A substructure combination strategy to create potent and selective transthyretin kinetic stabilizers that prevent amyloidogenesis and cytotoxicity

Article abstract of DOI:10.1021/ja908562q

Transthyretin aggregation-associated proteotoxicity appears to cause several human amyloid diseases. Rate-limiting tetramer dissociation and monomer misfolding of transthyretin (TTR) occur before its aggregation into cross-β-sheet amyloid fibrils. Small molecule binding to and preferential stabilization of the tetrameric state of TTR over the dissociative transition state raises the kinetic barrier for dissociation, imposing kinetic stabilization on TTR and preventing aggregation. This is an effective strategy to halt neurodegeneration associated with polyneuropathy, according to recent placebo-controlled clinical trial results. In three recent papers, we systematically ranked possibilities for the three substructures composing a typical TTR kinetic stabilizer, using fibril inhibition potency and plasma TTR binding selectivity data. Herein, we have successfully employed a substructure combination strategy to use these data to develop potent and selective TTR kinetic stabilizers that rescue cells from the cytotoxic effects of TTR amyloidogenesis. Of the 92 stilbene and dihydrostilbene analogues synthesized, nearly all potently inhibit TTR fibril formation. Seventeen of these exhibit a binding stoichiometry of >1.5 of a maximum of 2 to plasma TTR, while displaying minimal binding to the thyroid hormone receptor (<20%). Six analogues were definitively categorized as kinetic stabilizers by evaluating dissociation time-courses. High-resolution TTR · (kinetic stabilizer)₂ crystal structures (1.31-1.70 A) confirmed the anticipated binding orientation of the 3,5-dibromo-4-hydroxyphenyl substructure and revealed a strong preference of the isosteric 3,5-dibromo-4-aminophenyl substructure to bind to the inner thyroxine binding pocket of TTR.

Full text of DOI:10.1021/ja908562q

Published on Web 12/31/2009
A Substructure Combination Strategy To Create Potent and
Selective Transthyretin Kinetic Stabilizers That Prevent
Amyloidogenesis and Cytotoxicity
Sungwook Choi,† Nata`lia Reixach,‡ Stephen Connelly,§ Steven M. Johnson,†
Ian A. Wilson,§ and Jeffery W. Kelly*,†,‡
Departments of Chemistry, Molecular and Experimental Medicine, and Molecular Biology, and
The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey
Pines Road, La Jolla, California 92037
Received October 7, 2009; E-mail: jkelly@scripps.edu.
Abstract: Transthyretin aggregation-associated proteotoxicity appears to cause several human amyloid
diseases. Rate-limiting tetramer dissociation and monomer misfolding of transthyretin (TTR) occur before
its aggregation into cross-ꢀ-sheet amyloid fibrils. Small molecule binding to and preferential stabilization of
the tetrameric state of TTR over the dissociative transition state raises the kinetic barrier for dissociation,
imposing kinetic stabilization on TTR and preventing aggregation. This is an effective strategy to halt
neurodegeneration associated with polyneuropathy, according to recent placebo-controlled clinical trial
results. In three recent papers, we systematically ranked possibilities for the three substructures composing
a typical TTR kinetic stabilizer, using fibril inhibition potency and plasma TTR binding selectivity data. Herein,
we have successfully employed a substructure combination strategy to use these data to develop potent
and selective TTR kinetic stabilizers that rescue cells from the cytotoxic effects of TTR amyloidogenesis.
Of the 92 stilbene and dihydrostilbene analogues synthesized, nearly all potently inhibit TTR fibril formation.
Seventeen of these exhibit a binding stoichiometry of >1.5 of a maximum of 2 to plasma TTR, while
displaying minimal binding to the thyroid hormone receptor (<20%). Six analogues were definitively
categorized as kinetic stabilizers by evaluating dissociation time-courses. High-resolution TTR · (kinetic
stabilizer)2 crystal structures (1.31-1.70 Å) confirmed the anticipated binding orientation of the 3,5-dibromo-
4-hydroxyphenyl substructure and revealed a strong preference of the isosteric 3,5-dibromo-4-aminophenyl
substructure to bind to the inner thyroxine binding pocket of TTR.
Introduction
after which these diseases have been named, leading to the
degeneration of one or more tissues, often those composed of
Intrinsic and/or extrinsic challenges to the maintenance of
organismal protein homeostasis, in the absence of a biological
correction (e.g., induction of a stress-responsive signaling
pathway) or a chemical correction (a small molecule that binds
to and stabilizes a particular misfolding-prone protein) to
rebalance the proteostasis network, can lead to aging-associated
proteotoxicity and degenerative diseases.1-5 These include
Alzheimer’s disease, as well as the transthyretin and gelsolin
amyloidoses.3,5–10 These maladies are associated with the
accumulation of an insoluble protein(s), including amyloid fibrils
postmitotic cells, such as neurons or muscle cells.3,6,10 Whether
intra- or extracellular aggregates lead to degeneration, which
aggregate morphology is responsible, and by what mechanism
are key unanswered questions related to the amyloidoses.3–11
Transthyretin (TTR) is a homotetrameric protein composed
of 127-amino-acid, ꢀ-sheet-rich subunits.12-15 The established
physiological functions of TTR are to bind to and transport the
thyroid hormone thyroxine (T4) and holo-retinol binding protein
(6) Page, L. J.; Suk, J. Y.; Bazhenova, L.; Fleming, S. M.; Wood, M.;
Jiang, Y.; Guo, L. T.; Mizisin, A. P.; Kisilevsky, R.; Shelton, G. D.;
Balch, W. E.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,
11125–30.
† Department of Chemistry and The Skaggs Institute for Chemical
Biology.
‡ Department of Molecular and Experimental Medicine.
§ Department of Molecular Biology and The Skaggs Institute for
Chemical Biology.
(7) Coelho, T. Curr. Opin. Neurol. 1996, 9, 355–9.
(8) Dobson, C. M. Nature 2003, 426, 884–90.
(1) Balch, W. E.; Morimoto, R. I.; Dillin, A.; Kelly, J. W. Science 2008,
319, 916–9.
(9) Cohen, F. E.; Kelly, J. W. Nature 2003, 426, 905–9.
(10) Selkoe, D. J. Nature 2003, 426, 900–4.
(2) Powers, E. T.; Morimoto, R. I.; Dillin, A.; Kelly, J. W.; Balch, W. E.
Annu. ReV. Biochem. 2009, 78, 959–91.
(11) Johnson, S. M.; Wiseman, R. L.; Sekijima, Y.; Green, N. S.; Adamski-
Werner, S. L.; Kelly, J. W. Acc. Chem. Res. 2005, 38, 911–21.
(12) Monaco, H. L.; Rizzi, M.; Coda, A. Science 1995, 268, 1039–41.
(13) Blake, C. C.; Geisow, M. J.; Oatley, S. J.; Rerat, B.; Rerat, C. J. Mol.
Biol. 1978, 121, 339–56.
(3) Cohen, E.; Bieschke, J.; Perciavalle, R. M.; Kelly, J. W.; Dillin, A.
Science 2006, 313, 1604–10.
(4) Sekijima, Y.; Wiseman, R. L.; Matteson, J.; Hammarstrom, P.; Miller,
S. R.; Sawkar, A. R.; Balch, W. E.; Kelly, J. W. Cell 2005, 121, 73–
85.
(14) Hornberg, A.; Eneqvist, T.; Olofsson, A.; Lundgren, E.; Sauer-
Eriksson, A. E. J. Mol. Biol. 2000, 302, 649–69.
(5) Hammarstrom, P.; Wiseman, R. L.; Powers, E. T.; Kelly, J. W. Science
2003, 299, 713–6.
(15) Sacchettini, J. C.; Kelly, J. W. Nat. ReV. Drug DiscoVery 2002, 1,
267–75.
