTR in dog serum (∼4.6 μM) and found
it similar to that of healthy humans (Supporting Inforemation,
Figure 6).
To confirm the suitability of assays used with human-based
reagents for dog studies, the activity of AG10 and tafamidis
was evaluated in pooled dog serum using the same FPE and
Western blot assays used for the experiments described above.
The in vitro TTR binding and stabilization concentration−
effect relationships of AG10 and tafamidis in both assays
repeated using dog serum was similar to those observed in
human serum (Figure 6). These features made the healthy
canine a suitable system for subsequent investigations.
AG10 Potently and Selectively Binds to Canine TTR
Following Oral Administration. To explore the pharmaco-
kinetic−pharmacodynamic (PK−PD) relationship in vivo,
AG10 was administered to healthy beagle dogs daily by oral
gavage for 7 days. A total of 16 male (M) and 16 female (F)
beagle dogs made up four treatment groups: (i) 6M/6F at 0
mg/kg/d (vehicle control), (ii) 2M/2F at 50 mg/kg/d, (iii)
2M/2F at 100 mg/kg/d, and (iv) 6M/6F at 200 mg/kg/d.
Finally, the selectivity of AG10 and tafamidis for binding to
TTR in human serum was evaluated using a modified FPE
assay where human serum was replaced with purified human
TTR in buffer (PBS buffer, pH 7.4). In addition to purified
TTR (5 μM), four separate representative and abundant
plasma proteins were added to the FPE assay in buffer.
Addition of albumin, transferrin, fibrinogen, or immunoglobu-
lin (IgG) did not influence TTR occupancy by AG10 (>97%
TTR occupancy in the absence or presence of any of these
proteins, Figure 5c,d). Albumin, but not the other serum
proteins tested, interfered with TTR occupancy by tafamidis
(41.5 0.9% vs 68.2 0.1% in the absence of albumin; Figure
5e,f). Adding all of the tested plasma proteins simultaneously
yielded identical results for AG10. The higher selectivity of
AG10 for TTR could be attributed to a number of properties,
including the enthalpic binding and greater hydrophilicity of
AG10 (ClogP = 2.78) compared to the more lipophilic
tafamidis (ClogP = 4.2).
Healthy Beagle Dog is a Suitable Experimental
Model for Evaluating the Efficacy of TTR Stabilizers.
We then investigated if the high potency and selectivity of
AG10 for TTR can be maintained in vivo. Transgenic animal
models that faithfully reproduce the pathology of human
ATTR-CM are not yet available. Therefore, we took an
approach similar to that currently used in the clinic to examine
the efficacy of AG10 vs other TTR kinetic stabilizers. The
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Figure 7. Orally administered AG10 is effective in binding and stabilizing TTR in dogs. (a,b) Occupancy of TTR in beagle dogs after oral
■
●
administration (qd for 7 days) of escalating doses of AG10. Circles ( ) indicate predose day 1, squares ( ) indicate predose day 7 (AG10
▲
concentration at Cmin), and triangles ( ) indicate postdose day 7 (AG10 concentration at Cmax). Four groups of animals were dosed: (i) 0 mg/kg
(n = 12, 6 males/6 females), (ii) 50 mg/kg (n = 4, 2 males/2 females), (iii) 100 mg/kg (n = 4, 2 males/2 females), and (iv) 200 mg/kg (n = 12, 6
males/6 females). (b) Bar graph representing TTR occupancy at 3 h. Error bars indicate SD (n = 3). (c,d) Pharmacokinetic−pharmacodynamic
(PK−PD) analysis of AG10 in dogs receiving a single oral dose of AG10 at (c) 5 mg/kg and (d) 20 mg/kg. Scatterplot of concentration [AG10] vs
%TTR occupancy of serum samples obtained from dogs at various time points (n = 4, 2 males/2 females per dosing group). Error bars indicate SD
(n = 3). The significance of the differences were measured by One-Way ANOVA followed by Tukey’s multiple comparison test (n.s., not
significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).
Timed serum samples were collected predose on study day 1
(baseline), predose on study day 7 (representing trough
concentrations, or Cmin, at steady state), and at 1 h postdose on
study day 7 (representing peak concentrations, or Cmax, at
steady state). Binding occupancy of TTR by AG10 was
assessed by FPE assay (Figure 7a,b). All samples from dogs
treated with vehicle alone, and those collected from the active
treatment arms prior to exposure to AG10, showed zero TTR
occupancy. Serum from AG10-treated dogs displayed a dose-
proportional response in binding occupancy at the steady state
trough (day 7 predose; ∼81−94% TTR occupancy), and all
AG10 treated groups showed complete (>97%) TTR
occupancy at steady state Cmax (day 7 postdose). Lower
doses of AG10 were subsequently tested to further explore the
PK−PD (exposure−effect) relationship in order to identify a
minimally effective dose of AG10 that might still effectively
bind to and stabilize TTR. Eight dogs divided into two active
treatment groups received either 5 or 20 mg/kg AG10 as a
single oral dose. These results showed enhanced TTR
occupancy in the 20 vs 5 mg/kg dose groups (Figure 7c,d,
and Supporting Information, Figure 7). The %TTR occupancy
at Cmax was significantly higher (p ≤ 0.001) than at Cmin for
both doses. There was significantly higher (p ≤ 0.001) TTR
occupancy for the 20 mg/kg dose compared to the 5 mg/kg
dose at Cmin. The data also showed that circulating plasma
concentration of AG10 correlates well with %TTR occupancy.
