Schematic depiction of one of the two T₄ binding pockets within TTR occupied by a typical small molecule TTR aggregation inhibitor. Y represents a linker of variable chemical structure (e.g. NH, O, CH-CH, C(O)NH, etc.) joining the two aryl rings, which typically bear a combination of alkyl, carboxyl, halide, trifluoromethyl, or hydroxyl substituents (X and Z). Library 2, being the focus of this manuscript, explores the SAR of the linker-Y substructure. While the aryl-X substructure was initially envisioned to bind within the inner cavity of the thyroxine binding site, previous crystallographic data reveal that molecules can bind opposite to the expected orientation.26, 27, 29, 34, 36 Thus, it may not be the case that the aryl-X substructure of an inhibitor will occupy the inner binding cavity as illustrated. For conceptual simplicity in terms of integrating the data from the three inhibitor substructure optimization libraries, this initial schematic has been retained.

Efficacy scoring of the different linker-Y substructures calculated from the TTR aggregation inhibition and plasma binding stoichiometry data. Percent (%) values represent the extent of WT-TTR fibril formation in vitro in the presence of inhibitor (7.2 μM inhibitor, 3.6 μM TTR, pH 4.4, 37°C, 72 h) relative to aggregation in the absence of inhibitor (100%), with the best values shown in red (< 20% aggregation; errors are typically less than ± 5 percentage points). The binding stoichiometries of the most potent aggregation inhibitors bound to TTR in human blood plasma ex vivo are shown in italics (10.8 μM inhibitor incubated with 1.8−5.4 μM TTR; theoretical maximum binding stoichiometry = 2). Those exhibiting exceptional binding selectivity to TTR are boxed (errors are typically less than ± 0.1). The efficacies of the different linkers were quantitatively scored by entering the average % fibril formation (% F.F._ave) and average TTR plasma binding stoichiometry (P.S._ave) values into equation 1 (values averaged over the four inhibitors in each linker series – compounds displaying > 20% fibril formation were assigned TTR plasma binding stoichiometries of 0 for these calculations). Higher efficacy scores correspond to more potent and selective substructures.

X-ray crystallography structures and schematics of key features of homotetrameric WT-TTR co-crystallized with inhibitors 2c, 2d, 3d, 6d, and 10d (PDB ascession codes 3CN0, 3CN1, 3CN2, 3CN3, 3CN4, respectively). Individual TTR monomers in the three-dimensional ribbon diagrams have been color coded red, yellow, green, and blue for differentiation. Zoomed images show inhibitors bound in their two symmetry-related binding modes (orange and grey) in one of the two symmetrical thyroxine binding sites, appearing superimposed on one another owing to their positioning on the crystallographic C₂ axis. The ordered water molecules bridging the Ser117 and 117’ hydroxyls of adjacent TTR monomers in the TTR•(2c)₂ and TTR•(6d)₂ structures are shown as red spheres. Important hydrogen bonding and electrostatic interactions between protein side chains and the ligands (or H₂O, as in the case of TTR•(2c)₂ and TTR•(6d)₂ structures) are indicated by arrows, with distances shown in Å. Schematic representations of each bound inhibitor are presented as two-dimensional topology diagrams (generated using MOE (2006.08), Chemical Computing Group, Montreal, Canada). In these schematic diagrams, inhibitors are shown in only one of their symmetry-related binding modes for clarity. A graphical legend for interpretation of key binding site characteristics is displayed below the TTR tetramer ribbon diagram at the top left. Ligand exposure indicates specific portions of the ligand structures that are solvent accessible (i.e. not completely buried within the binding pocket). Residue exposure indicates those amino acids for which their side chains and/or peptide backbones are partially solvent accessible.

Crystal structure (2ROX) of thyroxine (T₄) bound within the two pockets created by the weak dimer-dimer interface of tetrameric TTR (the two dimer-dimer interfaces are bisected by the dashed lines).26 The expanded view of one site shows T₄ bound in its symmetry-related binding modes (green and yellow), with the binding site surface shown in grey (figure adapted from reference 26). Primed amino acids or halogen binding pockets (HBPs) refer to symmetry-related monomers of TTR. Two structural series of inhibitors are shown below, the bisaryloxime ethers28 and biphenyls29 (R = variable substitution patterns of fluorine, chlorine, trifluoromethyl, carboxyl, or hydroxyl substituents), many of which are excellent TTR kinetic stabilizers that display high plasma TTR binding selectivity.

Displayed are four potent TTR amyloidosis inhibitors (compounds 1a-d) reported previously in our aryl-X optimization study.27 The bisaryl scaffolds under evaluation in the current study are based on these four potent aryl-X substructures (a-d) to screen linker-Y substructures (2−10) for their ability to confer potent and selective inhibition of TTR amyloidogenesis, while minimizing COX-1 inhibition and thyroid hormone receptor binding.

Thyroid hormone receptor T₃ displacement and COX-1 inhibition for the most potent aggregation inhibitors. The extent of inhibitor that displaces T₃ from the thyroid hormone receptor is shown in red italics (errors are typically less than ±2 percentage points). COX-1 inhibition results are shown below in black, with values representing the % inhibition by the test compounds of COX-1 mediated conversion of arachidonic acid to prostaglandin-E₂ (errors are typically less than ±6 percentage points).