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Choi et al.
in the blood and cerebrospinal fluid (CSF).11,12,16-18 Although
TTR serves as the major carrier of thyroxine in the CSF, TTR
is a minor carrier in blood because of the presence of two other
T4 carrier proteins, thyroid binding globulin and albumin. Thus,
more than 99% of TTR’s T4 binding sites in the blood are
unoccupied.11
Amyloidogenesis of one of over 100 thermodynamically less
stable variants of TTR4,32,33 appears to cause familial amyloid
polyneuropathy (FAP), a peripheral neuropathy, often exhibiting
autonomic nervous system involvement.34 The most common
FAP variant is V30M-TTR, affecting largely the Portuguese,
Swedish, and Japanese populations.35-37 Central nervous system
selective amyloidosis (CNSA) appears to be a rarer disease
associated with the deposition of the most destabilized TTR
variants (e.g., A25T- and D18G-TTR) in the brain, but not in
the periphery.4,38,39 The choroid plexus secreting these variants
into the CSF appears to be a more permissive secretor of
unstable and misfolding-prone TTR variants than the liver,
which extensively degrades these highly destabilized variants
instead of secreting them into the blood, explaining why the
peripheral tissues are not subjected to A25T- and D18G-TTR
amyloidogenesis.4,38–40
There are currently no FDA-approved drugs or accepted
therapeutic strategies for treating SSA and FAC. The only
therapeutic strategy currently being practiced for ameliorating FAP
is gene therapy mediated by liver transplantation, wherein the
disease-associated TTR variant producing liver is replaced by a
WT-TTR producing liver, substantially reducing disease-associated
variant TTR levels in the blood.41-43 The drawbacks of utilizing
liver transplantation to treat FAP include the shortage of livers,
the significant surgical risk, life-long immune suppression require-
ments, and the expense. In addition, WT-TTR continues to deposit
in the heart post-transplantation, leading to cardiomyopathy,
suggesting an aging-associated decline in the proteostasis
capacity of the heart.1,2,44-46 Because of the limited applicability
of liver transplantation to treat the TTR amyloidoses (not
applicable to SSA, the disease with the most patients) and
especially the risk of death from transplant complications, it is
highly desirable to develop a general chemotherapeutic strategy
for ameliorating the TTR amyloidoses.11,47,48 Toward this end,
we have been developing kinetic stabilizers of TTR, small
molecules that differentially stabilize the native tetrameric structure
Transthyretin is one of more than 30 nonhomologous human
amyloidogenic proteins, whose misfolding and/or misassembly
appears to elicit the proteotoxicity and cell degeneration thought
to cause the amyloidoses.4,7,11,19 Amyloidogenesis from TTR
secreted by the liver appears to require rate-limiting tetramer
dissociation, which affords nonamyloidogenic folded monomers
that must undergo partial denaturation to misassemble into a
variety of aggregate structures, including cross-ꢀ-sheet amyloid
fibrils.20-25 TTR amyloidogenesis occurs by a thermodynami-
cally favorable or downhill aggregation reaction, and not by a
nucleated polymerization that governs many other amyloido-
genesis processes.26 Amyloidogenesis could also compete with
TTR monomer folding in the endoplasmic reticulum of hepa-
tocytes, although this source of proteotoxicity is still under
debate. The demonstrated effectiveness of a kinetic stabilizer
in a placebo-controlled clinical trial for polyneuropathy suggests
that dissociation of the TTR tetramer is the predominant process
that leads to TTR proteotoxicity.4 Accumulation of either wild
type (WT) TTR or mutant TTR aggregates outside of cells, and
possibly later inside certain cells, appears to cause the neuro-
degeneration and/or organ degeneration characteristic of the TTR
amyloidoses.
Amyloidogenesis of WT-TTR within the heart leads to the
sporadic amyloid disease, senile systemic amyloidosis (SSA),
a late onset cardiomyopathy that affects up to 20% of the aged
population.27-29 Familial amyloid cardiomyopathy (FAC) ap-
pears to be caused by the deposition of one of a few TTR
mutants within the heart, the most common variant deposited
being V122I-TTR, a mutation found in 3-4% of African
Americans that appears to confer complete penetrance of
FAC.30,31 Both SSA and FAC result from TTR proteotoxicity
in trans, as heart tissue does not synthesize TTR.
(32) Hammarstrom, P.; Jiang, X.; Hurshman, A. R.; Powers, E. T.; Kelly,
J. W. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16427–32.
(33) Hurshman Babbes, A. R.; Powers, E. T.; Kelly, J. W. Biochemistry
2008, 47, 6969–84.
(34) Andrade, C. Brain 1952, 75, 408–27.
(16) White, J. T.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
13019–24.
(35) Plante-Bordeneuve, V.; Said, G. Curr. Opin. Neurol. 2000, 13, 569–
73.
(17) Wojtczak, A.; Luft, J.; Cody, V. J. Biol. Chem. 1992, 267, 353–7.
(18) Soprano, D. R.; Herbert, J.; Soprano, K. J.; Schon, E. A.; Goodman,
D. S. J. Biol. Chem. 1985, 260, 1793–8.
(36) Coelho, T.; Chorao, R.; Sausa, A.; Alves, I.; Torres, M. F.; Saraiva,
M. J. Neuromusc. Disord. 1996, 6, 27.
(37) Saraiva, M. J. Amyloid 2003, 10, 13–6.
(19) Reixach, N.; Deechongkit, S.; Jiang, X.; Kelly, J. W.; Buxbaum, J. N.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 2817–22.
(20) Colon, W.; Kelly, J. W. Biochemistry 1992, 31, 8654–60.
(21) Lai, Z.; Colon, W.; Kelly, J. W. Biochemistry 1996, 35, 6470–82.
(22) Lashuel, H. A.; Lai, Z.; Kelly, J. W. Biochemistry 1998, 37, 17851–
64.
(38) Sekijima, Y.; Hammarstrom, P.; Matsumura, M.; Shimizu, Y.; Iwata,
M.; Tokuda, T.; Ikeda, S.; Kelly, J. W. Lab. InVest. 2003, 83, 409–
17.
(39) Hammarstrom, P.; Sekijima, Y.; White, J. T.; Wiseman, R. L.; Lim,
A.; Costello, C. E.; Altland, K.; Garzuly, F.; Budka, H.; Kelly, J. W.
Biochemistry 2003, 42, 6656–63.
(23) Liu, K.; Cho, H. S.; Lashuel, H. A.; Kelly, J. W.; Wemmer, D. E.
Nat. Struct. Biol. 2000, 7, 754–7.
(40) Gambetti, P.; Russo, C. Nephrol. Dial. Transplant 1998, 13, 33–40.
(41) Holmgren, G.; Ericzon, B. G.; Groth, C. G.; Steen, L.; Suhr, O.;
Andersen, O.; Wallin, B. G.; Seymour, A.; Richardson, S.; Hawkins,
P. N. Lancet 1993, 341, 1113–6.
(24) Jiang, X.; Smith, C. S.; Petrassi, H. M.; Hammarstrom, P.; White,
J. T.; Sacchettini, J. C.; Kelly, J. W. Biochemistry 2001, 40, 11442–
52.
(42) Tan, S. Y.; Pepys, M. B.; Hawkins, P. N. Am. J. Kidney Dis. 1995,
26, 267–85.
(25) Palaninathan, S. K.; Mohamedmohaideen, N. N.; Snee, W. C.; Kelly,
J. W.; Sacchettini, J. C. J. Mol. Biol. 2008, 382, 1157–67.
(26) Hurshman, A. R.; White, J. T.; Powers, E. T.; Kelly, J. W. Biochemistry
2004, 43, 7365–81.
(43) Suhr, O. B.; Herlenius, G.; Friman, S.; Ericzon, B. G. LiVer Transplant
2000, 6, 263–76.
(44) Olofsson, B. O.; Backman, C.; Karp, K.; Suhr, O. B. Transplantation
2002, 73, 745–51.
(27) Westermark, P.; Bergstrom, J.; Solomon, A.; Murphy, C.; Sletten, K.
Amyloid 2003, 10, 48–54.
(45) Dubrey, S. W.; Davidoff, R.; Skinner, M.; Bergethon, P.; Lewis, D.;
Falk, R. H. Transplantation 1997, 64, 74–80.
(28) Westermark, P.; Sletten, K.; Johansson, B.; Cornwell, G. G. Proc.
Natl. Acad. Sci. U.S.A. 1990, 87, 2843–5.
(46) Yazaki, M.; Tokuda, T.; Nakamura, A.; Higashikata, T.; Koyama, J.;
Higuchi, K.; Harihara, Y.; Baba, S.; Kametani, F.; Ikeda, S. Biochem.
Biophys. Res. Commun. 2000, 274, 702–6.
(29) Buxbaum, J. N.; Tagoe, C. E. Annu. ReV. Med. 2000, 51, 543–69.
(30) Jiang, X.; Buxbaum, J. N.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A.
2001, 98, 14943–8.
(47) Tojo, K.; Sekijima, Y.; Kelly, J. W.; Ikeda, S. I. Neurosci. Res. 2006,
56, 441–9.
(31) Jacobson, D. R.; Pastore, R. D.; Yaghoubian, R.; Kane, I.; Gallo, G.;
Buck, F. S.; Buxbaum, J. N. N. Engl. J. Med. 1997, 336, 466–73.
(48) Sekijima, Y.; Dendle, M. A.; Kelly, J. W. Amyloid 2006, 13, 236–49.
9
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Transthyretin Kinetic Stabilizers
A R T I C L E S
of TTR over the dissociative transition state, making the barrier
for TTR tetramer dissociation too high to surmount under physi-
ological conditions, thus preventing TTR amyloidogenesis.5,11,49-56
A phase II/III placebo-controlled clinical trial has recently been
completed by FoldRx Pharmaceuticals for one of the kinetic
stabilizers synthesized by our laboratory, demonstrating that it halts
neurodegeneration (www.foldrx.com). The demonstrated utility of
a TTR kinetic stabilizer to ameliorate a TTR amyloid disease is
further supported by human genetic evidence. The incorporation
of T119M-TTR trans-suppressor subunits into TTR heterotetramers
otherwise composed of FAP-associated subunits kinetically stabi-
lizes the TTR tetramer and ameliorates FAP amyloidogenesis in
Portuguese compound heterozygotes.5,36,49
optimize the three substructures comprising a typical TTR
kinetic stabilizer, the two aromatic rings (aryl-X and aryl-
Z), and the linker-Y connecting them (Figure 1B).58–60 In
each paper, we rank-ordered the numerous possibilities for
one substructure, based on their ability to inhibit TTR fibril
formation and to bind selectively to TTR in human plasma,
the remainder of the structure being constant. We also
evaluated the highly ranked substructures for their ability to
bind to the thyroid hormone receptor or cyclooxygenase.58–60
We created a so-called efficacy score, eq 1 (discussed in detail
below) integrating TTR amyloid inhibition efficacy data
(dependent on the extent of kinetic stabilization of TTR) and
plasma TTR binding stoichiometry data into one score.