been correlated with the stability of protein association
complexes.30,31 However, the effect of the enthalpic
component of binding of a small molecule on its potency in
stabilizing multimeric proteins has not been reported. To
account for structural features important for effective TTR
stabilization, we compared the TTR binding and stabilizing
activity of four ATTR clinical candidates and four AG10
analogues. We found that the Kd between a stabilizer and TTR
does not by itself correlate well with either potency or
selectivity (Table 1). Our results suggest that enthalpic forces,
in combination with Kd measurements in a purified protein, are
better predictors of both potency and selectivity for stabilizing
TTR in serum.
The trans-suppressor T119M−TTR is a superstabilizing
variant compared to TTRwt (reported T119M−TTR dis-
sociation rate is 40-fold slower than that of TTRwt).9 This
variant effectively ameliorates disease progression and
symptoms in compound heterozygous individuals carrying
the pathogenic, destabilizing V30M mutation.12 The T119M
substitution induces conformational changes that promote
hydrogen bonding between the hydroxyl groups of adjacent
S117 residues of T119M−TTR monomers and lead to closer
contacts between the two dimers within the tetramer.22 These
enthalpically favorable interactions and their consequences are
neither observed in TTRwt nor in the thermodynamically
stabilized, disease-protective R104H variant, suggesting that
they are important for the enhanced kinetic stabilization of the
TTR tetramer. Our studies show that AG10s enthalpically
driven mode of action is through kinetic stabilization of TTR,
which is similar to the kinetically stabilizing T119M variant
and not comparable to the thermodynamically stabilizing
DISCUSSION
■
Optimizing the binding enthalpy of small molecules has been
associated with the development of second-generation drugs
with improved selectivity.16,17,29 Enthalpic forces have also
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R104H. When comparing the T119M mutation to the
cocrystal structure of AG10 bound to the kinetically
destabilized V122I−TTR, the similarities of these critical
intramolecular interactions are evident and we conclude that
AG10s mode of binding, especially the two hydrogen bonds
with S117/S117′ that are buried in the low dielectric
macromolecular interior of the T4 pocket, may well explain
AG10s efficient binding and stabilizing of tetrameric TTR. The
experiments comparing closely related, structural analogues of
AG10 highlight the important role of the pyrazole ring of
AG10 in enthalpically driving the binding by forming two
hydrogen bonds with different subunits of TTR. These unique
interactions with the S117/S117′ residues at the bottom of the
T4 binding pocket of TTR are not observed in other
stabilizers. To the best of our knowledge, this is the first
report describing a correlation between enthalpic binding of a
ligand and enhanced potency in stabilizing multiprotein
complexes.
Similar to T119M−TTR, enthalpic forces have been
reported to play a dominant role in stabilizing the eye lens
crystallin proteins. Analysis of the association energetics of β-
crystallins (which associate into dimers, tetramers, and higher
order oligomers) showed that β-crystallins which associate by
predominately hydrophobic forces participate in a weaker
protein associations whereas the formation of stable tetramers
is dominated by enthalpically driven interactions between the
subunits (mediated by hydrogen bonds, salt bridges, and van
der Waals interactions).32 Hereditary cataracts are caused by
mutations that destabilize the crystallin proteins, leading to the
assembly of crystallins into amyloid-like fibers. For example,
the R120G mutation in αB-crystallin disrupts ionic interactions
that normally stabilize the αB-crystallin dimer (wild-type αB-
crystallin dimer is stabilized by a salt bridge between R120 and
D109).33 These studies with TTR (where the introduction of
an enthalpic interaction in the T119M variant hyperstabilize
the tetramer) and crystallins (where disrupting an enthalpic
interaction in the R120G variant destabilizes the dimer)
suggest that enthalpic forces controlling protein association
could be similar to forces influencing effective protein
stabilization by small molecules such as AG10. This hypothesis
would be further supported by additional site-directed
mutagenesis and structural studies to investigate if factors
other than enthalpy are also involved in the enhanced
stabilization of TTR.
exposed portion of the T4 pocket. There are limited examples
where the binding of a small molecule to a known disease-
causing target mimics a stabilizing mutation identified in that
target protein.34 In addition to stabilizing TTR, we hypothesize
that molecules with similar enthalpically driven binding mode
to that of AG10 could be useful in stabilizing multisubunit
protein complexes in certain diseases such as crystallins in
cataract and α-synuclein tetramer in Parkinson’s disease.35,36
EXPERIMENTAL SECTION
■
Experimental Animals. All experiments with male and female
beagle dogs were conducted in accordance with National Institutes of
Health guidelines for the care and use of live animals and were
approved by the Institutional Animal Care and Use Committee of
Covance and PreClinical Research Services.
