Potent Bivalent Amyloidosis Inhibitors
A R T I C L E S
Figure 1. Schematic representation of the tetrameric structure of transthyretin. There are two symmetry-related thyroxine binding sites per tetramer. The
two binding sites are connected by a central channel that runs through the TTR tetramer. Bivalent inhibitors are envisioned to bind with one substructure in
each of the two thyroxine binding sites connected by a linking group that threads the channel.
The two identical funnel-shaped thyroxine binding sites
located at the TTR dimer-dimer interface are interconverted
by two C2 axes oriented perpendicular to the crystallographic
2-fold axis (z-axis), Figure 1.9 The two binding sites are
connected by a very narrow channel centered on the z-axis.
Using both limited screening and structure-based design, we
have previously reported a large number of compounds that are
capable of inhibiting TTR fibril formation by binding to the
thyroxine sites.9,10 Previous studies demonstrate that the most
dramatic reduction of TTR fibril formation occurs when both
T4 binding sites are occupied by inhibitors that bind with high
affinity and exhibit slow dissociation rates.11 Ligands that bind
with high affinity to both sites (Kd1 and Kd2 < 10 nM) stabilize
the TTR‚inhibitor2 complex to the extent that the activation
energy for the rate-limiting step of amyloidogenesis (tetramer
dissociation) is no longer available under most denaturing con-
ditions, including acid-mediated amyloidogenesis conditions.11
inhibitors bind, there is a change in the second TTR binding
site as discerned from NMR titration experiments.13 One can
covalently link two different inhibitors, each known to have
high affinity for TTR’s T4 binding sites, in the search for potent
bivalent inhibitors. Optimally the linker should be fairly rigid
and designed to perfectly match the geometric requirements of
the central channel. This minimizes the conformational entropy
penalty associated with binding, allowing full advantage to be
taken of the fact that ∆Strans + ∆Srot only has to be paid once
for a bivalent inhibitor.12 Linkers with too much flexibility are
generally problematic because ∆Sconf g ∆Strans + ∆Srot; hence,
the added affinity derived from only having to pay ∆Strans
+
∆Srot once is lost.12 When ∆Sconf g ∆Strans + ∆Srot, it is generally
the case that inhibitors having the potential to bind in a bivalent
fashion bind to the receptor in a monovalent fashion. Mono-
valent binding of a potentially bivalent inhibitor should be
disfavored for the inhibitors synthesized herein because the
substructures selected to occupy the T4 sites have a preferred
orientation, which would need to be reversed for monovalent
binding.9,10
A single bivalent molecule that occupies both T4 sites
simultaneously could have several important advantages over
traditional monovalent inhibitors, including high binding affinity
and selectivity, as well as a slow dissociation rate. Most
monovalent TTR amyloidosis inhibitors display negatively
cooperative binding because ∆H2 is greater than ∆H1.9-11 There
is some evidence that when the first equivalent of these
Bivalent inhibitors can be administered at ca. half the dose
of traditional amyloidogenesis inhibitors, minimizing toxicity
caused by the unbound inhibitor. Recent examples of other
approaches to bivalent inhibitors include alkyl-linked benza-
midine inhibitors of human lung tryptase,14 bistetrahydro-
aminacrine inhibitors of acetylcholinesterase,15 and nonpeptide
inhibitors of the matrix metalloproteinase stromelysin.16
There are at least three potential mechanisms that would allow
bivalent inhibitors to bind both T4 binding sites simultaneously.
If one of the substructures envisioned to occupy a T4 site is
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