The energetically weaker dimer-dimer interface of tet-
rameric TTR,54 bisected by the crystallographic C2 axis
(Figure 1A), creates the two funnel-shaped T4 binding
pockets.11–14,17,57 To date, we have synthesized over one
thousand small molecules that exhibit structural complemen-
tarity to the T4 binding sites within TTR and bind with a
range of affinities and cooperativities.5,11,15,47,48,52,55–72 We
have recently conducted a systematic three-part study to
(100%-%F.F.ave) × (1 + P.S.ave
)
average efficacy score )
300%
(1)
While structure-based drug design principles were not
employed implicitly in these studies, we were aware that one
of the most highly ranked aryl-X substructures strongly prefers
to bind in the outer thyroxine binding subsite (Figure 1B, bottom
left) for reasons outlined below. In addition, we knew that the
trans -CHdCH- and -CH2-CH2- linkers maximized the
hydrophobic effect upon binding to TTR. Numerous TTR·(kinetic
stabilizer)2 crystal structures reveal the key molecular interac-
tions required for high affinity binding to the two T4
sites.57–60,62–67 Pertinent to this study, aryl-X rings bearing a
4-hydroxyl substituent flanked by bromides strongly prefer to
bind in the outer binding subsite, bridging subunits by making
salt bridging interactions with the Lys-15/15′ ε-ammonium
groups and by maximizing occupancy of halogen binding
pockets (HBPs) 1 and 1′.58 Electron-withdrawing substituents
at the 3 and 5 positions (e.g., Br, Cl) flanking a 4-hydroxyl
group result in phenolate formation (pKa lowering) at neutral
pH.58 Benzoxazole, biphenyl, biphenylether, and biphenylamide
TTR cocrystal structures all reveal that the 3,5-dibromo-4-
hydroxylphenyl substructure comprising them strongly prefers
to be placed in the outer binding site to make Lys15 salt
bridges.59,60 Hence, we expect the 3,5-dibromo-4-hydroxyphenyl
ring comprising the stilbenes and dihydrostilbenes used in this
study to occupy the outer T4 binding pocket. Moreover, we
expect the trans double bond linker and the -CH2-CH2- linker
used in this exercise to interact with the hydrophobic side chains
of Leu17/17′, Ala108/108′, Leu110/110′, and Val121/121′,
maximizing the hydrophobic effect and thus binding affinity,
(49) Hammarstrom, P.; Schneider, F.; Kelly, J. W. Science 2001, 293, 2459–
62.
(50) Lai, Z.; McCulloch, J.; Lashuel, H. A.; Kelly, J. W. Biochemistry 1997,
36, 10230–9.
(51) Foss, T. R.; Kelker, M. S.; Wiseman, R. L.; Wilson, I. A.; Kelly,
J. W. J. Mol. Biol. 2005, 347, 841–54.
(52) Wiseman, R. L.; Johnson, S. M.; Kelker, M. S.; Foss, T.; Wilson,
I. A.; Kelly, J. W. J. Am. Chem. Soc. 2005, 127, 5540–51.
(53) Wiseman, R. L.; Green, N. S.; Kelly, J. W. Biochemistry 2005, 44,
9265–74.
(54) Foss, T. R.; Wiseman, R. L.; Kelly, J. W. Biochemistry 2005, 44,
15525–33.
(55) Miroy, G. J.; Lai, Z.; Lashuel, H. A.; Peterson, S. A.; Strang, C.; Kelly,
J. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15051–6.
(56) Peterson, S. A.; Klabunde, T.; Lashuel, H. A.; Purkey, H.; Sacchettini,
J. C.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12956–60.
(57) Klabunde, T.; Petrassi, H. M.; Oza, V. B.; Raman, P.; Kelly, J. W.;
Sacchettini, J. C. Nat. Struct. Biol. 2000, 7, 312–21.
(58) Johnson, S. M.; Connelly, S.; Wilson, I. A.; Kelly, J. W. J. Med. Chem.
2008, 51, 260–70.
(59) Johnson, S. M.; Connelly, S.; Wilson, I. A.; Kelly, J. W. J. Med. Chem.
2008, 51, 6348–58.
(60) Johnson, S. M.; Connelly, S.; Wilson, I. A.; Kelly, J. W. J. Med. Chem.
2009, 52, 1115–25.
(61) Petrassi, H. M.; Johnson, S. M.; Purkey, H.; Chiang, K. P.; Walkup,
T.; Jiang, X.; Powers, E. T.; Kelly, J. W. J. Am. Chem. Soc. 2005,
127, 6662–71.
(62) Razavi, H.; Palaninathan, S. K.; Powers, E. T.; Wiseman, R. L.; Purkey,
H. E.; Mohamedmohaideen, N. N.; Deechongkit, S.; Chiang, K. P.;
Dendle, M. T.; Sacchettini, J. C.; Kelly, J. W. Angew. Chem., Int. Ed.
2003, 42, 2758–61.
57
based on crystal structures of WT-TTR ·(Resveratrol (1))2
(63) Purkey, H. E.; Palaninathan, S. K.; Kent, K. C.; Smith, C.; Safe, S. H.;
Sacchettini, J. C.; Kelly, J. W. Chem. Biol. 2004, 11, 1719–28.
(64) Oza, V. B.; Smith, C.; Raman, P.; Koepf, E. K.; Lashuel, H. A.;
Petrassi, H. M.; Chiang, K. P.; Powers, E. T.; Sachettinni, J.; Kelly,
J. W. J. Med. Chem. 2002, 45, 321–32.
(Figure 1A and C) and the complexes between WT-TTR and
other stilbenes having only one of the two aromatic rings bearing
substituents.59
On the basis of the data from the substructure optimization
studies,58–60 we tested the hypothesis that we could produce a
small molecule library that is rich in potent and highly selective
kinetic stabilizers by simply combining the most highly ranked
substructures. We utilized the highly ranked 3,5-dibromo-4-
hydroxy-substituted aryl-X ring and isosteric variants thereof
(replacing the hydroxyl group with functional groups like NH2),
a variety of highly ranked aryl-Z rings, as well as desirable
hydrophobic linkers (trans -CHdCH- and -CH2-CH2-) to
demonstrate that the substructure combination strategy affords
a library with a high proportion of potent and highly selective
(65) Adamski-Werner, S. L.; Palaninathan, S. K.; Sacchettini, J. C.; Kelly,
J. W. J. Med. Chem. 2004, 47, 355–74.
(66) Petrassi, H. M.; Klabunde, T.; Sacchettini, J. C.; Kelly, J. W. J. Am.
Chem. Soc. 2000, 122, 2178–92.
(67) Johnson, S. M.; Petrassi, H. M.; Palaninathan, S. K.; Mohamedmo-
haideen, N. N.; Purkey, H.; Nichols, C.; Chiang, K. P.; Walkup, T.;
Sacchettini, J. C.; Sharpless, K. B.; Kelly, J. W. J. Med. Chem. 2005,
48, 1576–87.
(68) Wojtczak, A.; Cody, V.; Luft, J. R.; Pangborn, W. Acta Crystallogr.,
Sect. D: Biol. Crystallogr. 1996, 52, 758–65.
(69) Baures, P. W.; Oza, V. B.; Peterson, S. A.; Kelly, J. W. Bioorg. Med.
Chem. 1999, 7, 1339–47.
(70) Baures, P. W.; Peterson, S. A.; Kelly, J. W. Bioorg. Med. Chem. 1998,
6, 1389–401.
(71) McCammon, M. G.; Scott, D. J.; Keetch, C. A.; Greene, L. H.; Purkey,
H. E.; Petrassi, H. M.; Kelly, J. W.; Robinson, C. V. Structure 2002,
10, 851–63.
(72) Green, N. S.; Palaninathan, S. K.; Sacchettini, J. C.; Kelly, J. W. J. Am.
Chem. Soc. 2003, 125, 13404–14.