Isothermal Titration Calorimetry (ITC). Binding experiments
were performed using MicroCal PEAQ-ITC at 25 °C. A solution of
ligand (25 μM in PBS pH 7.4, 100 mM KCl, 1 mM EDTA, 2.5%
DMSO) was prepared and titrated into an ITC cell containing 2 μM
of TTR in an identical buffer. Nineteen injections of ligand (2.0 μL
each) were injected into the ITC cell (at 25 °C) to the point that
TTR was fully saturated with ligand. Calorimetric data were plotted
and fitted using the standard single-site binding model. For control,
we tested the enthalpy change caused by titrating bank DMSO in
buffer into TTR and the resulting binding enthalpy was <0.4 kcal/
mol. We also used ITC to titrate tafamidis and AG10 against human
serum albumin (HSA). The Kd value for tafamidis (2.3 μM) was
similar to what has been reported earlier (Kd = 2.5 μM; EMA
assesment report EMA/729083/2011). The binding affinity of AG10
was calculated around 8 μM, which also fits with our data in Figure 5
(where AG10 has lower binding to albumin compared to tafamidis).
FPE Assay for Binding TTR in Buffer and Human or Dog
Serum. The binding affinity and selectivity of AG10 and other
stabilizers to TTR in buffer and serum was determined by their ability
to compete with the binding of a fluorescent probe exclusion (FPE
probe) binding to TTR in buffer and human serum.18 The FPE probe
is a thioester TTR ligand (Supporting Information, Figure 1) that is
not fluorescent by itself, however, upon binding to the T4 binding site
of TTR, it covalently modifies lysine 15 (K15), creating a fluorescent
conjugate. Ligands that bind to the T4 site of TTR will decrease FPE
probe binding as observed by lower fluorescence. The FPE assay was
also adapted for use with dog serum. FPE with TTR in buffer: An
aliquot of 98 μL of TTR in PBS (pH 7.4, final concentration 2.5 μM)
was mixed with 1 μL of test compounds (2.5 μM) and 1 μL of FPE
probe (0.18 mM stock solution in DMSO; final concentration 1.8
μM). The change in fluorescence (λex = 328 nm and λem = 384 nm)
were monitored using a microplate spectrophotometer reader
(SpectraMax M5) for 6 h at rt. FPE with TTR in human and dog
serum: An aliquot of 98 μL of pooled human serum (prepared from
human male AB plasma, Sigma; catalogue no. H4522; TTR
concentration 5 μM) or dog serum (Innovative Research, catalogue
no. IBG-SER; TTR concentration 4.6 μM) was mixed with 1 μL of
test compounds [all compounds were prepared as 10 mM stock
solutions in DMSO and diluted accordingly with DMSO (final
concentrations in serum were AG10 10 μM; diflunisal 200 μM;
tafamidis 20 μM; tolcapone 20 μM)] and 1 μL of FPE probe (0.36
mM stock solution in DMSO; final concentration 3.6 μM). In the
case of dog serum (after oral treatment with AG10), 1 μL of FPE
probe and 1 μL of DMSO were added to each well and mixed with 98
μL of the appropriate dog serum sample. The change in fluorescence
(λex = 328 nm and λem = 384 nm) were monitored using a microplate
spectrophotometer reader (SpectraMax M5) for 6 h at rt.
CONCLUSIONS
■
In summary, we have developed a preclinical in vivo approach
to evaluate the efficacy of TTR stabilizers. Beagle dogs
demonstrated that AG10 is orally available and achieves dose-
dependent plasma concentrations that potently and selectively
bind and stabilize tetrameric TTR. The similarity between dog
and human TTR and the expected similarity in PK behavior of
many small molecules between dog and human, suggest that
these data can serve as a guide for selecting a dosing regimen
for stabilizers to attain safe and effective TTR binding and
stabilization in ATTR-CM patients. By analyzing the molecular
interactions that stabilizers form with TTR and comparing it to
interactions within stabilizing TTR mutations, we suggest that
electrostatic interactions formed deep in the T4 site of TTR
(which could be similar to the hydrogen bonds between S117/
117′ of T119M−TTR and between the pyrazole of AG10 and
TTR) are more effective in stabilizing TTR compared to
hydrophobic interaction and interactions at the solvent
Stability Studies of TTR in Serum by Immunoblotting.
Western blotting was performed as reported earlier.15,37 All
compounds were prepared as 10 mM stock solutions in DMSO and
diluted accordingly with DMSO (final concentrations in serum were:
AG10 10 μM, diflunisal 200 μM, tafamidis 20 μM, tolcapone 20 μM).
Then 2 μL of each compound were added to 98 μL of human serum
(TTR concentration 5 μM). The samples were incubated at 37 °C for
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2 h, and then 10 μL of the samples were diluted 1:10 with
acidification buffer (pH 4.0, 100 mM sodium acetate, 100 mM KCl, 1
mM EDTA, 1 mM DTT). The Western blot assay was also performed
in urea buffer (pH 7.4) as reported earlier.19 The samples were
incubated at room temperature for 72 h, cross-linked with
glutaraldehyde (final concentration of 2.5%) for 5 min, and then
quenched with 10 μL of 7% sodium borohydride solution in 0.1 M
NaOH. All samples were denatured by adding 100 μL of SDS gel
loading buffer and boiled for 5 min. Then 10 μL of each sample was
separated in 12% SDS-PAGE gel and analyzed by immunoblotting
using anti-TTR antiserum (DAKO A0002, 1:10000 dilution for
human serum and 1:2000 for dog serum). The combined intensity of
TTR bands (TTR tetramer and tetramer bound to RBP) was
quantified by using an Odyssey IR imaging system (LI-COR
Bioscience) and reported as percentage of TTR tetramer relative to
TTR tetramer density of DMSO control at 0 h (considered 100%
stabilization) and 72 h (ranges between 10% and 35% TTR
remaining). The percentage tetramer stabilization is calculated as
100 × [(tetramer and tetramer + RBP density, 72 h)/(tetramer and
tetramer + RBP density of DMSO, 0 h)].