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Figure 1. Substructure combination strategy to identify potent and selective TTR kinetic stabilizers. (A) Ribbon diagram depiction of the crystal structure (PDB accession
code 1DVS) of resveratrol (1) bound to the two thyroxine (T4) binding pockets within the WT-TTR tetramer, with the monomer subunits individually
colored. The top portion represents an expanded view of one T4 binding site with 1 bound with a “Connelly” analytical molecular surface applied to residues
within 8 Å of ligand in the T4 binding pocket (green ) hydrophobic, purple ) polar). (B) Schematic depiction of the substructure combination strategy to
create TTR kinetic stabilizers (top). The aryl-X and aryl-Z rings as well as the linker-Y can be varied to generate candidate TTR kinetic stabilizers. The most
highly ranked aryl-X, aryl-Z, and linker-Y substructures from previous studies, based on potency and plasma TTR binding stoichiometry, are combined in
this substructure combination strategy to create potent and selective TTR kinetic stabilizers that nearly eliminate amyloidogenesis associated cytotoxicity.
The inner and outer T4 binding subsites within one schematically represented TTR T4 binding site are labeled in red font. (C) The innermost halogen binding
pockets (HBPs) 3 and 3′ (labeled in A) are composed of the methyl and methylene groups of Ser117/117′, Thr119/119′, and Leu110/110′. HBPs 2 and 2′
(labeled in A) are made up by the side chains of Leu110/110′, Ala109/109′, Lys15/15′, and Leu17/17′. The outermost HBPs 1 and 1′ (labeled in A) are lined
by the methyl and methylene groups of Lys15/15′, Ala108/108′, and Thr106/106′. This figure was generated using the program MOE (2006.08), Chemical
Computing Group, Montreal, Canada.
TTR kinetic stabilizers/amyloidogenesis inhibitors. Structural
studies reinforce prior data on the binding preference of the
3,5-dibromo-4-hydroxyphenyl substructure to the outer T4
binding subsite58–60 and, importantly, reveal the strong prefer-
ence of the 3,5-dibromo-4-aminophenyl substructure to bind to
the inner T4 binding subsite. The ability of all of the TTR kinetic
stabilizers tested in this study to nearly eliminate TTR-induced
cytotoxicity in the recently developed cell culture assay suggests
that they merit further study as potential drug candidates to
ameliorate the human TTR amyloidoses.
Results
Having established previously that a stilbene comprising one
3,5-dibromo-4-hydroxyphenyl ring and one unsubstituted aryl
ring exhibits a binding stoichiometry to TTR of 1.5 out of a
maximum of 2 in human plasma and completely inhibits TTR
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1362 J. AM. CHEM. SOC. VOL. 132, NO. 4, 2010

Transthyretin Kinetic Stabilizers
A R T I C L E S
Figure 2. Evaluation of the potency and selectivity of stilbene-based TTR kinetic stabilizers. Percent (%) fibril formation (F.F.) values are in black
font representing the extent of in Vitro WT-TTR (3.6 µM) fibril formation in the presence of inhibitor (7.2 µM) relative to aggregation in the absence
of inhibitor (assigned to be 100%). The TTR tetramer binding stoichiometry of potent aggregation inhibitors (defined as those exhibiting <10% F.F.)
bound to human plasma TTR ex ViVo is shown in blue font (10.8 µM kinetic stabilizer concentration, maximum binding stoichiometry is 2 due to the
two thyroxine binding sites per TTR tetramer). Extent of competitive binding of potent (based on % F.F. as defined above) and highly selective
(defined as those exhibiting a human plasma TTR binding stoichiometry >1.5) TTR kinetic stabilizers to the thyroid hormone receptor is shown in
red font. Individual efficacy scores (as defined by eq 2) of TTR kinetic stabilizers are shown in green font, whereas average efficacy scores (defined
by eq 1) are shown at the bottom of the columns (reflecting the average value in a column) and at the right side of the rows, reflecting the average
value of the compounds in that row.
amyloidogenesis (7.2 µM;59 see below), herein we first set out
to optimize the structure of stilbene-based TTR kinetic stabilizers
using previously determined aryl-Z ring efficacy scores to guide
this substructure combination strategy. Second, we used the
substructure combination strategy to evaluate the highly ranked
-CH2-CH2- linker, readily synthesized by hydrogenation of
the double bond of the stilbenes. Third, we used the substructure
combination strategy to demonstrate that by further optimizing
the aryl-X ring we could remove an off-target binding activity.
The chemical purity of all compounds used in this study was
g95%, Figures S1-S3.
Inhibition of Acid-Mediated WT-TTR Amyloid Formation
by Candidate Kinetic Stabilizers. The efficacy with which
stilbene and dihydrostilbene library members inhibit WT-TTR
amyloidogenesis was evaluated using the previously established
acid-mediated fibril formation assay.20,58–60 Briefly, each com-
pound (7.2 µM, the minimum concentration required to occupy
both T4 binding sites) was preincubated with a physiologically
relevant concentration of WT-TTR (3.6 µM) for 30 min, and
TTR amyloidogenesis was initiated by adjusting the pH to 4.4.
The extent of TTR fibril formation was quantified by measuring
turbidity, previously shown to be equivalent to thioflavin T
quantification. At this pH, a 90% yield of TTR fibril formation
is observed after a 72 h incubation period in the absence of a
TTR kinetic stabilizer. TTR amyloidogenesis in the presence
of a candidate kinetic stabilizer was expressed as a percentage
relative to that exhibited by WT-TTR in the absence of inhibitor
(assigned to be 100%). Potent TTR kinetic stabilizers allow
<10% of WT-TTR fibril formation at a concentration of 7.2
µM and <40% of WT-TTR fibril formation at a concentration
equal to that of WT-TTR (3.6 µM) (0% fibril formation ) 100%
inhibition).5,15,55,56,58–60,62,64–67,69–72 Of the 87 stilbene- and
dihydrostilbene-based kinetic stabilizers synthesized and evalu-
ated here, 80 are excellent amyloidogenesis inhibitors, allowing
<10% of WT-TTR fibril formation at a concentration of 7.2
µM (>90% inhibition, Figures 2-4, black font ) % fibril
formation). Compounds exhibiting more than 95% inhibition
were reevaluated at a concentration equal to the TTR tetramer
(3.6 µM). At this concentration, the library of compounds allows
between 15% and 30% of WT-TTR fibril formation (Figures
S4-S6), demonstrating that the substructure combination
strategy is capable of producing potent TTR kinetic stabilizers.
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A R T I C L E S
Choi et al.
Figure 3. Evaluation of the potency and selectivity of dihydrostilbene-based WT-TTR kinetic stabilizers. This figure is organized and defined strictly
analogous to the descriptions in the legend to Figure 2.
Binding Stoichiometry of Potent Kinetic Stabilizers to
TTR in Human Blood Plasma. The 80 stilbene and dihydro-
stilbene kinetic stabilizers that allowed <10% of WT-TTR fibril
formation in Vitro were further evaluated for their ability to bind
selectively to TTR in human blood plasma (3.6-5.4 µM) using
the established ex ViVo TTR plasma binding selectivity assay.73
Briefly, the candidate kinetic stabilizer (10.8 µM) was incubated
with human blood plasma at 37 °C for 24 h. Any unbound
inhibitors, endogenous small molecules, or macromolecules that
could bind to the resin used in the next step were removed by
additionofquenchedsepharoseresin.73 TheTTRandTTR ·(kinetic
stabilizer)n complexes in the plasma were immunocaptured using
a sepharose-resin-conjugated anti-TTR antibody. After extensive
washing, TTR and any TTR ·small molecule complexes bound
to the resin were dissociated by high pH treatment. The ratio
of TTR monomer to bound candidate kinetic stabilizer was
calculated from HPLC peak areas using standard curves.73 The
numbers in blue font in Figures 2-4 represent the average
candidate kinetic stabilizer binding stoichiometry to TTR in
human blood plasma, the maximum being 2, due to two
thyroxine binding sites per TTR tetramer. Of the 80 potent
(<10% fibril formation) TTR aggregation inhibitors evaluated,
Figure 4. Effect of replacement of a hydroxyl group by an amino group
in selected TTR kinetic stabilizers. Percent fibril formation (% F.F.) values
(black font), plasma TTR binding stoichiometry (blue font), T3 displacement
only three kinetic stabilizers display modest binding stoichi-
ometry (<1 equivalent bound), whereas 77 inhibitors exhibit
from thyroid hormone receptor (red font), as well as individual efficacy
scores (green font) are shown, precisely as defined in Figure 2, with the
exception that average efficacy scores are not shown.
(73) Purkey, H. E.; Dorrell, M. I.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A.
2001, 98, 5566–71.
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A R T I C L E S
average binding stoichiometries exceeding 1 equivalent bound
per tetramer. Notably, 37 of these display exceptional binding
selectivity to plasma TTR, exhibiting >1.5 equivalents bound
per TTR tetramer. It is reassuring that the substructure combina-
tion strategy yields stilbenes and dihydrostilbenes that generally
exhibit very high individual efficacy scores (eq 2), reflecting
desirable amyloid inhibition efficacy and plasma TTR binding
selectivity (Figures 2-4, numbers in green font, 1 being a
perfect kinetic stabilizer).