In Silico Structural and Modeling Studies. The analyses of the
crystal structures of TTR were carried out on four TTR crystal
structures obtained from the RCSB PDB site. Biological assemblies of
TTR tetramers were constructed using the X-ray crystallographic unit
cell information given in the pdb files. When multiple models were
suggested, the first choice model was used. The initial geometries of
the AG10 and its four derivatives (1, 2, 3, and 4) built with Molden38
were used, and geometry optimizations were carried out at the hybrid
density functional B3LYP level with 6-311+G(d) basis set using the
Gaussian’09 program package (Wallingford, CT, USA; Gaussian, Inc.,
2009). The Frequency calculations on the optimized geometries were
carried out to ensure they have no imaginary frequencies. Dock 6
program was used for the docking experiments. The crystal structure
of the V122I mutant TTR complex with AG10 (PDB 4HIQ)15 was
used as the receptor. Tetrameric TTR was built using the
crystallographic data, solvent and other heteroatoms were removed,
and one large docking grid was selected including the T4 binding
sites. For all the docking experiments, the same receptor and the grid
were used. The flexible ligand docking was carried out to allow the
rotation around the torsion angles. UCSF Chimera package was used
in visualization and analyses of the 3D structures.39
mL of assay buffer and stirred at room temperature. Samples from the
dialysis buffer were taken at different time points (0, 0.5, 1, 2, 6, and
24 h). After 24 h, the samples were removed from dialysis cassette and
the volume was measured and results normalized. The concentration
of TTR and AG10 obtained from the assay buffer were determined
using NanoDrop and LCMS, respectively.
Selectivity of AG10 and Tafamidis to TTR Compared to
Other Serum Proteins. The FPE assay was modified and performed
with purified human TTRwt (5 μM). Other serum proteins were
added either individually or in combination [fibrinogen (5 μM),
albumin (600 μM), IgG (70 μM), transferrin (25 μM)] to the TTR
and FPE mixture, and the fluorescence was monitored for 6 h as
described above. The percentage of FPE probe binding to TTR in the
presence of serum proteins measured after 3 h of incubation was used
to calculate % TTR occupancy.
Seven-Day Repeat Oral Dosing of AG10 to Dogs. Sixteen
male (M) and 16 female (F) beagle dogs, separated into four
treatment groups and orally dosed by gavage with vehicle (6M/6F at
0 mg/kg) or AG10 in 0.5% methylcellulose formulation (2M/2F at 50
mg/kg, 2M/2F at 100 mg/kg, and 6M/6F at 200 mg/kg) for a total of
32 dogs. Blood (approximately 1.5 mL) was collected from a jugular
vein into serum separator tubes on study day 1 (predose D1), predose
study day 7 (predose D7), and at 1 h postdose study day 7 (postdose
D7). These serum samples were analyzed for their TTR occupancy
using the FPE assay described above.
Single Oral Doses of AG10 to Dogs to Determine an
Exposure−Effect (PK−PD) Relationship with Respect to
Binding to and Stabilization of TTR. Four male and four female
beagle dogs, separated into two treatment groups (n = 2/sex/group)
for a total of eight dogs were evaluated to acquire simultaneous
pharmacokinetic (PK) and pharmacodynamic (PD) data for AG10
binding to and stabilization of TTR. Each animal received a single oral
gavage (PO) of AG10 at a single dose of either 5 or 20 mg/kg in 0.5%
methylcellulose. Blood was collected and analyzed predose and at 2, 4,
6, 8, 12, and 24 h postdose. The concentration of AG10 in these
serum samples was analyzed by LCMS and the TTR occupancy by
the FPE assay.
Statistical Analysis. All results are expressed as mean SD. All
statistical analysis was performed with GraphPad PRISM software.
The significance of the differences were measured by One-Way
ANOVA followed by Tukey’s multiple comparison test (n.s., not
significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).
Binding of AG10 and Tafamidis to Human Serum Albumin.
Test compounds (AG10 or tafamidis; both at 30 μM) were incubated
with human serum albumin (HSA, 600 μM; albumin from human
serum; Sigma-Aldrich, catalogue no. A3782) in assay buffer (10 mM
sodium phosphate, 100 mM KCl, and 1 mM EDTA, pH 7.6) for 1 h
at 37 °C. Then 500 μL of a solution of HSA and AG10 or tafamidis
mixture in assay buffer was subjected to gel filtration on PD Minitrap
G25 columns (GE Life Sciences, catalogue no. 45-001-529) by gravity
and the fractions containing HSA were identified by NanoDrop. The
concentration of HSA (i.e., concentration at time zero) was also
determined using NanoDrop (based on calibration curves of known
HSA concentrations). HSA concentration was 351 μM for the
tafamidis sample and 345 μM for AG10 sample. The concentration of
test compounds in these fractions (i.e., concn at time zero) was
evaluated using HPLC (based on calibration curves of known
concentration of test compounds). Then 500 μL of each HSA/test
compound samples was then added to a Slide-A-Lyzer dialysis cassette
G2 (3.5K MWCO, Thermo Scientific, catalogue no. PI87722). The
dialysis cassettes were placed in 100 mL of assay buffer and stirred at
room temperature. After 24 h, the samples were removed from
dialysis cassette and the volume was measured. The concentration of
HSA and test compounds were determined using NanoDrop and
HPLC as described above.