(100%-%F.F.) × (1 + P.S.)
individual efficacy score )
300%
(2)
Average efficacy scores (eq 1) are also depicted in bold black
font in Figures 2 and 3 in the bottom row and the rightmost
column. In this calculation, we average % fibril formation (%
F.F.ave) and plasma TTR binding stoichiometry (P.S.ave) values
in each column where the substituent(s) is constant but the
positioning on the aryl ring varies, and in each row where the
substituent(s) vary but the positioning on the aryl ring is
constant.
Evaluating TTR Kinetic Stabilizers for Thyroid Hormone
Receptor Binding. Because the majority of the members of our
stilbene and dihydrostilbene library are composed of the 3,5-
dibromo-4-hydroxyphenyl thyroxine-like substructure, we were
concerned about their binding selectivity to TTR over the thyroid
hormone receptor. Thus, TTR kinetic stabilizers were evaluated
for their ability to bind to the thyroid hormone receptor [analyses
carried out by Cerep laboratories in Redmond, WA (see
Experimental Section for details)]. The results are expressed as
percent displacement of the control radioligand (125I-labeled
triiodothyronine, [125I]-T3). Greater than 20% displacement of
Figure 5. The influence of kinetic stabilizer binding on the rate of TTR
tetramer dissociation. WT-TTR (1.8 µM) tetramer dissociation time courses
in 6 M urea without (9) and in the presence of putative kinetic stabilizers
9d, 13c, 20d, 23c, 24c, and 24e at a concentration of 3.6 µM (A) and 1.8
µM (B), evaluated by linking the slow tetramer dissociation process to rapid
and irreversible monomer denaturation in 6 M urea, as measured by far-
UV circular dichroism at 215-218 nm over a time course of 144 h.
[
125I]-T3 by candidate kinetic stabilizers indicates undesirable
binding selectivity in plasma (Figure 4, numbers in blue font).
Notably, none of these compounds bind to the thyroid hormone
receptor (Figure 4, percentage in red font), suggesting that
further aryl-X optimization may be an effective strategy to
reduce TTR kinetic stabilizer thyroid hormone receptor binding.
This limited series exhibits impressive individual efficacy scores
(Figure 4, numbers in green font). Moreover, as discussed in
detail below, the distinct inner binding subsite preference of
the 3,5-dibromo-4-aminophenyl substructure nicely comple-
ments the outer binding site preference of the 3,5-dibromo-4-
hydroxyphenyl substructure discussed above, providing another
substructure to include in future substructure combination
strategies.
Demonstrating That Potent Amyloidogenesis Inhibitors
Are Kinetic Stabilizers of WT-TTR. TTR fibril formation is rate
limited by tetramer dissociation.5,20–26 Thus, the imposition of
kinetic stabilization on TTR by small molecule binding to the
TTR tetramer can be measured by the rate of tetramer dissocia-
tion, which is assessed by linking the slow tetramer dissociation
step to rapid monomer unfolding, easily quantified using far-
UV circular dichroism (CD) or fluorescence spectroscopy.5,11,54
To demonstrate kinetic stabilization of TTR, we preincubated
WT-TTR (1.8 µM) with candidate kinetic stabilizers (1.8 or
3.6 µM) for 30 min. Dissociation was then accelerated and made
measurable on a convenient laboratory time scale by adding
urea to a final concentration of 6 M. The rate and extent of
dissociation was monitored by the thermodynamically linked
monomer unfolding of WT-TTR at 25 °C, followed by far-UV
CD over 144 h. All candidate kinetic stabilizers at a concentra-
tion of 3.6 µM dramatically slowed dissociation, allowing less
than 15% of the TTR to dissociate (Figure 5A). Tetramer
library members. Of the 41 TTR kinetic stabilizers evaluated,
nearly one-half (11 stilbene analogues and 10 dihydrostilbene
analogues) exhibit insignificant thyroid hormone receptor bind-
ing (<20%) (Figures 2-4, % values in red font). These
compounds are promising lead TTR kinetic stabilizers. Given
the rarity of thyroid hormone receptor binding in the previous
study that identified the 3,5-dibromo-4-hydroxy substructure as
optimal,58 it was disappointing, but perhaps not shocking, that
one-half of the stilbenes and dihydrostilbenes tested bound to
thyroid hormone receptor.
Replacing the Phenolic Group in 3,5-Dibromo-4-
hydroxyphenyl-Based Stilbenes. In a modest effort toward
further aryl-X substructure optimization, we asked whether we
could substitute the hydroxyl group in the context of a simple
stilbene composed of one unsubstituted aryl ring (aryl-Z) and
one ring bearing 3,5-dibromo-substituents and a variable 4-posi-
tion substituent without losing potency and selectivity. We
considered a variety of functional groups at the 4-position
(Figure S7). The amino group was equivalent to the hydroxyl
group in terms of potency, an encouraging result as anilines
are functional groups in FDA-approved drugs. However, some
anilines are carcinogenic in animals, and possibly in humans.
Motivated by this result, four stilbenes with aniline-based aryl-X
rings and substituted aryl-Z substituents were synthesized
(Figure 4, compounds 24b-e) to assess both potency and
selectivity. Given the limited number of compounds that were
synthesized and the diversity of Z-substituents, the potency of
these compounds (Figure 4, % F.F. numbers in black font) was
impressive, demonstrating the merit of further aryl-X optimiza-
tion. Moreover, three of these compounds exhibit excellent TTR
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A R T I C L E S
Choi et al.
dissociation was also clearly slowed at a kinetic stabilizer
concentration of 1.8 µM, equal to that of the TTR tetramer,
allowing 20-33% tetramer dissociation, indicating that the
binding of one small molecule to one of the two T4 binding
sites is sufficient to stabilize the ground state over the dissocia-
tive transition state, imposing kinetic stabilization on the entire
TTR tetramer (Figure 5B).
Inhibition of WT- and V30M-TTR-Induced Cytotoxicity
by Potent Kinetic Stabilizers. Previous studies demonstrate that
treatment of cell lines derived from tissues that are targets of
TTR deposition with recombinant WT-TTR or V30M-TTR
homotetramers results in proteotoxicity.19,74,75 Moreover, these
studies establish that TTR cytotoxicity can be inhibited by small
molecules that kinetically stabilize TTR against dissociation.19,74
Previous experiments conclude that partially folded monomers
and/or oligomers resulting from TTR dissociation and misas-
sembly are the major cytotoxic species in cell culture.19
Resveratrol (1) is known to bind to and kinetically stabilize WT-
and V30M-TTR and reduce their cytotoxicity, and therefore was
used as the positive control. WT-TTR and V30M-TTR were
preincubated without and with equimolar amounts of candidate
kinetic stabilizers or 1 at 4 °C for 18 h. The human neuroblas-
toma IMR-32 cells were treated with these solutions (8 µM final
concentration of TTR and compounds) for 24 h at 37 °C, after
which cell viability was assessed by the resazurin reduction
assay.76 Viable cells reduce the substrate resazurin to resorufin,
a fluorescent compound, which was monitored by fluorescence
emission at 590 nm. The results are expressed as the percentage
of fluorescence generated by viable cells treated with vehicle
only (100% viable). Cells treated with cytotoxic TTR in the
absence of kinetic stabilizers exhibit 45-65% viability. All of
the kinetic stabilizers tested nearly completely inhibit WT-TTR
and V30M-TTR-induced cytotoxicity at a final concentration
equimolar to that of TTR (Figure 6A). Importantly, none of
the compounds evaluated were cytotoxic to IMR-32 cells in
the absence of TTR (>80% cell viability (Figure 6B)). Thus,
the substructure combination strategy affords compounds that
protect against TTR proteotoxicity.
Figure 6. (A) Demonstration that kinetic stabilizers of WT-TTR and
V30M-TTR can prevent cytotoxicity associated with the process of TTR
amyloidogenesis. IMR-32 human neuroblastoma cells were treated with WT-
TTR (8 µM, white bar) or V30M-TTR (8 µM, gray bar), exhibiting
cytotoxicity that is prevented by preincubating WT-TTR (white bars) or
V30M-TTR (gray bars) with the stilbene and dihydrostilbene-based kinetic
stabilizers or resveratrol (1) (a kinetic stabilizer previously shown to be
effective) included as a positive control (8 µM each). Cell viability was
measured after 24 h by the resazurin reduction assay, and all of the
experimental conditions were compared to cells treated with vehicle only
(100% cell viability). (B) Cytotoxicity of selected compounds (8 µM) to
the human neuroblastoma cell line IMR-32. Columns represent the average
values of two independently performed experiments (six experimental
replicates). The error bars represent standard error.
stilbene 13c, composed of a 3,5-dibromo-4-hydrophenyl aryl-X
ring and a p-amino aryl-Z ring, a mixture of two binding
orientations is observed in the electron density. The 3,5-dibromo-
4-hydroxyphenyl ring occupies the T4 outer binding subsite
∼90% of the time for reasons described above, orientating the
p-amino group from the aryl-Z ring to make bridging hydrogen
bonds with Ser117/117′ side chains (Figure 7B). In the minor
binding orientation, the 3,5-dibromo-4-hydroxyphenyl ring of
13c occupies the inner binding subsite and the phenol/phenolate
makes hydrogen bonds with Ser117/117′ side chains, while the
bromides occupy HBPs 3 and 3′ (Figure 7B).