Chemistry: General. All reactions were carried out under argon
atmosphere using dry solvents under anhydrous conditions unless
otherwise noted. The solvents used were ACS grade from Fisher.
Reagents were purchased from Aldrich and Fisher and used without
further purification. Reactions were monitored by thin-layer
chromatography (TLC) carried out on 0.20 mm POLYGRAM SIL
silica gel plates (Art.-Nr. 805 023) with fluorescent indicator UV254
using UV light as a visualizing agent. Normal phase flash column
chromatography was carried out using Davisil silica gel (100−200
1
mesh, Fisher). H NMR and 13C NMR spectra were recorded on a
Jeol JNM-ECA600 spectrometer and calibrated using residual
undeuterated solvent as an internal reference. Coupling constants
(J) were expressed in hertz. The following abbreviations were used to
explain the multiplicities: s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet. High-resolution mass spectra (HRMS) were
recorded by JEOL Direct Analysis in Real Time (DART) AccuTOF.
HPLC analysis was performed on Agilent 1100 series HPLC system
connected to a diode array detector operating between the UV ranges
of 200−400 nm and quantified using Agilent Chemstation software.
The HPLC analysis was performed on both Waters XBridge C18
column with L1 packing (4.6 mm × 250 mm, 5 μm) and
SymmetricTM C4 (2.1 mm × 150 mm, 5 μm) at ambient
temperature upon injection of a 50 μL of each blank buffer, standard,
and/or sample to obtain the chromatogram. The mobile phase was
composed of solvent A consisting methanol−water (5:95, v/v)
containing 0.1% formic acid and solvent B consisting methanol−water
(95:5, v/v) containing 0.1% formic acid. The HPLC program was a
gradient separation method increasing linearly from 0% to 100%
Dialysis of AG10:TTR Complex. AG10 (10 μM) was incubated
with human wild-type TTR (5 μM, purified from human plasma;
Sigma-Aldrich, catalogue no. P1742) in assay buffer (10 mM sodium
phosphate, 100 mM KCl, and 1 mM EDTA, pH 7.6) for 1 h at 37 °C.
Then 500 μL of each AG10/TTR solution was then added to a Slide-
A-Lyzer dialysis cassette G2. The dialysis cassettes were placed in 100
L
DOI: 10.1021/acs.jmedchem.8b00817
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
solvent B from 0 to 20 min and then maintained 100% solvent B up to
30 min.
raphy (silica gel, 1−5% MeOH/EtOAc) to afford compound 3 (11
mg, 52% yield for two steps); (97.8% purity by HPLC) tR (column)
(C18) = 25.25 min; tR (C4) = 15.71 min. H NMR (CD3OD, 600
MHz) δ 7.58−7.51 (m, 2H), 7.10−7.06 (m, 1H), 3.92 (t, 2H, J = 6.0
Hz), 3.56 (s, 3H), 2.49 (t, 2H, J = 7.2 Hz), 2.05 (s, 3H), 2.01 (s, 3H),
1.83−1.88 (m, 2H). 13C NMR (CD3OD, 600 MHz) δ 168.1, 154.6,
146.8, 145.3, 137.2, 128.1, 122.8, 115.5, 115.4, 114.8, 67.4, 34.3, 29.4,
18.8, 10.1, 7.9. HRMS (DART) m/z: calcd for C16H19FN2O3 + H+
307.1458, found 307.1449 (M + H+).
1
Key Compounds Purity. HPLC analysis was performed on both
C18 and C4 reversed-phase columns. The purity for all key
compounds was >95%. Description of the purity analysis has been
included in the Experimental Section. Detailed HPLC information of
key compounds (traces, retention times, and %purity) are included in
the Supporting Information.
Synthetic Procedures. AG10 and tafamidis were synthesized as
reported earlier.15 Tolcapone and diflunisal were purchased from
Fisher. All AG10 analogues were prepared as described below.
3-(3-(3,5-Dimethyl-1H-pyrazol-4-yl)propoxy)-4-iodobenzoic Acid
(1). A solution of methyl 3-(3-bromopropoxy)-4-Iodobenzoate (5a)15
(834 mg, 2.1 mmol, 1 equiv) in benzene (3 mL) was added dropwise
to a solution of acetyl acetone (0.43 mL, 4.2 mmol, 2 equiv) and DBU
(0.627 mL, 4.2 mmol, 2 equiv) in benzene (7 mL). The reaction
mixture was stirred at room temperature for 3 days. The mixture was
filtered and concentrated. To a solution of this intermediate in
ethanol (5 mL) was added hydrazine hydrate (0.28 mL, 5.25 mmol,
2.5 equiv), and the reaction was heated under reflux for 4 h. The
reaction was concentrated and purified by flash column chromatog-
raphy (silica gel, 1−20% MeOH/CH2Cl2) to afford the methyl ester
of compound 1; sodium hydroxide (79 mg, 1.98 mmol, 2 equiv) in
water (2.5 mL) was added to a solution of ester intermediate (412
mg, 0.99 mmol) in methanol (10 mL), and the reaction was heated
under reflux for 4 h (50 °C). The reaction was concentrated and
purified by flash column chromatography (silica gel, 1−5% MeOH/
EtOAc) to afford compound 1 (183 mg, 22% yield for three steps);
(98.3% purity by HPLC) tR (column) (C18) = 25.72 min; tR (C4) =
16.06 min. 1H NMR (CD3OD, 600 MHz) δ 7.86 (d, 1H, J = 8.4 Hz),
7.41 (d, 1H, J = 1.2 Hz), 7.34 (dd, 1H, J = 1.2 and 8.4 Hz), 4.0 (t, 2H,
J = 6.0 Hz), 2.67 (t, 2H, J = 7.2 Hz), 2.13 (s, 6H), 1.97−1.93 (m,
2H). 13C NMR (CD3OD, 600 MHz) δ 168.5, 157.6, 142, 139.2,
133.2, 123.1, 114, 117.8, 91.7, 67.5, 29.6, 18.7, 9.3. HRMS (DART)
m/z: calcd for C15H17IN2O3 + H+ 401.0362; found 401.0347 (M +
H+).
3-(3-(3,5-Diethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzoic Acid
(4). Sodium hydroxide (3.2 mg, 0.08 mmol, 2 equiv) in water (0.5
mL) was added to a solution of 6 (13 mg, 0.04 mmol, 1 equiv) in
methanol (2 mL), and the reaction was heated under reflux for 4 h
(50 °C). The reaction was concentrated and purified by flash column
chromatography (silica gel, 1−5% MeOH/EtOAc) to afford
compound 4 (10 mg, 80% yield); (96.0% purity by HPLC) tR
(column) (C18) = 25.16 min; tR (C4) = 15.56 min. 1H NMR
(CD3OD, 600 MHz) δ 7.57−7.49 (m, 2H), 7.08−7.04 (m, 1H), 3.94
(t, 2H, J = 6.0 Hz), 2.51−2.43 (m, 6H), 1.87−1.82 (m, 2H), 1.06 (t,
6H, J = 7.8 Hz). 13C NMR (CD3OD, 600 MHz) δ 169.8, 157.9,
156.3, 149.3, 148.5, 124.6, 117.2, 117.1, 114.1, 69.4, 31.8, 20.1, 19.9,
14.7. HRMS (DART) m/z: calcd for C17H21FN2O3 + H+ 321.1614,
found 321.1601 (M + H+).
Methyl 3-(3-Bromopropoxy)-4-fluorobenzoate (5). Compound 5
was synthesized as reported earlier.15 To a solution of methyl 4-
fluoro-3hydroxybenzoate (1.0 g, 5.87 mmol, 1 equiv) and 1,3-
dibromopropane (3.0 mL, 29.4 mmol, 5 equiv) in DMF (15 mL) was
added K2CO3 (0.98 g, 7.1 mmol, 1.2 equiv). The reaction mixture was
stirred at room temperature for 16 h. The mixture was diluted with
EtOAc (500 mL), washed with brine (3 × 200 mL), and dried with
Na2SO4. The solution was filtered and concentrated. The residue was
purified by flash column chromatography (silica gel, 1−10% EtOAc/
hexanes) to afford compound 5 (1.3 g, 76% yield). 1H NMR
(CD3OD, 600 MHz) δ 7.67−7.61 (m, 2H), 7.14−7.07 (m, 1H), 4.21
(t, 2H, J = 5.89 Hz), 3.89 (s, 3H), 3.62 (t, 2H, J = 6.38 Hz), 2.38−
2.31 (m, 2H). ESI+ m/z: calcd for C11H12BrFO3 + H+ 290.00, found
290.01 (M + H+).
Methyl 3-(3-(3,5-Dimethyl-1H-pyrazol-4-yl)propoxy)-4-fluoro-
benzoate (2). A solution of methyl 3-(3-bromopropoxy)-4-
fluorobenzoate (5b)15 (780 mg, 2.69 mmol, 1 equiv) in benzene (3
mL) was added dropwise to a solution of acetyl acetone (0.552 mL,
5.38 mmol, 2 equiv) and DBU (0.804 mL, 5.38 mmol, 2 equiv) in
benzene (7 mL). The reaction mixture was stirred at room
temperature for 3 days. The mixture was filtered and concentrated.