In stilbenes 24c-e, the 3,5-dibromo-4-aminophenyl substruc-
ture was placed in the inner binding subsite in all cases. This
binding orientation appears to be stabilized by bridging hydrogen
bonds between the amino substituent and the Ser117/117′
hydroxyl side chains and by simultaneous placement of the
bromides into HBPs 3 and 3′ (Figure 7D-F). The p- or m-amino
substituent on the other ring in 24d and 24e does not appear to
interact with TTR, and thus the aryl-Z ring can be further
optimized by the substructure combination strategy. The
dimethoxyphenyl groups on 24c were able to interact with HBPs
1 and 1′ (Figure 7D).
Crystallographic
Analysis
of
WT-TTR ·(Kinetic
Stabilizer)2 Complexes. Crystal structures of WT-TTR in
complex with 3d, 13c, 17d, and 24c-e were determined to 1.70,
1.40, 1.40, 1.32, 1.40, and 1.48 Å resolutions, respectively (see
Table 1 for data collection and refinement statistics). In all six
structures, the electron density was clear and allowed unam-
biguous placement of the ligand (unbiased 2Fo - Fc electron
density maps contoured at 3σ shown in Figure S8). As
anticipated, the 3,5-dibromo-4-hydroxyphenyl rings of stilbene
3d and dihydrostilbene 17d contribute to binding by their
placement in the complementary T4 outer binding subsite,58,59
positioning the bromides in HBPs 1 and 1′. The phenolate anion
of 3d and 17d appears to engage in a salt bridge interaction
with the ε-ammonium group of Lys15/15′ (Figure 7A and C).
The 2,6-dichlorophenyl ring in 3d and 17d occupies the inner
binding subsite, directing the chloride atoms into HBPs 3 and
3′, stacking the face of the aryl ring between the two symmetry-
related Leu110 residues (Figure 7A and C). In the case of
(74) Reixach, N.; Adamski-Werner, S. L.; Kelly, J. W.; Koziol, J.;
Buxbaum, J. N. Biochem. Biophys. Res. Commun. 2006, 348, 889–
97.
In all six structures, the trans -CHdCH- or -CH2-CH2-
linker occupies the hydrophobic pocket created by residues
Leu17/17′, Ala108/108′, Leu110/110′, and Val121/121′. These
linkers afford the aryl rings some degree of rotational freedom
(75) So¨rgjerd, K.; Klingstedt, T.; Lindgren, M.; Ka˚gedal, K.; Hammarstro¨m,
P. Biochem. Biophys. Res. Commun. 2008, 377, 1072–8.
(76) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem. 2002,
267, 5421–6.
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A R T I C L E S
Table 1. Crystallographic Data and Refinement Statistics
WT-TTR·3d
WT-TTR·13c
WT-TTR·17d
Data Collection
APS GM/CA-CAT 23-IDB SSRL 11-1
WT-TTR·24c
WT-TTR·24d
WT-TTR·24e
beamline
wavelength (Å)
resolution (Å)
SSRL 9-2
0.9194
1.70 (1.70-1.76)
P21212
SSRL 11-1
0.9797
1.31 (1.31-1.36) 1.40 (1.40-1.45) 1.48 (1.48-1.53)
P21212 P21212 P21212
SSRL 11-1
0.9797
SSRL 11-1
0.9797
1.0333
0.9797
a
1.40 (1.40-1.45)
P21212
1.40 (1.40-1.45)
P21212
space group
a, b, c (Å)
42.92, 85.0, 64.50 42.26, 84.79, 63.24
42.50, 85.11, 63.32 42.8, 85.38, 64.39 42.7, 85.29, 64.2 42.78, 85.16, 64.16
no. of molecules in the au
no. of observations
no. of unique reflections
redundancy
2
2
2
2
2
2
a
322489 (31176)
44988 (4391)
7.2 (7.1)
98.8 (98.0)
5 0.0 (57.4)
28.3 (3.6)
165495 (16232)
45971 (4509)
3.6 (3.6)
99.7 (99.6)
4.0 (45.2)
26.1 (3.1)
268463 (20385)
56323 (4972)
4.8 (4.1)
97.6 (87.3)
3.1 (46.1)
37.5 (2.8)
220973 (18236) 192765 (18569)
46374 (4342)
4.8 (4.2)
98.2 (93.6)
2.9 (48.3)
45.5 (2.2)
376642 (30797)
26903 (2588)
14.0 (11.9)
99.5 (97.0)
5.7 (69.5)
39462 (3951)
4.9 (4.7)
97.7 (99.4)
3.4 (52.3)
36.5 (3.3)
a
a
completeness (%)
b
b
Rsym (%)
a
average I/σ
34.6 (7.3)
Refinement Statistics
resolution (Å)
no. of reflections (working set)
1.70-64.55
1.40-84.82
42702 (2843)
2257 (154)
16.3 (21.4)
19.0 (27.0)
1.40-63.37
43555 (3170)
2332 (177)
16.5 (19.8)
19.7 (25.8)
1.31-85.44
53423 (3720)
2869 (203)
16.7 (28.8)
19.7 (30.7)
1.40-85.13
44010 (2940)
2340 (142)
16.8 (25.3)
19.0 (32.7)
1.47-85.13
37452 (2478)
1981 (121)
16.7 (23.4)
20.0 (26.7
a
25229 (1781)
a
no. of reflections (test set)
1333 (93)
c
a c
,
Rcryst (%)
Rfree (%)
16.9 (17.9)
21.6 (26.4)
d
a d
,
Average B-Values
TTR
ligand
Wilson B-value
22.3
45.3
23.6
17.1
20.4R, 27.2F
19.0
14.7
15.9
17.1
18.6
18.4
18.2
20.5
28.6
22.0
19.6
26.4
21.7
Ramachandran Plot
most favored (%)
92.1
7.9
0
93.0
7.0
0
91.1
8.9
0
93.0
7.0
0
93.5
6.5
0
93.5
6.5
0
additionally allowed (%)
generously allowed (%)
disallowed (%)
0
0
0
0
0
0
RMS Deviations
0.018
bond lengths (Å)
angles (deg)
0.019
1.81
0.018
1.71
0.016
1.78
0.018
1.64
0.018
1.61
1.74
b
c
d
a Numbers in parentheses are for the highest resolution shell of data. Rsym ) ∑hkl|I - 〈I〉|∑hklI. Rcryst ) ∑hkl|Fo - Fc|∑hklFo. Rfree is the same as
Rcryst, but for 5% of data excluded from the refinement. F/R ) forward or reverse conformations of the ligand.
enabling optimal placement of the two substituted rings into
HBPs 1, 1′ and 3, 3′.
a concentration twice that of TTR, for which plasma binding
stoichiometry was not assessed, were assumed to have an
efficacy score <0.57.
Discussion
The individual efficacy scores displayed in Figure 3 reveal
that the linker substructure combination strategy was also very
effective. All of the dihydrostilbenes examined exhibit an
efficacy score >0.57 out of a maximum of 1. We did not prepare
quite as many dihydrostilbenes as we did stilbenes, and it could
be argued that we hydrogenated the stilbenes exhibiting high
efficacy scores; hence it is probably not appropriate to conclude
that dihydrostilbenes are superior to stilbenes. Nonetheless, we
sampled quite a range of substituents and substitution patterns
in both libraries, indicating that the substructure combination
strategy was quite effective in the context of both stilbenes and
dihydrostilbenes.
Surprisingly, only 30% of the promising stilbene compounds
evaluated displaced <20% of T3 from the thyroid hormone
receptor. This was unexpected because the compounds resulting
from the individual substructure optimization studies displayed
much less T3 displacement.58–60 However, the problem of T3
displacement seems to be easily remedied by a linker replace-
ment affected by simply hydrogenating the thyroid receptor
binding stilbenes. More than 76% of the promising dihydros-
tilbenes evaluated displaced <20% of T3 from the thyroid
hormone receptor. Another means of correcting the T3 displace-
ment problem could be to perform additional aryl-X substructure
optimization and combination strategies.
A substructure combination strategy was employed herein,
based on previous aryl-X, linker-Y, and aryl-Z substructure
rankings,58–60 to guide the synthesis of a library of stilbenes
and dihydrostilbenes. This effort afforded a high fraction of
potent and highly selective TTR kinetic stabilizers that rescue
cells from the cytotoxic effects of TTR amyloidogenesis
thought to cause the human amyloidoses, without exhibiting
cytotoxicity themselves. The stilbene and dihydrostilbene
libraries depicted in Figures 2 and 3 were made up of
combinations of substructural components exhibiting high
average efficacy scores in the aforementioned three-part
study.58–60 Even though more of the 3,5-dibromo-4-hydrox-
yphenyl-based stilbenes and dihydrostilbenes afforded thyroid
hormone binding capacity than the previous substructure optimiza-
tion studies would have predicted,58–60 a further aryl-X ring and/
or aryl-Z substructure optimization strategy may produce potent
and selective compounds lacking this property.
We calculated individual efficacy scores according to eq 2
for each compound synthesized and evaluated in this study,
Figures 2-4. The individual efficacy scores displayed in Figure
2 reveal that the substructure combination strategy was very
effective, in that 96% of the stilbenes examined exhibit an
efficacy score >0.57 out of a maximum of 1. The efficacy score
of 0.57 was chosen as a benchmark, because this is the efficacy
score of the compound that ameliorated familial amyloid
polyneuropathy in a placebo-controlled clinical trial (www-
.foldrx.com). Compounds exhibiting >10% fibril formation at
The structural data presented herein provide further evidence
for the strong preference of the 3,5-dibromo-4-hydroxyphenyl
ring to occupy the outer binding subsite of TTR in the context
of stilbenes and dihydrostilbenes (Figure 7A-C). In stark
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J. AM. CHEM. SOC. VOL. 132, NO. 4, 2010 1367

A R T I C L E S
Choi et al.
Figure 7. Crystal structures of homotetrameric WT-TTR in complex with inhibitors 3d, 13c, 17d, 24c, 24d, and 24e. Ribbon diagram depiction of a
close-up view of one of the two identical T4 binding sites (see Figure 1A). A “Connelly” analytical molecular surface was applied to residues within 8 Å
of ligand in the T4 binding pocket (green ) hydrophobic, purple ) polar). Polar residues K15 and S117/117′ are shown with bonds depicted where interactions
are observed. In the case of stilbene 13c, a mixture of two binding orientations is observed. The 3,5-dibromo-4-hydroxyphenyl ring occupies the thyroxine
outer binding subsite ∼90% of the time (see Results for a more complete description and explanation). This figure was generated using the program MOE
(2006.08), Chemical Computing Group, Montreal, Canada.
contrast, the structural data reveal that the 3,5-dibromo-4-
aminophenyl ring strongly prefers to occupy the inner binding
subsite in the context of stilbenes (Figure 7D-F). On the basis
of this result, we predict that stilbenes and dihydrostilbenes
composed of one 3,5-dibromo-4-hydroxyphenyl ring, which
prefers to bind in the outer thyroxine binding subsite, and one
3,5-dibromo-4-aminophenyl ring, which prefers to bind to the
inner thyroxine binding subsite, will exhibit high individual
efficacy scores. The substructure combination strategy used
herein to generate potent and selective stilbene and dihydros-
tilbene TTR kinetic stabilizers could be successfully applied to
generate potent and selective 2-aryl benzoxazole-based TTR
kinetic stabilizers as well. Not only is the heteroaromatic ring
of a benzoxazole a highly ranked linker,59 but previous studies
that were much more limited in scope demonstrate that
benzoxazoles are excellent TTR kinetic stabilizers.62
The kinetic studies carried out herein demonstrate that
selective binding of small molecules to the native tetrameric
state of TTR drastically slows the rate of tetramer dissociation,
by increasing the energy barrier for dissociation. We also
showed that the kinetic stabilization of TTR imposed by the
binding of these compounds precludes TTR-mediated cytotox-
icity using a recently introduced cytotoxicity assay, by prevent-
ing tetramer dissociation that leads to monomers and oligomers
previously shown to be the cytotoxic species. Because amy-
loidogenesis-linked neurotoxicity and cardiotoxicity is thought
to cause FAP and SSA, respectively, we envision that this assay,
not utilized in the prior substructure optimization studies, will
be useful for predicting compounds that will show efficacy in
human patients.
Conclusions
We have successfully utilized the data generated in three
previous substructure optimization papers to identify potent and
selective stilbene and dihydrostilbene kinetic stabilizers using
a substructure combination strategy. We also showed that this
strategy is useful for eliminating off-target activities, such as
thyroid hormone receptor binding. Notably, the majority of the
kinetic stabilizers emerging from the substructure combination
strategy exhibit high individual efficacy scores and prevent TTR
amyloidogenesis-associated cytotoxicity in a cell culture model
that we believe is predictive for kinetic stabilizer efficacy in
the human TTR amyloidoses. There is reason to be optimistic
that a subset of the potent and selective stilbene- and dihydros-
tilbene-based TTR kinetic stabilizers produced in this study will
exhibit the appropriate pharmacokinetic and pharmacodynamic
properties to motivate their further development.
Experimental Section
Wild Type Transthyretin Fibril Formation Assay. WT-TTR
was expressed and purified from an E. coli expression system as
described previously.77 To assess fibril formation, a test compound
(5 µL of a 1.44 mM solution in DMSO) was added to 495 µL of
(77) Lashuel, H. A.; Wurth, C.; Woo, L.; Kelly, J. W. Biochemistry 1999,
38, 13560–73.
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Transthyretin Kinetic Stabilizers
A R T I C L E S
a solution of TTR (0.4 mg/mL) in 10 mM phosphate, 100 mM
KCl, and 1 mM EDTA (pH 7.0) in a disposable cuvette. The
mixture was vortexed and incubated at room temperature for 30
min. An acidic buffer solution (500 µL of 100 mM acetate, 100
mM KCl, 1 mM EDTA, pH 4.2) was then added to decrease the
pH of assay solution to 4.4. The cuvettes were incubated at 37 °C
for 72 h without agitation. After the solution was vortexed to evenly
distribute any precipitate, the turbidity of the solution at 400 nm
was measured using a Hewlett-Packard model 8453 UV-vis
spectrophotometer.
TTR/test compounds (100 µL) were added to 900 µL of a 6.67 M
urea solution in 10 mM sodium phosphate buffer, 100 mM KCl,
and 1 mM EDTA (pH 7.0) to give a final concentration of 1.8 µM
for TTR and final concentrations of test compound of 1.8 µM (1X)
and 3.6 µM (2X), respectively. The mixtures were vortexed and
incubated in the dark at 25 °C without agitation. CD spectra at a
final urea concentration of 6 M were measured at 215-218 nm
(0.5 nm steps and five times scan) after 0, 5, 10, 25, 48, 72, 96,
120, and 144 h of incubation.
IMR-32 Cell-Based Assay. Cells. The IMR-32 human neuro-
blastoma cell line was maintained in Opti-MEM (cell culture
medium, Invitrogen), supplemented with 5% fetal bovine serum, 1
mM Hepes buffer, 2 mM L-glutamine, 100 units/mL penicillin,100
µg/mL streptomycin, and 0.05 mg/mL CaCl2 (complete cell
medium). The 2-3-day-old cultures (70% confluent) were used for
the cytotoxicity experiments.
Recombinant WT-TTR and V30M-TTR purified at 4 °C and
capable of amyloidogenesis were used as cytotoxic insults to IMR-
32 cells. The proteins were buffer exchanged in Opti-MEM at 10 °C
using a Centriprep device (10 kDa MWCO, Millipore).
Stocks of the TTR kinetic stabilizers were prepared at 1 mM in
0.5% DMSO/Opti-MEM and stored at -20 °C. For the experiments,
test compounds (32 µM in Opti-MEM) were mixed 1:1 with
filtered-sterilized TTR (32 µM in Opti-MEM) or with Opti-MEM
only, vortexed, and incubated for 18 h at 4 °C. TTR (16 µM) alone
and Opti-MEM alone containing the same amount of DMSO used
above were prepared in parallel and incubated under the same
conditions.
IMR-32 cells were seeded in black wall, clear bottom, 96-well
tissue culture plates (Costar) at a density of 6000 cells/well in
complete cell medium and incubated overnight at 37 °C. The
incubated TTR/test compound samples, TTR and Opti-MEM, were
diluted 1:1 with freshly prepared Opti-MEM supplemented with
0.8 mg/mL bovine serum albumin, 2 mM Hepes buffer, 4 mM
L-glutamine, 200 units/mL penicillin, 200 µg/mL streptomycin, and
0.1 mg/mL CaCl2. The medium from the cells was then removed
and replaced immediately by the TTR/test compound mixtures, TTR
or Opti-MEM. The 96-well plates were spun at 2000g for 30 min
at 4 °C to allow the IMR-32 cells to reattach to the bottom of the
wells. The cells were then incubated 24 h at 37 °C, after which
cell viability was measured. The final concentration of both TTR
and test compound was 8 µM.
Evaluating the Binding Stoichiometry of Candidate
Kinetic Stabilizers to TTR in Human Blood Plasma. The plasma
TTR binding selectivity assay that evaluates the binding stoichi-
ometry of a test compound to TTR in human blood plasma has
been previously described73 and is used with minor modifications.