The residue was purified by flash column chromatography (silica gel,
1−10% EtOAc/hexanes) to afford the alkylated intermediate which
was used in the next step directly. To a solution of this intermediate in
ethanol (5 mL) was added hydrazine hydrate (0.36 mL, 6.73 mmol,
2.5 equiv), and the reaction was heated under reflux for 4 h. The
reaction was concentrated and purified by flash column chromatog-
raphy (silica gel, 1−20% MeOH/CH2Cl2) to afford compound 2
(288 mg, 35% yield); (96.3% purity by HPLC) tR (column) (C18) =
Methyl 3-(3-(3,5-Diethyl-1H-pyrazol-4-yl)propoxy)-4-fluoroben-
zoate (6). A solution of 5b (100 mg, 0.35 mmol, 1 equiv) in
benzene (2 mL) was added dropwise to a solution of 3,5-
heptanedione (0.095 mL, 0.7 mmol, 2 equiv) and DBU (0.104 mL,
0.7 mmol, 2 equiv) in benzene (5 mL). The reaction mixture was
stirred at room temperature for 3 days. The mixture was filtered and
concentrated. The residue was purified by flash column chromatog-
raphy (silica gel, 1−10% EtOAc/hexanes) to afford the alkylated
intermediate which was used in the next step directly. Hydrazine
hydrate (0.047 mL, 0.875 mmol, 2.5 equiv) was added to the
alkylated intermediate in ethanol (4 mL), and the reaction was heated
under reflux for 4 h. The reaction was concentrated and purified by
flash column chromatography (silica gel, 1−5% MeOH/EtOAc) to
afford compound 6 (75 mg, 65% yield for two steps). 1H NMR
(CD3OD, 600 MHz) δ 7.59−7.54 (m, 2H), 7.15−7.11 (m, 1H), 3.98
(t, 2H, J = 6.0 Hz), 3.81 (s, 3H), 2.56−2.47 (m, 6H), 1.91−1.86 (m,
2H), 1.13 (t, 6H, J = 7.8 Hz). 13C NMR (CD3OD, 600 MHz) δ
167.9, 156.6, 156.2, 148.8, 148.7, 124.4, 117.5, 117.3, 116.9, 113.9,
69.5, 53.1, 31.8, 20.1, 14.7. HRMS (DART) m/z: calcd for
C18H23FN2O3 + H+ 335.1771, found 335.1773 (M + H+).
1
25.11 min; tR (C4) = 14.03 min. H NMR (CD3OD, 600 MHz) δ
7.63−7.58 (m, 2H), 7.19−7.15 (m, 1H), 4.00 (t, 2H, J = 6.0 Hz),
3.86 (s, 3H), 2.58 (t, 2H, J = 7.2 Hz), 2.12 (s, 6H), 1.97−1.92 (m,
2H). 13C NMR (CD3OD, 600 MHz) δ 168.1, 158.4, 156.7, 148.9,
128.5, 124.6, 117.6, 117.0, 115.6, 69.4, 53.3, 31.1, 20.2, 10.9. HRMS
(DART) m/z: calcd for C16H19FN2O3 + H+ 307.1458, found
307.1463 (M + H+).
4-Fluoro-3-(3-(1,3,5-trimethyl-1H-pyrazol-4-yl)propoxy)benzoic
Acid (3). A solution of 2 (21 mg, 0.07 mmol, 1 equiv) in DMF (3 mL)
was added sodium hydride (5 mg, 0.21 mmol, 3 equiv) and methyl
iodide (17 μL, 0.28 mmol, 4 equiv). The reaction mixture was stirred
at room temperature for 2 h. The mixture was extracted with brine,
filtered, and concentrated. The residue was purified by flash column
chromatography (silica gel, 0.5−2% MeOH/EtOAc) to afford the
alkylated intermediate which was used in the next step directly.
Sodium hydroxide (5.6 mg, 0.14 mmol, 2 equiv) in water (0.5 mL)
was added to a solution of alkylated intermediate in methanol (2 mL),
and the reaction was heated under reflux for 4 h (50 °C). The
reaction was concentrated and purified by flash column chromatog-
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.8b00817.
O−O distances between Ser117 residues in the
tetrameric TTR complexes; chemical structure, 1H
NMR, and HRMS mass spectrometry data for FPE
probe; Yasuda−Shedlovsky extrapolation curve for pKa
calculation of AG10; efficacy of stabilizers in stabilizing
M
DOI: 10.1021/acs.jmedchem.8b00817
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(5) Buxbaum, J. N.; Ruberg, F. L. Transthyretin V122I (pV142I)*
cardiac amyloidosis: an age-dependent autosomal dominant cardio-
myopathy too common to be overlooked as a cause of significant
heart disease in elderly African Americans. Genet. Med. 2017, 19,
733−742.
TTR in human serum in urea buffer; concentration−
effect relationship for TTR occupancy by AG10 and
tafamidis in whole human serum; correlation between
TTR occupancy and TTR stabilization for AG10 and
tafamidis; determination of TTR concentration in dog
serum; data used to generate the PK−PD scatterplot in
Figure 7c,d; HPLC analysis of AG10, tafamidis,
diflunisal, tolcapone, and AG10 analogues 1, 2, 3, and
4 (PDF)
(6) Alexander, K. M.; Falk, R. H. V122I TTR cardiac amyloidosis in
patients of African descent: recognizing a missed disease or the dog
that didn’t bark? Circ.: Heart Failure 2016, 9, e003489.
(7) Connors, L. H.; Doros, G.; Sam, F.; Badiee, A.; Seldin, D. C.;
Skinner, M. Clinical features and survival in senile systemic
amyloidosis: comparison to familial transthyretin cardiomyopathy.
Amyloid 2011, 18, 157−159.
Molecular formula strings of key compounds (CSV)
(8) Connors, L. H.; Lim, A.; Prokaeva, T.; Roskens, V. A.; Costello,
C. E. Tabulation of human transthyretin (TTR) variants, 2003.
Amyloid 2003, 10, 160−184.