Briefly, to a 1 mL sample of human blood plasma in a 2 mL
Eppendorf tube was added a test compound (7.5 µL of a 1.44 mM
solution in DMSO), and then the plasma solution was incubated at
37 °C for 24 h on a rocker plate (30 rpm). A 1:1 (v/v) slurry of
unfunctionalized sepharose resin in TSA (10 mM Tris, 140 mM
NaCl, pH 8.0) buffer was added, and the solution was incubated
for 1 h at 4 °C on a rocker plate (18 rpm). The solution was then
centrifuged, and the supernatant was divided into two 400 µL
aliquots, which were added to 200 µL of 1:1 (v:v) slurry of anti-
TTR antibody conjugated sepharose resin in TSA. The solution
was gently rocked (18 rpm) at 4 °C for 20 min, then centrifuged,
and the supernatant removed. The resin was washed three times
by shaking for 1 min with 1 mL of TSA containing 0.05% saponin
and then twice more with 1 mL of TSA. After centrifugation to
remove the supernatant, 155 µL of triethylamine (100 mM, pH 11.5)
was added to the sepharose resin to dissociate the TTR and bound
test compound from the resin, and the suspension was vortexed
for 1 min. After centrifugation, 144 µL of the supernatant containing
the test compound and TTR was neutralized by addition of 0.84
µL of glacial acetic acid before HPLC analysis. The supernatant
was analyzed by reverse phase HPLC, as described previously,73
on a Waters 600 E multisolvent delivery system, using a Waters
486 tunable absorbance detector, a 717 autosampler, and a
ThermoHypersil Keystone Betabasic-18 column (150 Å pore size,
3 µm particle size). The “A” mobile phase comprises 0.1% TFA
in 94.9% H2O + 5% CH3CN, and the “B” mobile phase is made
up of 0.1% TFA in 94.9% CH3CN + 5% H2O. Linear gradients
were run from 100:0 A:B to 0:100 A:B for 9 min.
Cell Viability Assay. The viability of the cells treated with TTR
or TTR/test compound mixtures was evaluated by resazurin
reduction assay. Briefly, 10 µL/well of resazurin (500 µM in PBS)
was added to each well and incubated for 2 h at 37 °C. Viable
cells reduce resazurin to the highly fluorescent resorufin dye, which
is quantitated in a multiwell plate reader (Exc/Em 530/590 nm,
Tecan Safire2, Austria). Cell viability was calculated as percentage
of fluorescence relative to cells treated with vehicle only (100%
viability) after subtraction of blank fluorescence (wells without
cells). All of the experimental conditions were performed at least
in triplicate. Averages and standard error corresponding to two
independently performed experiments were calculated using Graph-
Prism (San Diego, CA).
Binding of Potent TTR Amyloidogenesis Inhibitors to the
Thyroid Hormone Receptor. The potential binding of TTR kinetic
stabilizers to the thyroid hormone receptor was evaluated by Cerep
laboratories in Redmond, WA, using the following experimental
protocol. Membrane homogenates of liver (100 µg of total protein)
are incubated for 18 h at 4 °C with 0.1 nM [125I]T3 in the absence
or presence of a TTR kinetic stabilizer in buffer (20 mM Tris-
HCl, pH 7.6, 50 mM NaCl, 2 mM EDTA, 10% glycerol, and 5
mM ꢀ-mercaptoethanol). Nonspecific binding is corrected for in
the presence of 1 µM T3. Following the incubation period, the
samples are filtered rapidly under vacuum through glass fiber filters
(GF/B, Packard) and rinsed several times with an ice-cold buffer
containing 50 mM Tris-HCl and 150 mM NaCl (pH 7.4), employing
a 96-sample cell harvester (Unifilter, Packard). The filters are dried,
then counted for radioactivity in a scintillation counter (Topcount,
Packard) using a scintillation cocktail (Microscint 0, Packard).
Urea-Induced Dissociation Kinetics Study. Slow TTR tetramer
dissociation is not detectable by far-UV circular dichroism (CD)
spectroscopy; however, dissociation is linked to rapid (∼500 000×
faster) monomer unfolding under denaturing conditions, which is
easily detectable by far-UV CD spectroscopy. Test compounds (5
and 10 mM in DMSO) were diluted 10-fold in EtOH to give 0.5
and 1 mM stock solutions. Such stocks (7.2 µL) were added to
200 µL of TTR (18 µM in 10 mM sodium phosphate buffer, 100
mM KCl, 1 mM EDTA (pH 7.0)) in 2 mL Eppendorf tubes. These
mixtures were briefly vortexed and incubated for 30 min at 25 °C.
Crystallization and Structure Determination of the
WT-TTR/Ligand Complexes. The TTR protein was concentrated
to 4 mg/mL in 10 mM sodium phosphate buffer and 100 mM KCl
(pH 7.6) and cocrystallized at room temperature with a 5 molar
excess of each ligand using the vapor-diffusion sitting drop method.
All crystals were grown from 1.395 M sodium citrate, 3.5% v/v
glycerol at pH 5.5. The crystals were cryo-protected with 10% v/v
glycerol. Data for 13c were collected at beamline GM/CA-CAT
23-IDB at the Advanced Photon Source (APS) at a wavelength of
1.0333 Å. Data for 3d were collected at beamline 9-2 and data for
17d and 24c-e at 11-1 at the Stanford Synchrotron Radiation
Lightsource (SSRL) at wavelengths of 0.9194 and 0.9795 Å,
respectively. All diffraction data were indexed, integrated, and
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J. AM. CHEM. SOC. VOL. 132, NO. 4, 2010 1369

A R T I C L E S
Choi et al.
scaled using HKL200078 in space group P21212 with two subunits
observed per asymmetric unit. The structure was determined by
molecular replacement using the model coordinates of 2FBR72 in
the program Phaser.79 Further model building and refinement were
completed using Refmac.80 Hydrogens were added during refine-
ment, and anisotropic B-values were calculated. Final models were
validated using the JCSG quality control server incorporating
Molprobity,81 ADIT (http://rcsb-deposit.rutgers.edu/validate) WHA-
TIF,82 Resolve,83 and Procheck.84 Data collection and refinement
statistics are displayed in Table 1.
Protein Data Bank Accession Code. Atomic coordinates and
structure factors have been deposited in the Protein Data Bank and
are available under accession codes 3IMR (WT-TTR in complex
with 3d), 3IMT (WT-TTR in complex with 13c), 3IMS (WT-TTR
in complex with 17d), 3IMW (WT-TTR in complex with 24c),
3IMU (WT-TTR in complex with 24d), and 3IMV (WT-TTR in
complex with 24e).
X-ray diffraction data for 13c were collected at GM/CA-CAT 23-
IDB beamline at the Advanced Photon Source, Argonne National
Laboratory. Use of the Advanced Photon Source was supported
by the U.S. Department of Energy, Office of Basic Energy Sciences,
under Contract No. W-31-109-Eng-38. X-ray diffraction data
collection for 3d, 17d, and 24c-e was carried out at the Stanford
Synchrotron Radiation Laboratory, a national user facility operated
by Stanford University on behalf of the U.S. Department of Energy,
Office of Basic Energy Sciences. The SSRL Structural Molecular
Biology Program is supported by the Department of Energy, Office
of Biological and Environmental Research, and by the National
Institutes of Health, National Center for Research Resources,
Biomedical Technology Program, and the National Institute of
General Medical Sciences. We would also like to acknowledge Drs.
Stanfield, Dai, Yoon, Xu, and Ekiert for assisting with X-ray data
collection.
Acknowledgment. We thank the NIH (DK46335 to J.W.K. and
CA58896 and AI42266 to I.A.W.), the American Heart Association
(0865061F to N.R.), as well as the Skaggs Institute for Chemical
Biology and the Lita Annenberg Hazen Foundation for financial
support. Technical support from Mike Saure and Gina Dendle is
also greatly appreciated. We thank the General Clinical Research
Center of the Scripps Research Institute for providing human blood.
Note Added after ASAP Publication. Due to a production error
a typographical correction has been made to indicate “. . .87
stilbene- and dihydrostilbene-based kinetic stabilizers. . .” in the
section entitled Inhibition of Acid-Mediated WT-TTR Amyloid
Formation by Candidate Kinetic Stabilizers; the correct version
publish ASAP on January 6, 2010.
Supporting Information Available: Detailed synthesis, char-
acterization, and purities of stilbenes and dihydrostilbenes,
efficacy of stilbenes and dihydrostilbenes at a concentration of
3.6 µM, evaluation of a variety of related functional groups to
replace the hydroxyl group, unbiased 2Fo - Fc electron density
maps for the ligands in the WT-TTR·(kinetic stabilizer)2
complexes, and cytotoxicity of selected kinetic stabilizers to
the IMR-32 cell line. This material is available free of charge
via the Internet at http://pubs.acs.org.
(78) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307–26.
(79) Storoni, L. C.; McCoy, A. J.; Read, R. J. Acta Crystallogr., Sect. D:
Biol. Crystallogr. 2004, 60, 432–8.
(80) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallogr.,
Sect. D: Biol. Crystallogr. 1997, 53, 240–55.
(81) Lovell, S. C.; Davis, I. W.; Arendall, W. B., III; de Bakker, P. I.;
Word, J. M.; Prisant, M. G.; Richardson, J. S.; Richardson, D. C.
Proteins 2003, 50, 437–50.
(82) Vriend, G. J. Mol. Graphics 1990, 8, 52–6.
(83) Terwilliger, T. C. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2003,
59, 38–44.
(84) Laskowski, R.; MacArthur, M.; Moss, D.; Thornton, J. J. Appl.
Crystallogr. 1993, 26, 283–91.
JA908562Q
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