AUTHOR INFORMATION
Corresponding Authors
*For M.A.: E-mail, malhamadsheh@pacific.edu. Phone: +1-
209-946-3164.
■
̈
(9) Hammarstrom, P.; Jiang, X.; Hurshman, A. R.; Powers, E. T.;
Kelly, J. W. Sequence-dependent denaturation energetics: a major
determinant in amyloid disease diversity. Proc. Natl. Acad. Sci. U. S. A.
2002, 99, 16427−16432.
*For U.S.: E-mail, usinha@eidostx.com. Phone: +1-415-887-
1471.
́
(10) Adams, D.; Theaudin, M.; Cauquil, C.; Algalarrondo, V.; Slama,
ORCID
M. FAP neuropathy and emerging treatments. Curr. Neurol. Neurosci.
Rep. 2014, 14, 435.
Mamoun Alhamadsheh: 0000-0003-4325-7044
(11) Sekijima, Y.; Dendle, M. T.; Wiseman, R. L.; White, J. T.;
D’Haeze, W.; Kelly, J. W. R104H may suppress transthyretin
amyloidogenesis by thermodynamic stabilization, but not by the
kinetic mechanism characterizing T119 interallelic trans-suppression.
Amyloid 2006, 13, 57−66.
Author Contributions
M.M., A.P., and W.B. contributed equally, and U.S. and M.A.
are considered senior authors.
Notes
The authors declare the following competing financial
interest(s): Authors M.A. and I.G. are cofounders of Eidos
Therapeutics. R.Z, N.K., J.F., and U.S. are employees of Eidos
Therapeutics. The remaining authors declare noncompeting
financial interests.
(12) Coelho, T.; Chorao, R.; Sousa, R.; Alves, I.; Torres, M. F.;
̃
Saraiva, M. Compound heterozygotes of transthyretin Met30 and
transthyretin Met119 are protected from the devastating effects of
familial amyloid polyneuropathy. Neuromuscul Disord 1996, 6, S20.
(13) Hornstrup, L. S.; Frikke-Schmidt, R.; Nordestgaard, B. G.;
Tybjærg-Hansen, A. Genetic stabilization of transthyretin, cerebro-
vascular disease, and life expectancy. Arterioscler., Thromb., Vasc. Biol.
2013, 33, 1441−1447.
(14) Terazaki, H.; Ando, Y.; Misumi, S.; Nakamura, M.; Ando, E.;
Matsunaga, N.; Shoji, S.; Okuyama, M.; Ideta, H.; Nakagawa, K.;
Ishizaki, T.; Ando, M.; Saraiva, M. J. A novel compound heterozygote
(FAP ATTR Arg104His/ATTR Val30Met) with high serum
transthyretin (TTR) and retinol binding protein (RBP) levels.
Biochem. Biophys. Res. Commun. 1999, 264, 365−370.
ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Health
grant 1R15GM110677-01 (M.A.) and Eidos Therapeutics. The
support by a National Science Foundation Instrumentation
grant (NSF-MRI-0722654) is gratefully acknowledged. Thanks
to Vyacheslav Samoshin and Andreas Franz for NMR and mass
spectrometric analysis.
(15) Penchala, S. C.; Connelly, S.; Wang, Y.; Park, M. S.; Zhao, L.;
Baranczak, A.; Rappley, I.; Vogel, H.; Liedtke, M.; Witteles, R. M.;
Powers, E. T.; Reixach, N.; Chan, W. K.; Wilson, I. A.; Kelly, J. W.;
Graef, I. A.; Alhamadsheh, M. M. AG10 inhibits amyloidogenesis and
cellular toxicity of the familial amyloid cardiomyopathy-associated
V122I transthyretin. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 9992−
9997.
ABBREVIATIONS USED
■
TTR, transthyretin; ATTR, transthyretin amyloidosis; ATTR-
CM, transthyretin amyloid cardiomyopathy; TTRm, trans-
thyretin mutations; TTRwt, wild-type transthyretin; ATTRm-
CM, familial transthyretin amyloid cardiomyopathy; ATTRwt-
CM, wild-type transthyretin amyloid cardiomyopathy; ATTR-
PN, transthyretin peripheral polyneuropathy; T4, thyroxine;
ITC, isothermal titration calorimetry; NSAIDs, nonsteroidal
anti-inflammatory drugs; FPE assay, fluorescence probe
exclusion assay; PK-PD, pharmacokinetic−pharmacodynamic
̈
̈
(16) Iakovleva, I.; Brannstrom, K.; Nilsson, L.; Gharibyan, A. L.;
Begum, A.; Anan, I.; Walfridsson, M.; Sauer-Eriksson, A. E.; Olofsson,
A. Enthalpic forces correlate with the selectivity of transthyretin-
stabilizing ligands in human plasma. J. Med. Chem. 2015, 58, 6507−
6515.
(17) Freire, E. Do enthalpy and entropy distinguish first in class
from best in class? Drug Discovery Today 2008, 13, 869−874.
(18) Choi, S.; Kelly, J. W. A competition assay to identify
amyloidogenesis inhibitors by monitoring the fluorescence emitted
by the covalent attachment of a stilbene derivative to transthyretin.
Bioorg. Med. Chem. 2011, 19, 1505−1514.
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DOI: 10.1021/acs.jmedchem.8b00817
J. Med. Chem. XXXX, XXX, XXX−XXX